Mitochondria and Anaerobic Energy Metabolism in Eukaryotes: Biochemistry and Evolution 9783110612417, 9783110666779

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William F. Martin, Aloysius G. M. Tielens, Marek Mentel Mitochondria and Anaerobic Energy Metabolism in Eukaryotes

William F. Martin, Aloysius G. M. Tielens, Marek Mentel

Mitochondria and Anaerobic Energy Metabolism in Eukaryotes

Biochemistry and Evolution

Authors William F. Martin Institute of Molecular Evolution Heinrich-Heine-Universität Düsseldorf Düsseldorf Germany e-mail: [email protected]

Marek Mentel Department of Biochemistry Comenius University in Bratislava Bratislava Slovak Republic e-mail: [email protected]

Aloysius G. M. Tielens Department of Medical Microbiology and Infectious Diseases Erasmus MC University Medical Center Rotterdam Netherlands e-mail: [email protected]

ISBN 978-3-11-066677-9 e-ISBN (PDF) 978-3-11-061241-7 e-ISBN (EPUB) 978-3-11-061272-1 Library of Congress Control Number: 2020939095 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.dnb.de. © 2021 Walter de Gruyter GmbH, Berlin/Boston d|u|p Düsseldorf University Press is an imprint of Walter de Gruyter GmbH Cover Image: Dlumen / iStock / Getty Images Plus Typesetting: Integra Software Services Pvt. Ltd. Printing and binding: CPI books GmbH, Leck dup.degruyter.com

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Preface Views of eukaryote evolution continue to undergo significant change. The cell nucleus is the defining organelle of eukaryotes, the compartment that gave them their name. But in addition to the nucleus, mitochondria have become recognized as organelles ancestral to eukaryotic cells. The family of mitochondria now includes, however, reduced forms of the organelle that do not respire oxygen: hydrogenosomes and mitosomes. The discovery of hydrogenosomes in the 1970s, and the subsequent recognition that they are anaerobic forms of mitochondria, led to the finding that mitochondria – once synonymous with oxygen respiration – have a key role both in the aerobic and in the anaerobic energy metabolism of eukaryotes. Genome data have also impacted our views of phylogenetic relationships of eukaryotes and this has led to an improved understanding of the evolutionary significance of anaerobic metabolism in eukaryotes. In parallel, geochemical evidence has uncovered revolutionary new findings about the rise of oxygen in the Earth’s history. The new insights into Earth’s ancient habitats reveal that from the time of eukaryote origin roughly 1,600 million years ago and early lineage diversification of eukaryotes, up until about 500 million years ago, the Earth’s atmosphere contained a very low amount of oxygen corresponding roughly to 1% of the present atmospheric level, an oxygen level known as the Pasteur point. The Pasteur point is the level of oxygen where cells that are able to switch from oxygen respiration to anaerobic ATP synthesis and an anaerobic lifestyle, make that switch. Throughout much of that low oxygen past, the oceans were to a large extent anoxic and locally even rich in hydrogen sulfide, which is a strong inhibitor of oxygen respiration in mitochondria, hence a poison for cells that rely solely upon O2 for their ATP synthesis and redox balance. Up until about 450 million years ago, there was no appreciable life on land; all life was in the oceans, in the sediment, and in the Earth’s crust. Eukaryotes arose and diversified in anaerobic oceans. In the new view of Earth’s oxygen history, oxygen-independent pathways of eukaryotic energy metabolism in mitochondria reflect environmental conditions that dominated Earth’s history during eukaryote evolution. Those conditions were low oxygen or anaerobic. The mitochondria of eukaryotes have preserved the trace of that anaerobic past. We have many people to thank, too many to list, so we will make the list very short and specific. We wish to thank Miklós Müller for many years of friendship and dialogue on the physiology of eukaryotic anaerobes, Fred Opperdoes for many years of discussions about the biochemistry of mammalian parasites, John F. Allen for many years of discussions about oxygen and energy in evolution, Jaap van Hellemond, Sven Gould, and Sriram Garg for daily discussions on biochemistry and eukaryote evolution, and Rebecca Gerhards and Verena Zimorski for their invaluable help in preparing the manuscript.

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Preface

This book aims to provide an overview of the biochemistry and evolution of anaerobic energy metabolism in eukaryotes and, at the same time, strives to link the latest findings from biology, biochemistry, geochemistry, and biogeochemistry to form a general evolutionary picture. The work should serve as a source of information on this topic for students of biology and for faculty from various fields, including the earth sciences. William F. Martin, Aloysius G.M. Tielens, and Marek Mentel Düsseldorf, Rotterdam, and Bratislava, April 2020

Contents Preface

VII

List of figures

XIII

List of abbreviations

XV

Part I: Basics 1

Anaerobes and eukaryote origin

7

2

Eukaryotes in low oxygen environments

3

A modern context of atmospheric evolution

4

Energy metabolism and redox balance

5

Fermentation, glycolysis, and compartmentation

6

Respiration is not always aerobic

7

Using oxygen can be optional 42 7.1 An electron-transport chain with biosynthetic function 7.2 The Crabtree effect: fermentation in the presence of O2 7.3 The Warburg effect: aerobic glycolysis in cancer cells

13 18

23

36

8

The hypoxia-inducible factor (HIF)

9

O2 dependent fermentations in trypanosomes

10 Anaerobic mitochondria

26

48 52

58

11 Mitochondria with and without oxygen

61

12 Hydrogenosomes and H2-producing mitochondria 13 Mitosomes and microaerophilia

68

64

42 43 46

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Contents

14 Other organelles of mitochondrial origin 15 Genomes are not alive

73

78

Part II: Well-studied examples 16 Anaerobic use of the mitochondrial electron-transport chain

85

17 Naegleria gruberi, a strict aerobe with an “anaerobic genome” 18 Malate dismutation in the liver fluke Fasciola hepatica

89

92

19 The roundworms Ascaris suum and Ascaris lumbricoides

97

20 Animals in tidal zones, anaerobic sediments and sulfide 20.1 The mollusc Mytilus edulis 103 20.2 The polychaete annelid Arenicola marina 105 20.3 The peanut worm Sipunculus nudus 110 20.4 Diverse physiological functions of H2S in animals

101

21 Anaerobic respiration in eukaryotes, rare but there 115 21.1 Nitrate respiration in Fusarium and Cylindrocarpon 22 Enzymes of anaerobic energy metabolism in algae 23 Wax ester fermentation in Euglena gracilis

127

24 Chlamydomonas reinhardtii, a jack of all trades 25 Organisms with hydrogenosomes 25.1 Discovery 136 25.2 Trichomonads 139 25.3 Chytrids 145 25.4 Ciliates 150

124

131

136

26 Nyctotherus ovalis and H2-producing mitochondria 26.1 Blastocystis hominis 155

151

112

119

Contents

27 Energy metabolism in organisms with mitosomes 27.1 Entamoeba histolytica 160 27.2 Giardia intestinalis 164 28 Energy parasites

160

168

Part III: Evolution 29 Why did mitochondria become synonymous with O2? 30 Ubiquitous mitochondria among anaerobes

176

180

31 Differential loss from a facultative anaerobic ancestral state 32 Oxygen availability in early eukaryote evolution: the Pasteurian 33 Evolution with mitochondrial energy metabolism 34 Envoi

208

Bibliography Index

249

211

201

186 190

XI

List of figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Figure 24 Figure 25 Figure 26 Figure 27 Figure 28 Figure 29 Figure 30 Figure 31 Figure 32 Figure 33 Figure 34 Figure 35

Oxygen in Earth’s history 19 A hydrogenosome 30 A mitochondrion 37 Hypoxia-inducible factor 50 Energy metabolism in bloodstream forms of trypanosomes 53 Energy metabolism in procyclic forms of trypanosomes 54 Classification of organelles of mitochondrial origin 59 The mitochondrion and the hydrogenosome head to head 65 Energy metabolism in the brain-eating amoeba Naegleria gruberi 90 Energy metabolism in the liver fluke Fasciola hepatica 94 Changes in complex II in the roundworm Ascaris suum 98 Energy metabolism in the roundworm Ascaris lumbricoides 99 Energy metabolism in the blue mussel Mytilus edulis 104 Energy metabolism in the lugworm Arenicola marina 106 The electron-transport chain in the annelid Urechis unicinctus 109 Energy metabolism in the peanut worm Sipunculus nudus 111 Mitochondrial sulfide oxidation and sulfide production 114 Steps in dissimilative nitrate reduction 116 Proposed pathways of denitrification in Fusarium oxysporum 120 Mixed acid fermentation and ammonia fermentation in Fusarium oxysporum Wax ester fermentation in Euglena gracilis 129 Energy metabolism in Chlamydomonas reinhardtii 132 Energy metabolism in Tritrichomonas foetus 138 Energy metabolism in Trichomonas vaginalis 141 The arginine dihydrolase pathway 144 Energy metabolism in Piromyces sp. E2 147 Energy metabolism in Nyctotherus ovalis 152 Possible energy metabolism in Blastocystis hominis 158 Energy metabolism in Entamoeba histolytica 163 Energy metabolism in Giardia intestinalis 166 Origin of the terms procaryotes [sic] and eucaryotes [sic] in 1925 178 Anaerobic mitochondria across eukaryote supergroups 183 Distribution of genes for energy metabolism in eukaryotes 187 Oxygen in evolution: the Pasteurian 193 Transitions in mitochondrial evolution 206

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List of abbreviations AAC ACK ACS [ADP]-ACS ADH ADHE ADP ALDH AMP AN aNar aNir AOX APS ASCT ASCT IA ASCT IB ASCT IC ATP ATP-PEPCK BIF CBS CoA CSE DHAP DHOD DNA dNar dNir ETF ETF: RO FAD FADH2 Fdh FDP Fdx Fe Fe-S cluster Fe-S protein FMN FRD G3P GAP GDP GOE GPDH GTP

ADP/ATP translocase (some authors use ANT) Acetate kinase Acetyl-CoA-synthetase Acetyl-CoA-synthetase (ADP-forming) Alcohol dehydrogenase Bifunctional alcohol/aldehyde dehydrogenase E Adenosine diphosphate Aldehyde dehydrogenase Adenosine monophosphate Alternative NADH dehydrogenase (rotenone-insensitive) Assimilatory nitrate reductase Assimilatory nitrite reductase Alternative oxidase Adenosine-5´-phosphosulfate Acetate:succinate CoA-transferase Acetate:succinate CoA-transferase of the IA protein subfamily Acetate:succinate CoA-transferase of the IB protein subfamily Acetate:succinate CoA-transferase of the IC protein subfamily Adenosine triphosphate ATP-dependent phosphoenolpyruvate carboxykinase Banded iron formation Cystathionine-β-synthase Coenzyme A Cystathionine-γ-lyase Dihydroxyacetone phosphate Dihydroorotate dehydrogenase Deoxyribonucleic acid Dissimilatory nitrate reductase Dissimilatory nitrite reductase Electron-transferring flavoprotein Electron-transferring flavoprotein:rhodoquinone oxidoreductase Flavin adenine dinucleotide Reduced flavin adenine dinucleotide Formate dehydrogenase Flavodiiron proteins Ferredoxin Iron Iron−sulfur cluster Iron−sulfur protein Flavin mononucleotide Membrane-bound fumarate reductase Glycerol-3-phosphate Glyceraldehyde-3-phosphate Guanosine diphosphate Great oxidation event Glycerol-3-phosphate dehydrogenase Guanosine triphosphate

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List of abbreviations

GTP-PEPCK hDNA HIF HydEFG ISC pathway ITP kDNA LDH LECA LGT MDH NAD+-MDH ME MLOs MOB-CoA MOP-CoA MQ mRNA MROs mtDNA NAD+ NADH NADP+ NADPH NoR NTT OMO P450nor PAL PAPS PAP PAT Pi PPi PDH PDC PEPCK ITP/GTP-PEPCK PPi-PEPCK PFL PFL-AE PFO, PFOR PGA PNO PNT pO2 PPDK PPi PTA

GTP-dependent phosphoenolpyruvate carboxykinase Hydrogenosomal DNA, hydrogenosomal genome Hypoxia-inducible factor [FeFe]-hydrogenase maturases Biosynthetic pathway of iron−sulfur cluster formation Inosine triphosphate Kinetoplast DNA, mitochondrial genome of trypanosomatids Lactate dehydrogenase Last eukaryotic common ancestor Lateral (horizontal) gene transfer Malate dehydrogenase NAD+-dependent malate dehydrogenase Malic enzyme (decarboxylating malate dehydrogenase) Mitochondria-like organelles 2-Methyl-3-oxobutanoyl-CoA 2-Methyl-3-oxopentanoyl-CoA Menaquinone Messenger RNA Mitochondria-related organelles Mitochondrial DNA, mitochondrial genome Nicotinamide adenine dinucleotide Reduced nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide phosphate Reduced nicotinamide adenine dinucleotide phosphate Nitric-oxide reductase (alternative abbreviation NO-reductase P450nor) Nucleotide transporter Organelles of mitochondrial origin Cytochrome P450 NO-reductase Present atmospheric level of oxygen 3´-Phosphoadenosine-5´-phosphosulfate Adenosine-3´,5´-bisphosphate Phosphate:acetyltransferase (phosphotransacetylase) Inorganic phosphate Pyrophosphate (inorganic diphosphate) Pyruvate dehydrogenase complex or pyruvate dehydrogenase Pyruvate decarboxylase Phosphoenolpyruvate carboxykinase ITP/GTP-dependent Phosphoenolpyruvate carboxykinase PPi-dependent phosphoenolpyruvate carboxykinase Pyruvate formate lyase Pyruvate formate lyase activation enzyme Pyruvate:ferredoxin oxidoreductase 3-phosphoglycerate Pyruvate:NADP+ oxidoreductase Pyridine nucleotide transhydrogenase Partial pressure of oxygen in the environment Pyruvate:orthophosphate dikinase (pyruvate phosphate dikinase) Pyrophosphate Phosphotransacetylase (phosphate acetyltransferase)

List of abbreviations

Q QFR MCF RQ RNA ROS rRNA SCS SD SQR ST STK SUF TIM TOM TPP tRNA UQ VDAC WHO

Generic quinone of unknown structure Quinol:fumarate reductase Mitochondrial carrier family Rhodoquinone Ribonucleic acid Reactive oxygen species Ribosomal RNA Succinyl-CoA-synthetase (succinate thiokinase) Sulfur dioxygenase Sulfide:quinone oxidoreductase Sulfurtransferase Succinate thiokinase (succinyl-CoA-synthetase) A pathway of iron−sulfur cluster formation Translocator inner Membrane Translocator outer Membrane Thiamine pyrophosphate Transfer RNA Ubiquinone Mitochondrial porin, voltage-dependent anion channel World Health Organization

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Part I: Basics

Eukaryotes are important for understanding the history of life on the blue planet. Cells on Earth are classified as prokaryotes which have no nucleus, or eukaryotes which have a cell nucleus. Eukaryotes encompass all the genuinely complex organisms, like plants, fungi, and animals. In comparison with eukaryotic cells, prokaryotic cells are less complex from the cytological, genetic, and genomic point of view. Prokaryotes encompass archaea (called archaebacteria until the 1990s) and bacteria. Prokaryotes lack a nucleus. They also do not have a complex cytoskeleton, peroxisomes, vacuoles/lysosomes, or any organelles of endosymbiotic origin − mitochondria and plastids. The flagella of prokaryotes are not homologous with eukaryotic flagella, which are based on microtubules composed of tubulin. The flagella of prokaryotes represent analogous structures: even the flagella of archaea and bacteria are not homologous, for which reason the archaeal flagellum is called an archaellum. Homologous in this book means similar by virtue of common ancestry. In general, eukaryotic cells are more complex than prokaryotic cells. Their linear chromosomes are localized in the nucleus. The nuclear membrane is contiguous with the endoplasmic reticulum, which is in contact with the Golgi apparatus through membrane vesicles. As complex as the eukaryotic nucleus might seem, it can be induced to form spontaneously in cell-free extracts. Newport (1987) showed that a mixture containing 100 µL of Xenopus egg extract, 2 mM ATP (along with 20 mM creatine phosphate and some creatine kinase to provide continuous ATP supply), and 1.6 µg of bacteriophage lambda DNA incubated at 22 °C will spontaneously form nuclei around the lambda DNA, with two lamina (inner and outer leaves of the nuclear membrane) along with nuclear pores, in about an hour. Another specific feature of eukaryotes is the presence of mitochondria in different biochemical variations: as aerobic mitochondria, (facultative) anaerobic mitochondria, hydrogenosomes, and mitosomes (Müller et al. 2012). Complex structures in eukaryotic cells require ATP for their formation; ATP synthesis in eukaryotes is the legacy of mitochondria. Eukaryotes and prokaryotes have followed different trajectories in evolution. Prokaryotes have boundless biochemical diversity, but they are always packaged into small single celled units of life. Prokaryotes can grow with a broad diversity of energy sources (chemotrophic or phototrophic), carbon sources (autotrophic or heterotrophic), and electron sources (organotrophic or lithotrophic) (Madigan and Martinko 2006). Prokaryotes can colonize environments with temperatures ranging from ca. −30 °C to ca. +115 °C and pH varying between ~3 and ~12. Eukaryotes persist in more restricted ranges of temperature (from ca. −20 °C to ca. +50 °C) and pH (from ~1 to ~10) (Nealson and Conrad 1999). In terms of carbon and energy metabolism, eukaryotes are far narrower in scope than prokaryotes. In fact, the full breadth of energy metabolic diversity known in eukaryotes is less than that found in a single, generalist bacterium like members of the genus Rhodobacter (Müller et al.

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Part I: Basics

2012), with the exception of course, of oxygenic photosynthesis in plants, which is an inheritance from cyanobacteria via the endosymbiotic origin of plastids (Zimorski et al. 2014). However, the cellular complexity of eukaryotes comes at a cost of being less robust, that is, less resistant to extreme or changing external conditions. Diversity in eukaryotic energy metabolism encompasses a narrow spectrum of electron donors and acceptors compared to prokaryotes. Moreover, the energy metabolic diversity that eukaryotes have is linked to mitochondria. The narrow sample of prokaryotic energy metabolic diversity in eukaryotes, together with the association of anaerobic energy metabolism with the mitochondrion, reflects the single endosymbiotic origin of mitochondria, the event that gave rise to eukaryotes roughly 1.5 billion years ago. Students or specialists might wonder why this book is necessary. One can just turn to the internet, google some papers on “evolution of mitochondria” or “mitochondrial evolution” or “anaerobic mitochondria” and get the goods, everything one needs to know, maybe. It is not that simple. What we actually know about energy metabolism in mitochondria comes from biochemical laboratories where people measure enzyme activities, purify enzymes, reconstitute systems, and (very important for energy metabolism) measure end products of metabolism so that when one puts it all together one has an idea of what the organism might be doing, enzymatically, in order to generate ATP from a growth substrate en route to excreting the observed end products as waste. Doing that kind of work takes years or decades before one knows what is going on in the organism (its cells, its cytosol, and its mitochondria). In the old days, the enzymes needed to be highly purified and microsequenced (Edman degradation) in order to get information about the amino acid sequence or the gene. The assignment to the function of such purified proteins is certain because an enzymatic activity copurifies with a physical entity, the chemically sequenced protein, that was present at high activity in the organism. For eukaryotes with anaerobic mitochondria, there are very few organisms where the physiological measurements have been done, and where we know what end products the organism is producing with the 10–100 genes that are devoted to energy metabolism (redox balanced ATP synthesis) out of the 30,000 genes that might be in the genome sequence (Müller 2003). The organisms that we cover in this book have physiology behind the maps. Why is that important? In the age of genomes (2020) one can obtain an automatically annotated genome sequence for a given organism at a modest price in a couple of weeks. The annotations are made by automated sequence comparisons to genome sequences in the databases that were often annotated by automated sequence comparisons as well. However, metabolic maps generated from genomes without supporting physiology can foster very misleading results. An example is the case of Naegleria gruberi, a relative of the brain-eating amoeba, Naegleria fowleri, which causes a deadly brain infection in humans that only a handful of people have ever survived and

Part I: Basics

5

that is so rapid (about a week) that the diagnosis often comes post mortem. The genome sequence (Fritz-Laylin et al. 2010) presented a map of N. gruberi metabolism based on some genes identifiable in the genome, suggested that the parasite might have an elaborate and sophisticated anaerobic metabolism of the type commonly found in anaerobic and microaerophilic eukaryotes such as Entamoeba, Giardia and Trichomonas. When one however goes to the effort of trying to grow N. gruberi anaerobically, one will see that it does not grow (Mach et al. 2018; Bexkens et al. 2018). It shuns sugars and amino acids, the substrates that the named protists use for ATP synthesis. But it devours the stuff that nerve cells have in abundance (lipids) and it has a high demand for O2 (like the brain), which goes a long way to explaining why the brain-eating amoeba is a strict aerobe and why it feeds on brain tissue (Bexkens et al. 2018). We make this point to underscore the fact that the stories that can be read from, and into, genome sequences can differ substantially from the actual physiology of the organism. The organisms that we cover in this book have some physiology behind the maps that we present, which represent the results of measurements and experiments, not merely data from genome sequencing projects. The main reactions of energy metabolism (core ATP synthesis) are catalyzed by proteins that are abundant in the cell and that are present in very high activities. Many glycolytic and Calvin cycle enzymes in eukaryotes (core carbon metabolism) can only be purified about 250-fold because they each typically constitute around 0.4% of the soluble protein in the cell. Energy metabolism is the chemical reaction that keeps us alive. A human adult who is not doing much exercise consumes about 500 liters of O2 per day and generates about a bodyweight of ATP in the process. A 60 kg runner doing 20 km/h will consume roughly 50 ml O2 per kg bodyweight per minute (Joyner and Coyle 2008), which translates to about 180 liters O2 per hour. For 500 liters of O2 consumed we generate about 500 liters of CO2, which contain about 250 grams of carbon. A marathon runner burns about 500 grams of fat from start to finish to make the ATP to move the muscles to run the race, and synthesizes close to a bodyweight of ATP in the process. We will hear a lot about redox balance in this book. Energy metabolism in eukaryotes involves redox reactions, typically generating CO2 from organic substrates. The 500 liters of O2 that a human consumes per day corresponds to about 22 moles of O2, or about 5·1025 electrons that have to change hands, most of them via NAD+ to reduced NADH that has to be reoxidized to NAD+ again in a human, daily, to keep life going. There is a strict requirement for NADH to be reoxidized, the electrons from oxidations have to be excreted as end products, otherwise life comes to a halt. That is the meaning of redox balance. In heterotrophs, the chemical reactions that release energy to support ATP synthesis involve the removal of electrons from food substrates and the deposition of those electrons onto products that can be excreted from the cell (or the organism). If the flow of electrons stops, the synthesis of ATP stops, and the chemical reaction that is life ultimately stops as a consequence. The

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Part I: Basics

evolution of energy metabolism is the evolution of staying alive. In eukaryotes, energy metabolism has everything to do with mitochondria, but in many lineages of modern eukaryotes, mitochondrial ATP synthesis does not involve O2. How that works and why it is important for understanding evolution are the subject of the book. The book is divided into three parts. Part I deals with basics and some general principles, Part II covers biochemically well-studied examples, and Part III covers evolutionary aspects. Earlier books dealing with aspects of the eukaryotic anaerobe evolution include Metazoan Life Without Oxygen edited by Bryant (1991) with an excellent overview of the metabolism of some of the animal groups that we discuss, Surviving Hypoxia edited by Hochachka et al. (1993) covering a wide range of physiological responses to low oxygen in animals, and Biochemical Adaptation by Hochachka and Somero (2002) with a comprehensive treatise on physiological responses to hypoxia in higher animals. Ecology and Evolution in Anoxic Worlds by Fenchel and Finlay (1995) is a rich source of information about modern anaerobic environments and the organisms that live there, couched in a context that however interprets photosynthetic lineages as ancient and anaerobes as recent, a view upon which Origin and Early Evolution of Life by Fenchel (2002a) is also based but that runs counter to our current interpretations. (For a differing perspective on physiological evolution linking anaerobes and photosynthesis, see Martin, Bryant and Beatty 2018.) The volume Anaerobic Fungi by Mountfort and Orpin (1994) provides an excellent overview of biology and ecology in the chytrids. Origin of Mitochondria and Hydrogenosomes (Martin and Müller 2007) contains a good collection of chapters on eukaryotic anaerobes, as does the much more recent volume Hydrogenosomes and Mitosomes: Mitochondria of Anaerobic Eukaryotes (Tachezy 2019). The Vital Question by Lane (2015) provides insights on the evolutionary significance of mitochondria. Life on a Young Planet by Knoll (2003) remains a very readable overview of early life with modern perspectives on symbiosis. Oxygen by Canfield (2015) reports information about O2 in the Earth’s history. Here we have aimed for a comparative approach across all eukaryotic lineages, couched in a modern Earth’s history context.

1 Anaerobes and eukaryote origin The first cells on the Earth were prokaryotes: bacteria and archaea. They evolved via direct filiation from the last universal common ancestor of all cellular forms of life, which according to genomic reconstructions was anaerobic, arose and lived in a habitat closely resembling a hydrothermal vent, and had a H2-dependent metabolism (Weiss et al. 2016). Eukaryotes arose later in evolution, via symbiosis of prokaryotic cells (McInerney et al. 2014; Betts et al. 2018). The symbiotic origin of eukaryotes is supported by many lines of evidence. In terms of gene origins, eukaryotes are chimaeras. Eukaryotes have archaeal ribosomes in the cytosol, archaeal histones, archaeal RNA polymerases, archaeal proteasomes or, in brief, a eukaryotic information processing machinery; by contrast, the majority of genes in eukaryotic genomes are bacterial in origin, including genes for energy metabolism, vitamin cofactor biosynthetic pathways, lipid, and amino acid metabolism (Esser et al. 2004; Brueckner and Martin 2020). Eukaryotes also possess a number of genes that are specific to the eukaryotic lineage, these are eukaryote specific inventions. The most direct evidence for the chimeric nature of eukaryotes is the persistence of mitochondria, mitochondrial DNA, and mitochondrial ribosomes, in all eukaryotic lineages studied to date (Maier et al. 2013). The archaeal nature of eukaryotic ribosomes and the bacterial nature of mitochondria combined with the circumstance that the archaeal ribosomes and bacterial mitochondria of eukaryotes each trace to one single eukaryote common ancestor leave no room for doubt that the mitochondrion was present in the eukaryote common ancestor. The mitochondrial DNA leaves no doubt that the mitochondrion derived from an endosymbiotic event. Thus, all eukaryotes we know descend from that symbiotic encounter. Eukaryotes are chimeric organisms, formed by a symbiosis of bacteria and archaea. There are many theories that address the possible selective pressure and mutual interactions of prokaryotes that might have led to the origin of eukaryotes; these have been reviewed extensively elsewhere (Martin et al. 2001; Embley and Martin 2006; Martin et al. 2015; Zachar and Szathmary 2017). The theories differ in various aspects, such as number of prokaryotic partners, taxonomic nature of the host, metabolic flexibility of the endosymbiont that gave rise to the mitochondrion, or the kind of environment in which the endosymbiosis that resulted in the appearance of eukaryotes took place. The latter factor (environment) bears upon the physiological interactions that put host and symbiont together in the first place. At the most basic level, the multitude of theories for the origin of eukaryotes can be divided into two classes. (i) Autogenous theories that envisage the host for the origin of mitochondria as a complex, nucleated cell that had already attained the organizational complexity of a eukaryote (an amitochondriate eukaryote), and that the acquisition of mitochondria added little or nothing to the eukaryotic lineage beyond

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1 Anaerobes and eukaryote origin

respiration. (ii) Symbiogenic theories posit that the host for the origin of mitochondria was a simply organized prokaryote (an archaeon) and that the acquisition of mitochondria precipitated the process that gave rise to eukaryote complexity. For more detailed reviews and comparison of the two theories, see Archibald (2015) and Martin et al. (2015). Autogenous theories, sometimes called classical or traditional theories, presume the existence of primitively amitochondriate eukaryotes. According to this class of theories, primitively amitochondriate eukaryotes were eukaryotic cells that contained a nucleus as the defining organelle, as well as some other eukaryotic traits, but they had no mitochondria. The bacteria that evolved into mitochondria entered into a symbiotic association with the host as an undigested meal through phagocytosis − a specific eukaryotic cellular process of food uptake. The presumed existence of primitively amitochondriate eukaryotes is associated with the idea that the host was an anaerobe, while the phagocytosed endosymbiont was, in typical formulations, strictly aerobic, consuming the oxygen through its energy metabolism (Andersson and Kurland 1999; de Duve 2007). The original benefit from such a symbiosis is sometimes seen as detoxification of oxygen, toxic to the host, via aerobic respiration of the endosymbiont. This concept, however, has several weak spots. First, it is the aerobic respiration in mitochondria that produces, during electron transport in the electron-transport chain, the reactive oxygen species (ROS) that are toxic for the cell. ROS are removed by cells via antioxidant enzyme systems (Temple et al. 2005). Such ROS include the superoxide anion (O2•−), hydroxyl radicals (HO•), various organic radicals, and hydrogen peroxide (H2O2) (Sies et al. 2017). Second, a logical consequence of these theories is the existence of eukaryotic organisms whose cytosol never harbored mitochondria. Such primitively amitochondriate eukaryotes were once called archezoa and were deemed to represent organisms or lineages descended from the host that acquired the mitochondrion. But they were sooner or later all found to contain mitochondria in some form (Embley and Martin 2006). Such candidate archezoan lineages with surprising remnant mitochondria included the unicellular human intestinal parasites Entamoeba histolytica (Mai et al. 1999; Tovar et al. 1999), Giardia intestinalis (syn. Giardia lamblia; Tovar et al. 2003), and the obligate intracellular energy parasite, the microsporidian fungus Trachipleistophora hominis (Williams et al. 2002). After the identification of mitochondria in a presumably amitochondriate flagellate Trimastix pyriformis (syn. Paratrimastix pyriformis), Hampl et al. (2008) felt confident in concluding that “[. . .] all known living eukaryote lineages descend from a common ancestor that had mitochondria.” To accommodate the unexpected ubiquity of mitochondria, autogenous theories had to tack on an additional assumption that all primitively amitochondriate eukaryotic lineages (direct descendants of the hypothetical host) went extinct in the course of evolution and the only descendants of the original eukaryotes to survive were those that possessed or had possessed mitochondria. Third, the

1 Anaerobes and eukaryote origin

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autogenous theories tend to focus on oxygen, oxidative phosphorylation, and increased ATP yield from substrate oxidation with the origin of mitochondria, hence they do not provide an explanation for the existence of anaerobic forms of mitochondria, hydrogenosomes, and mitosomes. Therefore, they require additional corollary assumption and conceptual extensions to account for the existence of different anaerobic forms of mitochondria as well as their presence across all the diverse eukaryotic lineages. A eukaryote was recently described that was reported to lack mitochondria altogether, Monocercomonoides sp. (Karnkowska et al. 2016), but it belongs to a larger group of eukaryotes that possess mitochondria, hence its evolutionary ancestors possessed mitochondria, which were lost in its immediate lineage. Symbiogenic theories posit that primitive amitochondriate eukaryotes never existed. These theories suppose that the eukaryotic cell originated via the endosymbiotic association of two prokaryotic cells (Martin and Müller 1998; Vellai et al. 1998), but without involving phagocytosis. Mitochondrial origin without phagocytosis is doubted for supporters of autogenous theories, but several precedents of such symbioses have been observed in nature. Cells of the mealybug Planococcus citri harbor a prokaryotic endosymbiont, a β-proteobacterium Tremblaya princeps, whose cells host another bacterium Moranella endobia, which belongs to the group of γ-proteobacteria (von Dohlen et al. 2001). Another example of endosymbiosis of a prokaryotic cell living inside other prokaryote’s cytosol was an unclassified microorganism Parakaryon myojinensis that harbors an endosymbiont which exhibits morphological resemblance to a spirochaete (Yamaguchi et al. 2012). There is also the older example of a cyanobacterium harboring a prokaryotic endosymbiont (Wujek 1979). Symbioses involving a prokaryotic endosymbiont within a prokaryotic host can be found today in nature, hence there is no reason to doubt that similar prokaryote–prokaryote symbioses could have occurred at the time of the origin of eukaryotes as well. Theories of the origin of eukaryotes differ not only concerning the nature of cells that gave rise to eukaryotes (the mitochondriate lineage) but also concerning different selective pressures that led to formation of eukaryotic cell complexity. Here too, opinions are divided into two major views. The classical view is based on oxygen and ATP, a view that Lynn Margulis championed (Margulis 1970), that a subsequent generation embraced (Doolittle 1998; Andersson and Kurland 1999; de Duve 2007; Cavalier-Smith 2002; Lenton and Watson 2011), and that resides at the heart of the phagotrophic theory. In the phagotrophic view, the host was an anaerobic fermenting eukaryote and could gain about 2 ATP per glucose from glycolysis and that could phagocytose prokaryotic cells. Adding mitochondria, the phagotrophic theory, improved ATP yield from glucose from about 2 ATP per glucose to about 30 ATP per glucose. At the same time, aerobic respiring α-proteobacteria removed oxygen in the vicinity of the host, detoxifying the environment (Andersson and Kurland 1999).

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Theories supposing a phagotrophic origin of mitochondria have a number of problems, explained elsewhere in detail by us (Martin et al. 2017; Zimorski et al. 2019) and by Speijer (2020a; 2020b). Three of those problems need to be mentioned here. Problem one is that eukaryotic anaerobes do not acquire endosymbionts to avoid or remove oxygen. When confronted with oxygen, eukaryotic anaerobes do not enslave bacteria, they use and express oxygen detoxifying enzymes, such as soluble NADH oxidases that convert O2 to water without conserving energy or they just stop growing until anaerobic conditions return. The conjuring of oxygen-consuming symbionts to save an anaerobic host is an example of evoking the evolutionary acquisition of endosymbionts in order to fulfill a simple physiological function that eukaryotes perform with a single enzyme (Garg and Martin 2018). Problem two is that the 30 versus 2 ATP per glucose calculation for with versus without oxygen is way off the mark, but not for the reasons one might think. Let us start with the number 30. ATP yields are traditionally calculated per glucose (Rich and Maréchal 2010) because blood glucose is the mainstay of mammalian physiology (whence most knowledge about mitochondria stems) and blood glucose is also the source of carbon for most human parasites. In the environment, particularly the ancient environments in which eukaryotes arose, there is (was) no free glucose sitting around waiting to be digested. The main source of carbon for heterotrophic microbes in nature is amino acids from dead or lysed cells, because cells are about 50–60% protein by dry weight. Aerobic amino acid breakdown in mitochondria delivers about 22 ATP per amino acid (Bender 2012) or 21 ATP per amino acid if we normalize for the fact that amino acids are not equally common in proteins; 30 ATP versus 2 then becomes closer to 20 versus 2. We also have to look at the number 2. The idea of 2 ATP per glucose is based on fermentations in yeast (ethanol fermentation) or human muscle (lactate fermentation), both organisms are aerobes. Eukaryotic anaerobes gain 2–5 ATP per glucose (Müller et al. 2012). ATP yields from amino acid fermentations are not well-studied in eukaryotes, but 1 ATP per amino acid can typically be gleaned by substrate-level phosphorylation alone (Martin et al. 2017). Still at problem two, the main flaw in the 30 versus 2 ATP yield is, however, even more severe in magnitude while being very subtle in nature. It is thermodynamic (Zimorski et al. 2019), and it is this: There is an important but not widely recognized relationship between oxygen and cellular energy. The synthesis of chemical constituents of cells (amino acids, bases, and lipids) from glucose and ammonium demands about 13 times more energy per cell in the presence of O2 than in the absence of O2 (McCollom and Amend 2005; Lever et al. 2015). The reason is that the synthesis of the chemicals that comprise cells is thermodynamically much more favorable under anaerobic conditions than it is in the presence of oxygen (McCollom and Amend 2005; Lever et al. 2015), because equilibrium in the reaction of cell mass with O2 lies very far on the side of CO2 and H2O. Organic compounds are far more stable in the absence of O2 than in the presence of O2.

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Thus, although the ATP yield from oxidizing glucose or amino acids is about 10-fold higher in the presence of O2, the energetic cost of living in an atmosphere containing 21% oxygen is 13-fold higher than for anaerobic environments. Life on O2 is energetically much more expensive than life without it. In the presence of O2, it costs more energy to assemble the components of cells than in the absence of O2, and life in the presence of a strong oxidant comes at a steep energetic price that is easily overlooked (Zimorski et al. 2019), and that has been overlooked in 50 years of literature on the origin of mitochondria. What mitochondria did for eukaryotes, energetically, was not to boost the energy yield per glucose, but to provide internalized bioenergetic membranes that boost the energy available per gene (Lane and Martin 2010). Problem three with the phagotrophic theory is the most severe of all, though, and it is that phagocytosis only affords physiological, energetic, or other selective benefits if the cell already possesses mitochondria (Martin et al. 2017). In other words, there is no energetic benefit to be gained in evolving phagocytosis if the cell lacks mitochondria, but if the cell possesses mitochondria, phagocytosis can yield an energetic benefit. The reason is twofold: (i) phagocytosing cells internalize their plasma membrane and (ii) fermentations (the starting point of phagocytosing host theories) in prokaryotes typically involve ATP synthesis via ATPases at the plasma membrane (Buckel and Thauer 2013). Those are the reasons why no prokaryotes are phagocytotic, why the only cells that evolved phagocytosis possessed mitochondria, and why no eukaryotes synthesize ATP at their plasma membrane. The origin of mitochondria was concomitant with the loss of ion pumping and ATP synthesis at the host’s plasma membrane (Gould et al. 2016) and the transition to energy metabolism involving internalized bioenergetic membranes at the outset of the eukaryotic lineage. If not phagocytosis, ATP, and oxygen, what other selective pressure could have been the physiological bond between host and symbiont at the mitochondrial origin? Molecular hydrogen, H2, is a frequent currency of electrons and energy in anaerobic environments (Schink 1997; Nealson 2010). It has been an important source of energy and electrons since the first cells existed on Earth (Sleep et al. 2011; Martin and Russell 2007). An anaerobic environment, not an oxic environment, is the context assumed by the hydrogen hypothesis (Martin and Müller 1998) for the origin of mitochondria and eukaryotes. The hydrogen hypothesis entails a symbiosis based on metabolic syntrophy between a symbiont, an α-proteobacterium that produced H2 as an end product of anaerobic fermentation from its facultatively anaerobic energy metabolism, and a host, an archaeon that is dependent upon H2 produced by the symbiont as a source of energy and electrons. This symbiosis, anaerobic syntrophy, led to physical association and eventually internalization of the α-proteobacterium, which ultimately gave rise to the mitochondria, hydrogenosomes, and mitosomes of eukaryotic lineages. In that view, the aerobic and anaerobic forms of mitochondria represent different forms of

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mitochondria, which underwent evolutionary specialization to different environmental conditions during the evolution of eukaryotic lineages. Therefore, energy metabolism in all types of mitochondria – aerobic and anaerobic – comes from an originally facultative anaerobic state. Mitosomes, which appear to not participate directly in energy metabolism, are a prediction of the hypothesis among eukaryotes in which all energy metabolic pathways have been relocated to the cytosol. By virtue of its premise that eukaryotes formed from a symbiosis of two prokaryotic species, the hydrogen hypothesis (Martin and Müller 1998) directly explains both (i) the presence of mitochondria in all known eukaryotic linages, and (ii) the existence of anaerobic forms of mitochondria, hydrogenosomes, and mitosomes. In symbiogenic theories, the hallmark of eukaryotic cell organization, the endomembrane system, arises in the wake of symbiosis (Gould et al. 2016; Sugiura et al., 2017), not a prerequisite. In summary, debates about the origin of eukaryotes have been ongoing for over 100 years as we will see in more detail in Part III, and like most major evolutionary issues, they will no doubt continue for years (or decades) to come. The most recent development in the debate favored the symbiosis view, however, in that Imachi et al. (2020) reported the isolation and enrichment of an archaeon, Prometheoarchaeum syntrophicum, that (in ribosomal protein phylogenetic trees) branches closer to the eukaryotic host lineage than any archaeon studied so far. That archaeon was tiny (0.5 μm in diameter), it was not phagocytotic, it was prokaryotic in organization, and, of note, it required a bacterial symbiotic partner in order to grow.

2 Eukaryotes in low oxygen environments We live in an atmosphere that contains 21% O2. We humans need O2 because of respiration in our mitochondria and it is somehow natural to assume that all other eukaryotes have similar needs, due at least in part to our everyday observation of environments, colonized by multicellular eukaryotic organisms, such as animals, plants, and fungi, with oxygen as ubiquitous component. The view that oxygen is essential for eukaryote survival in general is deeply engrained in classical theories of eukaryote origin, which, following Margulis’ suggestion in 1967, links the origin of mitochondria with the first appearance of molecular oxygen on Earth ~2.4 billion years ago (Campbell and Allen 2008). We will see in this book that the initial appearance of O2 was not the origin of a fully oxic atmosphere. Even though we live in an atmosphere with 21% [v/v] O2, the mitochondria in our tissues never see 21% oxygen. They operate at concentrations (partial pressures) corresponding to 0.1–1% [v/v] O2. Because of the oxygen cascade in blood, O2 levels drop continuously from air, to the lung, to arteries, to capillaries, to cells, to mitochondria, to cytochrome c oxidase (complex IV) of the respiratory chain, where the O2 is consumed (Keeley and Mann 2019; Nanadikar et al. 2019). The amount of O2 present in human mitochondria in vivo is not terribly different from the Km of human cytochrome c oxidase in vivo (the O2 concentration at which the enzyme has half-maximal activity), which is on the order of 0.1 to 10 µM, though many reports have it much lower (Gnaiger et al. 1998), corresponding to roughly 0.01–1% [v/v] O2. The affinity of human cytochrome c oxidase for O2 is in roughly the same range as that of the corresponding A1-type heme copper oxidases in α-proteobacteria (1–10 µM), the group from which mitochondria arose (Degli Esposti et al. 2019) over 1.5 billion years ago. Yet the A1-type terminal oxidases have the lowest affinity for O2 as a substrate among the αproteobacterial oxygen reductases. The bd type, Cio type, bo3 type, A2 type, and C type oxygen reductases of α-proteobacteria have higher affinities for O2, suggesting that the low affinity A1 (mitochondrial) type arose last in evolution (but before mitochondria arose), when there was more environmental O2 available than when the other types arose (Degli Esposti et al. 2019). The reader might now wonder: does that mean that there was only 0.1–1% O2 [v/v] in the atmosphere at the time when mitochondria arose? Findings over the last 20 years from the geochemical record say that yes, that is indeed the case. The data furthermore say that O2 remained more or less constant at that low level for about a billion years after the origin of mitochondria. We find that to be significant because it allows only one conclusion: the physiology of eukaryotic anaerobes provides a window into ancient life. For reference, ~0.1% O2 corresponds to many microbiological definitions of anoxia and ~1% O2 corresponds to the Pasteur point, the O2 level at which facultative

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anaerobes tend to switch between aerobic respiration and anaerobic energy metabolism. An underlying theme of this book is that mitochondria arose in a world of borderline anoxia and that the first one billion years of eukaryote evolution took place in borderline anoxic conditions. That means that anaerobic eukaryotes can be seen as harboring biochemical relics of the anaerobic past. It also means that the low O2 concentrations at which our own mitochondria operate can also be seen as relics from our low oxygen ancestry. Understanding how eukaryotes synthesize ATP using little or no oxygen can help us better understand our place in the larger scheme of things. Understanding that our own mitochondria operate at 0.1–1% [v/v] O2 is a good place to start. There are different kinds of environments on Earth that have less than the “normal” amount of oxygen that we breathe. Typically, when we think of low O2 environments we think of sediment, stratified lakes, rumen, waterlogged soils, and the like. But even the air can harbor low O2. The atmosphere is 21% O2, more or less everywhere. Near the top of Mount Everest, the air also has 21% O2, but there is much less air up there. At about 8,000 m altitude, the air is so thin that its oxygen content corresponds to about the same amount of available O2 as if the air only had about 6% O2 at sea level. Low oxygen also impairs judgement and decision making, which are severe life threatening risks for alpine climbers (and for divers). The altitude above 8,000 m is called the “death zone” for that reason. If a human were to step out of a pressurized airplane at 8,000 m, that is, make the transition to the 8,000-m atmosphere without low oxygen acclimatization, that human would survive for about a minute, because of a lack of oxygen (suffocation). The environment limits the altitude where animals can live, dictated by physiological capacities for tolerating hypoxia, in addition to extreme cold and food availability. The highest-dwelling mammals were discovered at the summit (6,739 m) of Volcán Llullaillaco in Chile (Storz et al. 2020). It is well known that climbing Mount Everest poses lethal risks. Humans need to become acclimated to high altitude as it can, and does, present lethally low oxygen availability if encountered without acclimatization. Low oxygen in the environment results in hypoxia (low oxygen in the body). Acclimatization takes days and weeks. How, one wonders, is it then possible that humans like Reinhold Messner were able to climb Mount Everest (8,848 m) without an oxygen mask? Acclimatization is a physiological response that involves hypoxia induced effects in both blood and muscle (Schuler et al. 2005; Bigham and Lee 2014). Low oxygen induces a response that is mediated to a very significant extent by a protein called hypoxia-inducible factor (HIF) that induces a number of physiological changes (Semenza 2012). These include increased blood vessel formation via induction of VEGF, the vascular endothelial growth factor, and induction of erythropoietin, EPO, which in turn increases the number of red blood cells (Haase 2013). The role of HIF in high altitude acclimatization is not cut and dried, however, as some effects seem to indicate its participation while others are less clear (Schuler et al. 2005). We will revisit HIF in somewhat more detail in the subsequent section.

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Today’s atmosphere is 21% O2. In aquatic environments, O2 is often reported as µM concentrations. Ice cold well-aerated fresh water at sea level has about 460 µM O2, and at 25 °C the value drops to about 250 µM O2 and for seawater the corresponding values are 350 and 200 µM, accordingly. In Table 1, we have collected a sample of useful numbers relating to O2 as expressed in units that will confront us in this book. Reports concerning low oxygen environments sometimes refer to hypoxic, suboxic, or anoxic conditions. Anoxic conditions entail 0 μM O2, suboxic conditions entail on the order of 0−9 μM O2, hypoxic conditions can be 10–22 μM O2, intermediate hypoxia might be in the range 22−45 μM O2, and mild hypoxia corresponding to 45–90 μM O2. Eukaryotes are common in low oxygen environments, though in anoxic environments large animals are rare if present at all (Bryant 1991; Fenchel and Finlay 1995; Judson 2017). Environmental sequencing from anoxic sediments uncovers eukaryotes, presumably alive (Edgcomb et al. 2009; Edgcomb et al. 2011b). The rumen of cows and the termite gut are unquestionably alive and teeming with eukaryotic anaerobes including ciliates and anaerobic fungi (Fenchel and Finlay 1995). Moreover, from the taxonomic point of view, weakly oxygenated and anoxic environments harbor representatives from every currently recognized eukaryote group (Hampl et al. 2009; Mentel and Martin 2010; Müller et al. 2012). Anaerobic eukaryotes are hardly mysterious, phylogenetically limited, or evolutionary primitive organisms – they are commonly found as sibling species to aerobic lineages within their respective taxonomic groups (Mentel and Martin 2008). Eukaryotes inhabit diverse environments with limited oxygen concentrations or the complete absence of oxygen, such as deep-sea brine pools, marine and freshwater sediments, oxygen minimum zones, lakes with lack of regular water circulation (stratified meromictic lakes), tidal zones, dry land, and microbial mats, whether floating or benthic (Anbar and Knoll 2002; Fenchel 2002b; Dasgupta et al. 2012). Depending on their life cycle stages, symbiotic eukaryotic species, both parasites and mutualists, can be exposed to insufficient oxygen in different body parts of the host, such as inside the digestive system (Tovar et al. 2003). Organisms that inhabit hypoxic environments can be exposed to the absence of oxygen for different amounts of time. Anoxia can be temporary or permanent with the periodicity varying from diurnal cycles (on dry land) to monthly cycles (in tidal zones) to anoxic conditions lasting thousands of years in deep-sea brine pools. Hypoxia or anoxia is induced when O2 availability cannot compensate for oxygen consumption during aerobic respiration (Meuser et al. 2009). Oxygen input to aquatic environments, whether in equilibrium with the atmosphere or from local photosynthesis, depends on mixing rates, convection, light intensity, and depth. Oxygen gradients can be very steep in aquatic environments. During the day, the sandy sediments of seas and oceans, a completely oxygenated spot can be as close as 2 mm from a spot without O2 (Fenchel 2002b). The same thing happens in termite guts. The periphery can harbor aerobes, and the central lumen strict anaerobes. For animal studies, environmental hypoxia should be distinguished from functional hypoxia. Environmental hypoxia occurs when an organism is naturally present

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Table 1: Some miscellaneous values regarding oxygen. Solubility of O (µM) in water saturated with air at .% O and at  atm

O partial pressure at high altitudes relative to .% O at sea level

O expressed as % present atmospheric level (% PAL)

Water Fresh Sea temp water water °C ( g/L) ( g/L)

Meters above sea level

Mean air pressure (atm)

Relative pO (%)

% PAL

pO (%)

 , , , , , , , ,

. . . . . . . . .

. . . . . . . . .

    . . .

. .  . . . .

        

        

        

Max[O] in sea water at  °C (µM)     . . .

Common units of pressure:  atm = . bar = , Pa = . kPa =  Torr (mm Hg) Some other miscellaneous values (sea level, µM concentrations for seawater at 25 °C): Pasteur point (O2 level at which facultative anaerobes switch) pO2 ca. 0.2% = 2 µM = 1% PAL O2 concentration in operating human mitochondria: pO2 ca. 1% or ca. 10 µM Free O2 concentration in blood is very low because ca. 98% of O2 in blood is bound to hemoglobin Km of human cytochrome c oxidase (O2 conc. that results in half-max. respiratory rate): ca. 10 µM Km of α-proteobacterial A1 type cytochrome c oxidases (the mitochondrial type): ca. 0.2 - 10 µM Km of α-proteobacterial AOX type quinonol:O2 oxidoreductase for O2: ca. 20 µM Km of α-proteobacterial A2 type cytochrome c oxidases for O2: ca. 0.8 µM Km of α-proteobacterial A1-bo3 type quinonol:O2 oxidoreductase for O2: ca. 0.2 µM Km of α-proteobacterial bd type quinonol:O2 oxidoreductase for O2: ca. 0.02-0.1 µM Km of α-proteobacterial Cio type quinonol:O2 oxidoreductase for O2: ca. 0.5 µM Km of α-proteobacterial C type cytochrome c oxidase for O2: ca. 0.08 µM pO2 content of inhaled air in kPa: 20.9 pO2 content of exhaled air in kPa: 15.9 pCO2 content of inhaled air in kPa: 0.03 pCO2 content of exhaled air in kPa: 4.2 O2 consumption of an adult with normal activity: ca. 500 liters = 22.3 moles = 0.714 kg per day CO2 production of an adult with normal activity: ca. 250 grams = 127 liters per day O2 consumption of a running adult: ca. 180 liters per hour CO2 production of a running adult: ca. 90 grams per hour Km values from Degli Esposti et al. (2019) and Krab et al. (2011), physical data from technical sourcebooks, Berg et al. (2015), and Wenger et al. (2015) and Encyclopedia Brittanica.

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in an environment with limited oxygen supply. Examples are worm parasites inside host bodies (Tielens et al. 1984). Functional hypoxia refers to animal physiological conditions associated with insufficient O2 required for aerobic energy metabolism of a tissue with the lack of oxygen being usually caused by very intense muscle activity. This type of hypoxia is experienced by diving whales (del Castillo Velasco-Martinez et al. 2016). Abrupt muscular exercise can be related, for instance, to an effort of prey to escape from predator and logically, also occurs in the predator chasing its potential prey. In humans, insufficient oxygen supply is commonplace in sports such as short sprints or bodybuilding, when electrons obtained through oxidative degradation of energy-rich substrate contribute to lactate formation, thus maintaining redox balance inside cells (Neumann et al. 2005). Lactate produced in this way induces the acute muscle soreness during relatively short and intense exercise. This is because when demand for ATP exceeds the amount that can be covered by aerobic respiration, the organism starts anaerobic production of ATP through substrate-level phosphorylation, which is the only form of ATP production when glucose is degraded to lactate via glycolysis. The concept of sufficient O2 is noteworthy in its own right though, because what starts out as 21% O2 in the air inhaled into a human lung drops to about 1% O2 in our mitochondria. Metabolic repression is another physiological process that various species use for protection during hypoxic and anoxic periods. In this case, however, focus is on the rate of ATP consumption rather than its synthesis. During metabolic repression, energy metabolism is heavily slowed down in order to reduce the consumption of ATP, thereby limiting the need of its synthesis. Metabolic repression has been observed in invertebrates as well as vertebrates, mostly in marine animals that dive following inhalation, such as earless seals and sea turtles (Hochachka et al. 1996).

3 A modern context of atmospheric evolution The evolution of eukaryotic anaerobes needs to be seen in the context of Earth’s history. In particular, the availability of O2 in the Earth’s history sets the scene for appreciation of eukaryote anaerobe evolution. A search of the literature for papers on the evolution of eukaryotic anaerobes will return a large number of contributions. At the time we write this book, there are two main schools of thought about the evolution of eukaryotic anaerobes. One view has it that eukaryotic anaerobes have preserved ancient biochemical relicts of the evolutionary past. That is our view. The alternative view is that eukaryotic anaerobes are recently arisen newcomers on the stage of evolution, an interpretation that is usually based on evidence from branching patterns in gene trees usually involving lateral gene transfer (for the interested reader, the exchange between Martin (2017c) and Leger et al. (2018) provides an introduction to that debate). One reason we hold that eukaryotic anaerobes have preserved relicts from the past is summarized in Figure 1, which presents a general overview of eukaryotic evolution in the context of oxygen evolution in Earth’s history. In modern accounts of Earth’s atmospheric evolution, the atmosphere contained only no free O2 at all until ~2.4 billion years ago. That view was also current in the 1960s. A big change in the picture of Earth’s atmospheric oxygen has occurred over the last 20 years, however. That change is this: In contrast to the prevailing view in Margulis’s time and well into the 1990s (Kasting 1993), the advent of O2 ~2.4 billion years ago did not immediately result in modern O2 levels of 21% [v/v]. Rather, newer data have it that the first appearance of O2 was followed by a “long” period of O2 stasis during which atmospheric O2 stayed constant at about 0.02% [v/v] or roughly 1% present atmospheric levels (Lyons et al. 2014; Catling and Zahnle 2020). When we say “long” we mean long, because that long period lasted for more than a billion years, almost 2 billion years in fact, up until roughly the beginning of the Phanerozoic ~540 million years ago, around the beginning of the Cambrian when the first animal fossils start to appear (Lyons et al. 2014; Catling and Zahnle 2020). Two billion years represent fully half of the evolution of life. This low oxygen phase in the Earth’s history corresponds very closely to what geologists call the Proterozoic. From the standpoint of evolutionary ecology, however, one could call this period the Pasteurian (Figure 1), because oxygen stayed very near the Pasteur point for almost 2 billion years. The Pasteur point is the level of oxygen where cells that are able to switch from oxygen respiration to anaerobic ATP synthesis and an anaerobic lifestyle, make that switch. Part III will deal with the evolution of atmospheric oxygen in more detail. The view that eukaryotic anaerobes have preserved relicts of a low oxygen past fits very well with modern accounts of oxygen accumulation in Earth’s history, because eukaryotes arose more than 1.5 billion years ago, a billion years before O2 levels rose to roughly their current values (Figure 1). Consequently, the diversification of eukaryotes

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Figure 1: Eukaryotes in a modern context of Earth’s oxygenation history. The upper panel summarizes current data on oxygen accumulation in Earth’s history. The main contours of oxygen accumulation are redrawn from several references: (Müller et al. 2012; Lyons et al. 2014; Fischer et al. 2016; Allen et al. 2019; Catling and Zahnle 2020). See also Part III, which covers information on O2 accumulation more fully. The Pasteur point is the oxygen level at which facultative anaerobes switch from O2 respiration to fermentation and vice versa. The main message of the upper panel is that initial O2 accumulation at the great oxidation event (GOE) ~2.4 billion years ago

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into the main eukaryotic lineages that biologists now call supergroups (Adl et al. 2012) took place long before there was enough oxygen to support the life of animals on land. Why do we mention land animals? The anaerobic physiology in animals (seals and turtles) that Hochachka and colleagues (Hochachka et al. 1996) studied in great detail arose in lineages that were fully O2 dependent land-dwelling ancestors that had already specialized to life in the atmosphere we know (Gould et al. 2019). The anaerobic capacities of reptiles and mammals represent evolutionarily recent secondary adaptations to low oxygen environments. Indeed, the oldest mammals are only 160 million years old (Luo et al. 2011). They arose long after the Earth had already experienced an atmosphere of ~ 30% O2 during the Carboniferous (360–300 million years ago), where dragonflies came to be as big as seagulls, with wingspans of up to 70 cm (Nel et al. 2018). In this book we are dealing with evolutionary processes that took place during the time 1600 million years ago to 600 million years ago, long before there was a high O2 atmosphere to which to adapt. The reader might wonder whether 0.2% O2 is enough to support eukaryotic life. The answer is yes, but not all eukaryotes can survive such low oxygen levels and the only ones that are known to survive such oxygen levels permanently over generations are protists (unicellular eukaryotes). Protists represent the early stages of eukaryotic evolution (Figure 1). The biochemical means by which eukaryotes from different supergroups that inhabit low oxygen environments survive low O2 (how they make ATP and maintain redox balance in the absence of O2) is the main topic of this book. Given that eukaryotes survived a billion years at the Pasteur point, the reader might also wonder whether the respiratory chain in the mitochondria of different eukaryotic groups is descended from one and the same event. There too, the

Figure 1 (continued) was followed by a long period of O2 stasis during which atmospheric O2 stayed constant at about 0.02% [v/v] or roughly 1% present atmospheric levels (PAL) for more than a billion years up until roughly the beginning of the Phanerozoic ~540 MY ago, the beginning of the Cambrian (see geological time scale in the bottom panel). The blue vertical bars labeled “Whiffs” indicate reports suggesting intermittent availability of small amounts of atmospheric oxygen (whiffs of oxygen) preceding the GOE (Fischer et al. 2016). The lower panel summarizes important events in biological evolution that pertain to this book. The fossil record for cyanobacteria goes back as far as the evidence for O2 accumulation. Yet the fossil evidence for heterocysts, specialized cells of cyanobacteria that protect nitrogenase from O2, goes only as far back as the Rhynie chert, or 415 million years ago. That makes heterocysts younger than land plants (see text, Part III, and Allen et al. 2019). The tree represents a simplified view of eukaryote evolution with an approximate time scale, summarizing data from Parfrey et al. (2011) for the age of eukaryotes and protist groups, dos Reis et al. (2015) for the age of animal groups and Zimorski et al. (2014) for the number of secondary symbiotic events (red and green lines connecting members of SAR and Excavata with members of Archaeplastida). Note that the first eukaryotes might well have been syncytial rather than the canonical “protist” type as morphology indicated (Garg and Martin 2016; Tria et al. 2019). Eukaryote origin and the first billion years of eukaryote evolution took place in anaerobic environments.

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answer is yes, because the components of the respiratory chain of O2-dependent mitochondria are homologous across all supergroups. (As an appetizer, we note that the components of O2-independent ATP synthesis are also homologous across all supergroups, as we will see in subsequent sections.) Many of those respiratory chain components are still encoded in mitochondrial DNA and mitochondria have a single origin. We can thus be sure that the ancestor of mitochondria could respire O2. But the affinity of the mitochondrial terminal oxidase (the enzyme that converts O2 into H2O during respiration) is low, on the order of 0.1 to 10 µM O2, even in human mitochondria. That is, an oxygen level that corresponds to 0.04 to 4% of present atmospheric levels (Zimorski et al. 2019) in equilibrium with water or an atmosphere of roughly 0.01 to 1% O2 [v/v], again, right at the Pasteur point. That probably also represents a relict of the low oxygen past. A long look at Figure 1 is worthwhile, as it clearly reveals that eukaryotes spent the vast majority of their evolutionary history in low oxygen environments. They arose and evolved on an effectively anaerobic planet. During animal development, stem cells have been observed to show a clear tendency to prefer very low oxygen niches (Ivanovich and Vlaski-Lafarge 2019), which would make sense if the early ‘bauplan’ of animal development was laid down before high oxygen levels had arisen. Before we move on to the next section, let us consider heterocysts, which are specialized N2 fixing cells of some cyanobacteria. The first fossil appearance of heterocysts is indicated in Figure 1. Why is that noteworthy? Cyanobacteria and their descendants, the plastids of plants and algae, are the source of the O2 in our atmosphere. Representatives of all major cyanobacterial lineages can fix N2 but only some filamentous cyanobacterial lineages develop heterocysts, larger, thick-walled cells within the filament that contain the N2 fixing enzyme, nitrogenase. Nitrogenase is very O2 sensitive because O2 oxidizes one of the essential iron-sulfur clusters in the nitrogenase enzyme at O2 levels greater than ~ 1% atmospheric O2 (Allen et al. 2019). Heterocysts express nitrogenase but do not express photosystem II, the site of O2 synthesis in the photosynthetic chain. That helps them to keep the O2 low. Heterocysts have always been thought of as indicators for the rise of O2. It is therefore surprising that the oldest fossil heterocysts are only 415 million years old, younger than land plants, which are about 450 million years old. The cyanobacterial lineages that make heterocysts are much older, as old as plastids probably, but the heterocysts themselves are young, indicating that they arose in response to the late rise of oxygen (Allen et al. 2019). One wonders whether endogenous and local O2 production by cyanobacteria might outweigh the effects of atmospheric O2 on nitrogenase. The converse is true. Kihara et al. (2014) determined that the amount of O2 that is generated by photosynthesis in illuminated cyanobacterial cells corresponds to a steady state O2 concentration of 0.25 to 0.025 µM O2. That in turn corresponds to an ambient O2 level of an atmosphere with 0.02 to 0.002% [v/v] O2. In other words, photosynthesizing cyanobacterial cells do not produce enough endogenous O2 from their own ongoing photosynthesis to inhibit nitrogenase, because nitrogenase is inhibited at about 2% [v/v] O2, again right at the

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Pasteur point (Allen et al. 2019). That means that the selective pressure to bring forth heterocysts came from atmospheric O2 accumulation, not endogenous O2 production. As we will see in Part III, the late appearance of heterocysts fits very well with the independent geochemical evidence for O2 accrual over the Earth’s history (Figure 1). The measurements of O2 levels inside cyanobacteria by Kihara et al. (2014) have another very far reaching consequence for constraining the ecology and habitats of early eukaryotes. It is this: Proponents of the view that eukaryotes arose and evolved initially as aerobes, with anaerobes and anaerobiosis coming late, will often argue that there were “oxygen oases” that supported aerobic growth eukaryotic during the deeper anaerobic past of the Pasteurian Proterozoic. What Kihara et al. (2014) show is that O2 diffusion to the environment in photosynthesizing cyanobacteria outpaces endogenous O2 production within the cell. That means that even cyanobacterial colonies under an atmosphere of 0.1 to 1% O2 would not be able to boost O2 to local concentrations of 10% O2 or 21% O2 because diffusion outpaces O2 production on thylakoids. That means in turn that there were no oxygen oases into which either aerobes could retreat physically, or into which proponents of aerobic eukaryote origins and early evolution could retreat philosophically. The world was anaerobic, even around cyanobacteria. We will see, however, that eukaryotic anaerobes come fully equipped with enzymes to remove O2 (it inactivates some of their important enzymes of energy metabolism) and that these O2 detoxifying enzymes are the same across eukaryotic supergroups. That in turn indicates that eukaryotes were confronted with trace amounts of O2 from the very inception of their lineage. That should hardly surprise anyone. Trace amount of O2 also provided an evolutionary rationale for the retention of the enzymes of the respiratory chain in organism lineages that spent most of their time (on average 50% of each day at least) over millennia in anaerobic environments (Martin and Müller 1998). Just like enzymes of anaerobic energy metabolism, the enzymes of O2 metabolism in eukaryotes, both O2 detoxification and O2 utilization as an electron acceptor, trace to the eukaryote common ancestor. Given the familiar, strict dependence of humans on O2, one might wonder how it was possible for eukaryotes to survive for a billion years at about 1/100th the amount of O2 we have today. The answer to the question of how eukaryotic anaerobes survive is straightforward. It is the biochemistry of their O2-independent energy metabolism (redox balanced ATP synthesis). So if you want to know how eukaryotic anaerobes survive, you have the right book in hand.

4 Energy metabolism and redox balance Organisms stay alive by keeping their metabolism far from equilibrium. Reaching chemical equilibrium is the end of the life process. To stay far from equilibrium, organisms must continuously extract energy from endogenous or environmental sources using their energy metabolism, but while simultaneously maintaining redox balance (Atteia et al. 2013). Nature offers energy sources, in the form of photons (phototrophy) and in the form of chemical energy (chemotrophy). Chemotrophy entails harnessing the energy of redox reactions (oxidation−reduction reactions) (Madigan and Martinko 2006). With the exception of eukaryotes that possess functional plastids, eukaryotes are chemoorganoheterotrophs: they obtain energy from chemical reactions (chemotrophy) rather than from light (phototrophy), they obtain their electrons from organic substances (organotrophy) rather than from inorganic sources (lithotrophy), and they obtain their carbon from organic compounds (heterotrophy) rather than from CO2 (autotrophy). When discussing electron donors as energy sources, it has to be noted that it is not the compound per se that contains energy, but the energy is released in the process of a chemical reaction during which the electron donor becomes oxidized (Madigan and Martinko 2006). As in all exergonic (energy releasing) chemical reactions, the reactions of energy metabolism lose a fraction of their energy in the form of heat but the portion of energy called Gibbs free energy (abbreviated as G) is available to perform work. The change in free energy, ΔG, during a reaction run at standard conditions and pH 7 is denoted as ΔG0′. If the value of ΔG for the reaction under the conditions that exist in the cell is 0, the reaction requires energy input to go forward and this kind of reaction is called endergonic. Some reactions in metabolism are endergonic, but the overall bioenergetic reaction of the cell needs to be exergonic, otherwise metabolism comes to a halt. Organisms obtain energy from exergonic chemical reactions, store it in chemical form (usually ATP) and spend it on endergonic reactions. The fundamentals of thermodynamics in energy metabolism of anaerobes were set down in the landmark paper by Thauer et al. (1977) that is freely available from the publisher and that readers are encouraged to consult, along with its predecessor (Decker et al. 1970). Chemical substances differ by their tendency to donate or accept electrons and such electron transport potential is expressed as a standard potential, E0′, or redox potential (Nicholls and Ferguson 1992). Reducing agents such as H2 have a tendency to donate electrons and they are characterized by a negative redox potential, while oxidizing agents such as O2 have a high affinity for electrons and a positive redox potential. The amount of free energy released from a redox reaction depends on the redox properties of both reactants; the higher the difference in redox potentials of the

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electron donor and the electron acceptor, the more free energy release is involved in the redox reaction (Madigan and Martinko 2006). The term energy metabolism designates the set of reactions that a cell employs to obtain ATP. There are two fundamental mechanisms of ATP synthesis: substrate-level phosphorylation and chemiosmotic coupling, which requires ion gradients across membranes in membrane bounded compartments. Eukaryotes employ both mechanisms, and both mechanisms can be found among eukaryotic anaerobes. Substratelevel phosphorylation involves the synthesis of a high energy organophosphate bond and transfer of the activated phosphate to ADP to form ATP (Decker et al. 1970). In eukaryotes, such high-energy organophosphate bonds are typically formed during glycolysis as 1,3-bisphosphoglycerate (the acyl phosphate bond) and phosphoenolpyruvate (an enol–phosphate bond) but also in the Krebs cycle, in which succinyl phosphate occurs as an intermediate. In addition, energy metabolism in eukaryotic anaerobes commonly entails propionyl phosphate, as well as acetyl phosphate and carbamoyl phosphate in some lineages. Many eukaryotes can also store the high energy bond in ATP by reversibly phosphorylating creatine or arginine to generate phosphocreatine (Brosnan et al. 2007) or phosphoarginine (termed phosphagens) as rapidly accessible energy reserves (Beis and Newsholme 1975). In eukaryotic heterotrophs, chemiosmotic ATP synthesis occurs in mitochondria, involving the generation of proton gradients across the inner mitochondrial membrane during the oxidation of reduced carbon compounds and the reentry of protons into the mitochondrial matrix through the mitochondrial rotor-stator ATP synthase. This process of the flow of electrons through the electron-transport chain coupled to the production of ATP is called oxidative phosphorylation, sometimes abbreviated as OXPHOS. Redox balance: At the heart of energy metabolism is the requirement for redox balance. The heterotrophic energy metabolism of eukaryotes is based on exergonic chemical reactions in which reduced organic substrates are oxidized. The oxidation reactions need not involve oxygen, nor need they involve any other inorganic acceptor. Anaerobic fermentations also involve oxidation steps. The fermentation reactions in which energy is conserved as ATP involve oxidations of carbon atoms. In glycolysis, energy is conserved during the conversion of glyceraldehyde-3-phosphate to 3-phosphoglycerate, whereby C1 of the substrate is converted from an aldehyde to a carboxyl group. Electrons are extracted from the carbon backbone at the step catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferred to NAD+ to yield NADH. In the enzyme mechanism, the C1 aldehyde carbon in glyceraldehyde-3-phosphate is attacked by the active site cysteine thiol of GAPDH forming a covalently enzyme-bound hemithioacetal. Hydride removal from the thiol bonded carbon atom of the hemithioacetal by NAD+ (the oxidation step) generates an energy-rich thioester bond (Lipmann 1941) with a free energy of hydrolysis under standard physiological conditions of –32 kJ/mol (Buckel and Eggerer 1965). The thioester has a carbonyl carbon, which is a target for nucleophilic attack. The enzyme-substrate thioester

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bond is cleaved by nucleophilic attack of the C1 carbonyl, not by water (hydrolysis), but by phosphate instead (phosphorolysis), resulting in the free thiol and the formation of the high energy acyl phosphate bond in 1,3-bisphosphoglycerate. The acyl phosphate can then phosphorylate ADP in the step catalyzed by 3phosphoglycerate kinase (PGK), providing energy conservation via substrate-level phosphorylation (Martin and Cerff 2017). The key to this energy-conserving reaction sequence is that electrons are extracted from the substrate to form NADH, and this NADH has to be reoxidized so that the next GAPDH reaction can work. This is essential, because if NADH is not reoxidized, energy metabolism comes to an immediate halt and with it the life process. For every energy metabolic food substrate that enters the cell and is oxidized, the extracted electrons have to eventually leave the cell. That is the meaning of redox balance. Redox balance is integral to energy metabolism. Maintaining redox balance is not just good for the cell, it is absolutely essential for the life process. In respirations, NAD+ from glycolysis or other oxidations is regenerated by donating the electrons to environmentally available acceptors, usually inorganic compounds (e.g., O2 or nitrate). This generates reduced products (e.g., H2O or nitrite) that accumulate in the environment. In fermentations, NAD+ is regenerated by donating the electrons to compounds that are generated within the cell during metabolism. These can be organic acceptors such as acetaldehyde, which is reduced to ethanol as an end product, or fumarate, which is reduced to succinate as an end product, or inorganic acceptors such as the conversion of protons, which arise during the reduction of NAD+ to NADH, and H+ to H2. In order to maintain redox balance in energy metabolism, each oxidation must be coupled with a reduction. Like thermodynamics, redox balance is not just a good idea it is compulsory.

5 Fermentation, glycolysis, and compartmentation ATP synthesis can occur by substrate-level phosphorylation or by chemiosmotic coupling; the underlying reactions can entail fermentation or respiration. Fermentations involve the breakdown of reduced carbon compounds and the excretion of end products in the absence of exogenous electron acceptors obtained from the environment. Fermentations are typically disproportionation reactions in which substrate carbon is converted into a more oxidized and a more reduced state; for example, the conversion of glucose to ethanol (reduced) and CO2 (oxidized), or glucose to lactate, in which the reduced carbon moiety (the methyl group) and the oxidized moiety (carboxylate) occur in the same molecule. In order to maintain redox balance in energy metabolism, each oxidation must be coupled with reduction. Typical endogenous electron acceptors used in eukaryotic fermentations are (i) pyruvate, which is reduced to lactate and/or opines, (ii) acetaldehyde, which becomes ethanol, (iii) fumarate, giving rise to succinate, (iv) the acetyl moiety in acetyl-CoA, being precursor to fatty acids and derived compounds, and (v) protons, which accept electrons to generate hydrogen gas (Müller et al. 2012). Sugars are fermentable substrates because of their intermediate carbon oxidation state. Lipids are an example of nonfermentable substrates because they are too reduced. Energy released during redox reactions of energy metabolism has to be stored in a form that can be later utilized by the cell to power endergonic reactions when necessary. ATP is the universal energy currency of eukaryotes, though other energy-rich compounds including thioesters and reduced ferredoxin also play an important role in eukaryotic energy conservation. ATP is such an efficient energy currency because it has a high phosphoryl-transfer potential, the tendency to transfer a phosphoryl group to an acceptor molecule. The high phosphoryl potential of ATP stems from structural differences between ATP and its hydrolysis products, which result in a high standard free energy of hydrolysis (ΔG0′). Hydrolysis reduces the electrostatic repulsion of negative charges on the phosphate groups of ATP. Furthermore, the product, inorganic phosphate (Pi), has greater resonance stabilization than the phosphates in ATP and the greater solvation (hydration) of the products Pi and ADP compared to ATP also favors ATP hydrolysis (van Beek et al. 2019). In the cell, ATP is present as ATP-Mg2+ complexes which serve as the true substrate for essentially all ATP-dependent enzymes. The three phosphate groups of ATP are designated as α, β, and γ, with γ being the most distal. The divalent magnesium ion binds to ATP4− as a β, γ chelate. This serves to promote nucleophilic attack at the γ-phosphate during phosphoryl transfer reactions (Cowan 2002). ATP, acyl phosphates, and the phosphoenolate bond all have a high free energy of hydrolysis: the values of ΔG0′ are –31.5 kJ/mol for ATP, –43 kJ/mol for acetyl phosphate, –52 kJ/mol for 1,3-bisphosphoglycerate, and –62 kJ/mol for phosphoenolpyruvate (PEP). Similar applies to the thioester bond in acetyl-CoA, with a free energy of hydrolysis of –32 kJ/mol. https://doi.org/10.1515/9783110612417-006

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In classical glycolysis, energy is invested to activate glucose by phosphorylation and isomerization to fructose-1,6-bisphosphate and energy is conserved at two steps: at the GAPDH reaction that we just discussed earlier and at the conversion of 3-phosphoglycerate to pyruvate. We said that in eukaryotic heterotrophs, ATP is synthesized during the oxidation of reduced carbon compounds. We explained carbon oxidation in the GAPDH reaction in detail earlier. What happens during the conversion of 3-phosphoglycerate to pyruvate? A carbon atom is oxidized but no NADH is produced. How does that work? In glycolysis, 3-phosphoglycerate is converted to 2-phosphoglycerate by phosphoglyceromutase and water is eliminated at the enolase step to yield PEP. At the enolase step, the phosphoester bond in 2-phosphoglycerate (not energy rich) is converted to an enolphosphate bond, which is energy rich. PEP can phosphorylate ADP via substrate-level phosphorylation at the pyruvate kinase step, generating pyruvate. But no NADH is generated, so where is the oxidation step? The carbon oxidation step occurs in the conversion of the hydroxy group at C2 of 3-phosphoglycerate to the keto group of pyruvate. The hydride (a proton with an electron pair) that was extracted from C2 in that oxidation step ends up (formally) on C3 of 3-phosphoglycerate, which becomes the methyl group in pyruvate. The reaction sequence in the 3-phosphoglycerate to pyruvate conversion is an intramolecular disproportionation, a pair of organic hydroxyl groups are converted to a keto group (oxidized) and a methyl group (reduced) with water elimination. The energy-conserving enolphosphate reaction of PEP is atypical. Substrate-level phosphorylation in eukaryotes almost always involves acyl phosphates generated by thioester intermediates, as in the ubiquitous GAPDH reaction, or in the reaction catalyzed by succinate thiokinase, which is almost ubiquitous, or in the reaction catalyzed by acetyl-CoA synthase (ADP forming), which is common in eukaryotes with exclusively cytosolic ATP production, or in the reaction catalyzed by phosphotransacetylase (PTA), which so far is rare in eukaryotes. Compartmentation of the enzymes of core carbon metabolism in eukaryotes can vary across lineages and species (Schnarrenberger et al. 1995; Müller 1996; Martin and Schnarrenberger 1997; Hannaert et al. 2003). Fermentation in eukaryotes can be localized exclusively in the cytosol or partially in the cytosol and partially in mitochondria. Human erythrocytes are completely devoid of mitochondria, with all ATP synthesis stemming from glycolysis in the cytosol (Bratosin et al. 2001). ATP synthesis is also confined to the cytosol in the unicellular parasitic species Giardia intestinalis and Entamoeba histolytica, whose mitochondria have been evolutionarily reduced to the form of mitosomes, which do not participate in energy metabolism (Müller 2003). In the parasitic protist Trichomonas vaginalis, fermentation is partly localized in hydrogenosomes, which are mitochondria that lack an electron-transport chain (Müller 1993). In many parasitic worms and marine invertebrates, the process of fermentation involves malate dismutation, which is coupled with electron transport in an electrontransport chain, localized in the mitochondria (Tielens et al. 2002). Some aspects of pathway compartmentation in eukaryotes are the direct result of the endosymbiotic origin of organelles, for example, the Krebs cycle in mitochondria. Energy metabolic

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pathways consisting of soluble enzymes can be relocated during evolution from one compartment to another (Martin 2010). However, no examples are known so far where membrane-associated energy metabolic pathways (respiration or photosynthesis) have been relocated from one organelle to another during evolution. Glycolysis: The backbone of energy metabolism in eukaryotes and their main fermentation pathway is glycolysis (Müller 2003), the Embden–Meyerhof pathway. During glycolysis, oxidation of one mole of glucose produces two moles of pyruvate with two moles of ATP and two moles of the reduced NADH. As we saw earlier, in order to maintain redox balance, continuous oxidation of glyceraldehyde-3-phosphate with formation of reduced NADH requires its reoxidation to NAD+. In eukaryotes specialized to aerobic environments, such as terrestrial plants and vertebrates, the most common fermentation pathways that regenerate NAD+ are ethanol fermentation and lactate fermentation (Livingstone 1991; Plaxton 1996; Loreti et al. 2018). The end product is generated in two successive steps: Pyruvate is decarboxylated by pyruvate decarboxylase (PDC) to acetaldehyde, which is subsequently reduced via alcohol dehydrogenase (ADH), to ethanol, alongside the oxidation of NADH to NAD+. Ethanol and CO2 are the typical fermentation products in plants, but goldfish can also survive weeks of anoxia through ethanol fermentation (see the following). Many eukaryotes including animals and fungi use lactate dehydrogenase for the synthesis of lactic acid, which accumulates in human muscles during exercise (Neumann et al. 2005). The crucian carp (Carassius carassius) can use ethanol fermentation to survive anoxia conditions at 2 °C for several months via ethanol fermentation using the PDC and ADH (van Waarde 1993; Fagernes et al. 2017). At 10 °C, freshwater goldfish (Carassius auratus) can survive without the use of oxygen in anoxic conditions for more than a week (van den Thillart et al. 1983). Neither in the carp nor in goldfish do mitochondria directly participate in ATP synthesis during anoxia, the fermentation is purely cytosolic (Fagernes et al. 2017). There is more variation of pyruvate catabolism observed among unicellular eukaryotes − protists. They harbor pathways of anaerobic energy metabolism that are not found in multicellular plants or animals. Plants and animals that have adapted to the modern high oxygen environments found on land have often retained the genes for many of the enzymes that their protistan relatives use in anaerobic energy metabolism, but use these enzymes for other purposes, for example, serving as oxygen sensors in plants or as components of iron–sulfur cluster assembly in animals (Gould et al. 2019). Diversity of protist anaerobic fermentation pathways was initially characterized in parasites because of their medical relevance (Müller et al. 2012). More recently, the fermentative pathways of parasites have been found in many photosynthetic eukaryotes, which often need to periodically deal with hypoxic and anoxic conditions in their natural environments, such as soils, microbial mats, and freshwater or marine sediments (Atteia et al. 2013), or which simply need to maintain redox balance (Gould et al. 2019).

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Pyruvate metabolism in anaerobic protists is often localized in mitochondria: anaerobic mitochondria and hydrogenosomes. Hydrogenosomes are anaerobic forms of mitochondria that lack cytochromes and quinones and produce ATP via fermentations. Fermentations in photosynthetic eukaryotes can reside in chloroplasts or can be dually localized in both chloroplasts and mitochondria (Mus et al. 2007; Meuser et al. 2009; Atteia et al. 2013). The enzymes of glycolysis are usually localized in the cytosol. But there are many exceptions. In trypanosomatids, a major portion of glycolysis is localized in glycosomes, specialized microbodies that are integral to the lifestyle of the parasite, which infects the human bloodstream and obtains energy solely from human blood sugar (Opperdoes and Michels 1993; Pieuchot and Jedd 2012). In diatoms, the enzymes of glycolysis are localized in the mitochondrion (Río Bártulos et al. 2018). In the green plant lineage, enzymes of glycolysis can be localized in the plastid (Klein 1986; Schnarrenberger et al. 1994). The glycolytic pathway starts with glucose; its product is pyruvate. The majority of variation in energy metabolism in eukaryotes concerns differences in the fate of pyruvate. The paradigm for pyruvate metabolism is the textbook situation found in mammals. Pyruvate enters the mitochondrion via the pyruvate carrier (Herzig et al. 2012) and oxidative decarboxylation via the pyruvate dehydrogenase (PDH) complex converts the pyruvate into acetyl-CoA, which is then oxidized to CO2 in the Krebs cycle with the released energy being stored in ATP (or GTP) as well as in NADH and FADH2. In most oxygen respiring mitochondria, pyruvate is oxidized to acetyl-CoA by the multienzyme complex PDH. Though typical of aerobic mitochondria, PDH also operates in some anaerobic forms of mitochondria. Examples include the common liver fluke Fasciola hepatica (Tielens and van Hellemond 1998). In eukaryotic anaerobes, pyruvate oxidation to acetyl-CoA is typically catalyzed by pyruvate:ferredoxin oxidoreductase (PFO or PFOR) (Steinbüchel and Müller 1986b; Charon et al. 1999) with release of CO2 and transfer of electrons to ferredoxin (Figure 2). The paradigms for PFO are the eukaryotic parasites Trichomonas vaginalis, Giardia lamblia and Entamoeba histolytica (Müller 2003). In Trichomonas, PFO is localized in hydrogenosomes, in Giardia and Entamoeba it is localized in the cytosol. Some facultative anaerobic protists like Euglena gracilis use both PDH and PFO in mitochondria, whereby the PFO of Euglena occurs as a protein fusion with a C terminal domain that transduces one-electron transfer to two-electron transfer, such that the electrons from pyruvate oxidation do not generate two reduced ferredoxins but one NADPH (Inui et al. 1982; Tucci et al. 2010). The same pathway can be localized to different compartments in eukaryotic cells. This is somewhat more of an evolutionary puzzle than it might seem. Not only glycolysis and pyruvate metabolism can occur in different compartments, there are many examples. The biosynthesis of terpenoids and starch metabolism in algae can be localized in different compartments (Lichtenthaler 1999; Deschamps et al. 2008)

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Malate ME

NAD+ NADH

CO2

2H+

HYD

Fd+ Fd

Pyruvate

PFO

CO2

H2

Acetyl-CoA

ASCT

Acetate

Succinate Succinyl-CoA CoA

SCS

Pi

ATP ADP

Figure 2: Oxidative pyruvate degradation in hydrogenosomes. In hydrogenosomes of some eukaryotic anaerobes, formation of acetyl-CoA is catalyzed by pyruvate:ferredoxin oxidoreductase, which is sensitive to the presence of oxygen. ATP is formed by substrate-level phosphorylation coupled with formation of acetate. End products are framed in dashed rectangles. Abbreviations: ASCT, acetate:succinate CoA-transferase Fd, ferredoxin; HYD, [FeFe]-hydrogenase; ME, malic enzyme; PFO, pyruvate:ferredoxin oxidoreductase; SCS, succinyl-CoA synthetase.

or β-oxidation of fatty acids, which can occur in either mitochondria or peroxisomes in animal tissues (Speijer 2011). How do pathways get from one compartment to another? Most proteins in eukaryotes are encoded in the nucleus. Chloroplasts and mitochondria contain more than 1,000 proteins each, but they harbor few genes in their organelle genomes. During the course of evolution, the genes for most proteins that were once encoded by the organelles have been relocated to the nucleus (Timmis et al. 2004). The genes are transcribed in the nucleus; the proteins are synthesized in the cytosol as precursors with an N-terminal extension that targets them back to the organelle where the gene came from. This is called endosymbiotic gene transfer and it requires the existence of a protein import apparatus that is specific to each respective organelle and that recognizes the N-terminal extension. If a gene is transferred to the nucleus, it can also be expressed as a cytosolic protein without an N-terminal extension. This simple mechanism can transfer organelle encoded pathways to the nucleus (Martin and Müller 1998). By transferring whole organelle genomes to the nucleus, which happens in evolution (Huang et al. 2005), whole pathways can end up in the cytosol, provided that all of the enzymes for the pathway become expressed. Before the origin of the protein import machinery of mitochondria, the TIM and TOM complex, genes transferred from the ancestral mitochondrial endosymbiont resulted in novel pathways being expressed in the host’s cytosol. These pathways might have competed with one another, a situation called functional redundancy (Martin and Schnarrenberger 1997), with the result that either the host’s pathway or the symbiont’s pathway survived, the other being displaced or lost or reprogrammed in function. An example of

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reprogramming is lipid biosynthesis. The archaeal host’s lipid biosynthetic pathway (the mevalonate pathway for isoprenes) was not lost, it was retained but not to synthesize membrane lipids, rather to synthesize isoprenes like dolichol phosphate or the isoprene tail of ubiquinone (Lange et al. 2000). Once expressed in the cytosol, the organelle localized genes (the original gene copies) can become lost. This process can lead to a nuclear encoded organelle pathway or a nuclear encoded cytosolic pathway. Once a pathway has become established, in order to become recompartmentalized, the whole pathway has to reach the new target compartment, for example, targeting glycolysis to the glycosome in trypanosomes. If a mutation sends one enzyme to the new organelle, then one component of the pathway is in the new location, yes, but one enzyme of a pathway is useless in a new compartment because it has no selectable function for lack of substrates and product conversion by the neighboring enzymes of the pathway. In order to maintain function, the whole pathway has to move, but that would require a huge number of simultaneous mutations all directed toward the same result (new localization for a dozen or so proteins). That is not the way that mutation operates, because mutation is blind. However, if an entire metabolic pathway, even with low amounts of the individual enzymes, finds its way into a novel compartment by chance or by imperfection in protein import specificity, it could readily become subject to natural selection. Recall that very small amounts of enzymes can provide very large amounts of product, a few percent wild-type enzyme activity is often sufficient to rescue a null mutant for a metabolic enzyme. Natural variation and selection could then increase or decrease the amount of novel pathway in the compartment. For this to operate, a basal level of minor mistargeting of pathways is required (Martin 2010). This is not unlikely, dual targeting of proteins to two compartments is extremely common in eukaryotes, up to one-third of mitochondrial proteins in yeast have dual localization (Burak et al. 2013). Here a warning is in order. This book will present a number of energy metabolic maps for eukaryotes indicating sites and amounts of ATP synthesis. Glycolysis is the backbone of eukaryotic energy metabolism and almost all variation in eukaryote energy metabolism has to do with the fate of pyruvate (Müller 1996). Glycolysis can yield two ATP from glucose in almost all species studied (an exception are the energy parasites that can lack even glycolysis), whereby we note that the ATP yield from amino acids, the main food source for most free-living eukaryotes, has not been extensively studied across lineages (Martin et al. 2017). The metabolic maps that we present in this book do not specifically indicate glycolytic ATP synthesis. That was the warning. Glycolysis is usually there but we do not discuss it because it does not differ between aerobes and anaerobes. Free-living eukaryotes rarely, if ever, encounter free glucose in the environment. Cellulose (a glucose polymer) is abundant but cellulose is not free glucose. Moreover, it was only a common substrate after the origin of land plants hence it is not particularly relevant for early eukaryote evolution. Glycolysis is usually there if glucose is. The converse is true

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for parasites of mammals. Parasites rarely encounter or utilize anything but glucose, because glucose is one of the main currencies of energy storage and distribution in humans. We will however see an example in this book of a voracious human facultative parasite that has specialized to consuming lipids instead of glucose. Again, almost all variation in eukaryote energy metabolism has to do with the fate of pyruvate. Returning to pyruvate metabolism, though, consider Figure 2, which shows the anaerobic energy metabolism of pyruvate breakdown in hydrogenosomes. Cleavage of the carbon–carbon bond between the carbonyl and carboxyl groups of pyruvate by PFO requires thiamine pyrophosphate. The enzyme has several FeS clusters and is readily deactivated by atmospheric oxygen. PFO in eukaryotes was first discovered in Tritrichomonas foetus in the early 1970s (Lindmark and Müller 1973), localized in a previously unknown organelle producing hydrogen gas (H2) as one of its fermentation products, the hydrogenosome. PFO is medically relevant because it reduces and thereby activates the drug metronidazole, which is used to treat infections with anaerobic or microaerophilic protozoa and bacteria (Rasoloson et al. 2002). Metronidazole is the drug most frequently used to treat patients infected with the parasites Trichomonas vaginalis and Giardia intestinalis or Entamoeba histolytica (Leitsch et al. 2009). The NADP+ reducing form of PFO is the fusion enzyme pyruvate:NADP+ oxidoreductase (PNO), which catalyzes the oxidative decarboxylation of pyruvate in hydrogenosomes and anaerobic mitochondria in several species of (facultative) anaerobic eukaryotes. PNO was first reported from Euglena gracilis (Inui et al. 1984a), which possesses two alternative pathways for the formation of acetyl-CoA from pyruvate (Müller et al. 2012). PNO consists of an N-terminal PFO protein domain fused with a C-terminal flavoprotein domain (Rotte et al. 2001; Nakazawa et al. 2003). The flavoprotein domain, also called NADPH:cytochrome P450 reductase domain, interacts with NAD+, FAD, and FMN cofactors and plays the role of a shuttle between singleelectron transfer from FeS clusters of the PFO domain and two-electron transport provided by the NADPH cofactor. PNO in the facultative anaerobic Euglena gracilis provides sufficient supply of acetyl-CoA and reduced NADPH cofactor needed for the formation of wax esters − end products of its fermentative metabolism (Inui et al. 1982; Tucci et al. 2010). Genes for PNO were also found in the apicomplexan Cryptosporidium parvum (Rotte et al. 2001) and homologs were later discovered in diverse eukaryotic lineages (Hug et al. 2010), such as Blastocystis sp. subtype 7 (Lantsman et al. 2008). Enzymatic activity is only known for the Euglena enzyme. Besides PDH, PFO, and PNO, eukaryotes also contain another alternative pathway of pyruvate conversion to acetyl-CoA, which is catalyzed by pyruvate:formate lyase (PFL). PFL provides pyruvate degradation that is independent from redox reactions, thereby avoiding formation of reduced NADH or NADPH cofactors. This can be important to maintain redox balance under some growth conditions. AcetylCoA and formate are formed through homolytic cleavage of pyruvate in a mechanism that requires a free radical (Wagner et al. 1992; Gelius-Dietrich and Henze

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2004; Stairs et al. 2011). Posttranslational activation of PFL is necessary, occurring by introduction of a free radical onto a glycine residue in PFL by the PFL activating enzyme (PFL-AE), which is dependent on iron. PFL-AE also requires an S-adenosyl methionine cofactor and reduced ferredoxin or flavodoxin. Like PFO, also PFL is very sensitive to the presence of oxygen because O2 causes its irreversible inactivation by cleavage at the position of the glycine residue. Based on the currently known distribution of PFL in eukaryotes, it seems that PFL is found more frequently in photosynthesizing eukaryotes than in heterotrophic eukaryotes such as fungi, which more often use PFO. Acetyl-CoA produced from pyruvate using PDH, PFO, PNO, or PFL is subsequently used by the cell to either (i) synthesize ATP from ADP and Pi by substrate-level phosphorylation, giving rise to acetate as product of fermentation, or to (ii) maintain redox equilibrium in the cell, forming an alternative fermentation product − ethanol (Atteia et al. 2013). So far, three different eukaryotic metabolic pathways in eukaryotes are known that produce acetate from acetyl-CoA, which yield ATP by substrate-level phosphorylation: (i) The first uses a pair of enzymes acetate:succinate CoA-transferase (ASCT) and succinyl-CoA-synthetase (SCS; also known as succinate thiokinase, abbreviated as STK) being sometimes descriptively referred to as ASCT/SCS cycle. ASCT first transfers coenzyme A from acetyl-CoA to succinate, catalyzing conversion of one thioester to another. Energy of the energy-rich thioester bond in the formed succinyl-CoA is then deposited into ATP using SCS, which is a classic Krebs cycle enzyme, thereby regenerating the succinate for repeated binding of coenzyme A (Hamblin et al. 2008). There are three types of ASCTs with limited sequence homology: ASCT protein subfamilies IA, IB, and IC (Tielens et al. 2010). The ASCT/SCS cycle is used for fermentative generation of ATP in anaerobic eukaryotes that contain hydrogenosomes as well as in species with (facultative) anaerobic mitochondria and even in some aerobic eukaryotes. (ii) The second pathway for formation of acetate and ATP from acetyl-CoA utilizes a single enzyme, acetyl-CoA synthetase (ADP-forming; abbreviated as [ADP]ACS). The enzyme generates an enzyme bound acetyl phosphate intermediate (Weiße et al. 2016). So far, the enzyme does not seem to be as common in eukaryotes as the pair of ASCT/SCS cycle enzymes. For instance, [ADP]-ACS is found in two parasitic protists Giardia intestinalis and Entamoeba histolytica, being involved in fermentative metabolism localized exclusively in the cytosol, as their mitochondria, reduced to mitosomes both in size and function, do not participate in energy metabolism (Reeves et al. 1977; Sánchez and Müller 1996). (iii) The third route of anaerobic degradation of acetyl-CoA coupled with ATP synthesis is again catalyzed by a pair of enzymes. In the first step, inorganic phosphate (Pi) is utilized by phosphate:acetyl transferase (PAT; also known as phosphotransacetylase, PTA) to liberate coenzyme A from acetyl-CoA, giving

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rise to acetyl-Pi. The energy-rich anhydride bond in acetyl phosphate acts as source of energy for substrate-level phosphorylation of ADP and ATP formation, catalyzed by acetate kinase (ACK, sometimes referred to as AK). So far, this third method of storing energy from the thioester bond of acetyl-CoA has been observed, in eukaryotes, only in the green alga Chlamydomonas reinhardtii (Atteia et al. 2006) and its relatives. The ASCT/SCS cycle is the most common route of ATP synthesis from acetyl-CoA through substrate-level phosphorylation in eukaryotes. Although glycolysis is the backbone of eukaryotic energy metabolism, some species possess glycolysis-independent metabolic pathways of ATP synthesis. Examples are the methylotrophic, strictly aerobic yeast species (e.g., Hansenula polymorpha (syn. Ogataea polymorpha) and Pichia pastoris) capable of using methanol as the sole source of carbon and energy for their growth (van Zutphen et al. 2010). It has been shown that when growing on methanol, the key role, besides mitochondria, is played by peroxisomes, which are enlarged and more numerous in such a condition. The reason is the peroxisomal localization of enzymes of methanol catabolism. Methanol is oxidized to carbon dioxide gas (CO2) and the electrons are utilized by an O2-dependent mitochondrial electron-transport chain for ATP synthesis via oxidative phosphorylation. Another exception, rarely found in eukaryotes, is represented by chemolithotrophic metabolism using inorganic electron donors in energy metabolism. Mitochondria localized in gills of Arenicola marina, annelid of the Polychaeta class, which lives buried in sands of littoral zones of seas and oceans, periodically exposed to euxinic conditions, anoxia combined with a high concentration of hydrogen sulfide (Lyons et al. 2009), are capable of oxidative degradation of toxic hydrogen sulfide (H2S). This powers proton pumping across the inner mitochondrial membrane and ATP synthesis by oxidative phosphorylation using an electron-transport chain (Grieshaber and Völkel 1998). Relatively rare is also the arginine dihydrolase pathway, used by some anaerobic eukaryotes to augment energy metabolism. The arginine dihydrolase pathway has a role analogous to the urea cycle (also known as ornithine cycle) in aerobic eukaryotes as it provides disposal of excessive nitrogen by microorganisms (Morada et al. 2011). In contrast with the urea cycle, the pathway includes ATP synthesis by substratelevel phosphorylation − one mole of ATP is produced from one mole of substrate, the amino acid arginine. In the first step, arginine releases ammonia, giving rise to citrulline, which, assisted by inorganic phosphate (Pi), undergoes phosphorolytic degradation to ornithine and carbamoyl phosphate. The energy-rich bond in carbamoyl phosphate is subsequently used by carbamate kinase to synthesize ATP, yielding CO2 and NH3 as end products (Yarlett et al. 1996). An extreme example of eukaryotes that are capable of obtaining ATP independently from glycolysis are intracellular parasitic fungi called microsporidia, whose

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mitochondria have evolved into mitosomes, during evolution of their parasitic lifestyle. Microsporidia are also known as energy parasites because they steal ATP from their host through their bacterial-type nucleotide transporter with homologs found in plastids of eukaryotes and inside obligate intracellular pathogenic bacteria of the genera Rickettsia and Chlamydia (Tsaousis et al. 2008). In order to make their survival inside host cells even more comfortable, microsporidia are able to manipulate the structure of the infected cell to such an extent that they gather host mitochondria in their vicinity, maintaining abundance of nutrition − they always dine at a richly laid table set by the host (Scanlon et al. 2004).

6 Respiration is not always aerobic Respiration is classically defined as O2 consumption with CO2 production. That is why peroxisomes were once considered as respiring organelles, which they are under the classical definition. In the context of energy metabolism, a more utilitarian definition of respiration is the use of external electron acceptors for redox balance (whether O2 or other compound) as opposed to the use of endogenously generated electron acceptors, which defines fermentations. In eukaryotes, respiration entails oxidative degradation of organic substrates, electron transfer through the mitochondrial electron-transport chain to a terminal electron acceptor, usually oxygen (aerobic respiration), but sometimes a different acceptor with sufficiently positive redox potential (anaerobic respiration). Respiration is usually, but not always, associated with the generation of ion gradients across the mitochondrial membrane and ATP synthesis via the mitochondrial ATPase. Both fermentation and respiration utilize the energy of redox reactions to power ATP synthesis. Respiration requires exogenous electron acceptors, which are obtained by organisms from their environment. Compared to fermentation, respiration is more efficient in terms of higher yield of ATP from the same substrate. However, in the environment, selection does not drive all organisms in the direction of energy efficiency, for were that so, each environment would harbor only greedy winners. Microbial communities are not greedy. They are characterized by trophic interactions that tend to be inclusive and inherently diverse, rather than inherently dominated by the most energy efficient or fastest growing species. Fermentation is not as energy efficient as respiration, but it is much faster when it comes to the rate of ATP synthesis. Sprinters fuel their muscles via substrate-level phosphorylation while long distance runners rely on respiration (Ross and Leveritt 2001). In mammalian mitochondria, pyruvate is oxidatively decarboxylated to acetylCoA, which enters the Krebs cycle (also known as the citrate cycle, tricarboxylic acid cycle, or citric acid cycle), in which the acetyl moiety is oxidized to CO2, with electrons entering the electron-transport chain. The complete oxidation of pyruvate yields three CO2, four NADH, and one FADH2 (Figure 3). Complex I is the proton pumping NADH dehydrogenase (Zhu et al. 2016), complex II is succinate dehydrogenase which donates electrons from FADH2 to ubiquinone (UQ) (Cecchini 2003), complex III is the cytochrome bc1 complex (Solmaz and Hunte 2008), and complex IV is cytochrome c oxidase (Tsukihara et al. 1996). Complex I and complex II transfer electrons to complex III via quinones (abbreviated by the letter U), which are soluble in the hydrophobic lipid bilayer of the membrane. Quinones accept electrons in pairs (2e−) together with two protons (2H+). Complex III transfers electrons to complex IV via cytochrome c. Protons are pumped at complex I, III, and IV. During respiration, protons are pumped from the inside of the mitochondrion (the matrix) to the intermembrane space. By moving charged particles (protons) across the https://doi.org/10.1515/9783110612417-007

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CO2

Pyruvate

PDH

Acetyl-CoA DNA

CO2

NADH

Krebs cycle

CO2

NAD+

O2 I

II e-

H+

U

III e-

C

ADP H 2O

ATP

IV

A

H+

H+

e-

H+

Figure 3: Pyruvate metabolism via O2 respiration in aerobic mitochondria. The Krebs cycle and respiratory chain using oxygen as the terminal electron acceptor oxidize pyruvate to carbon dioxide and water. ATP is produced via oxidative phosphorylation utilizing a proton-motive force generated by the electron-transport chain. Abbreviations: I−IV, respiratory chain complexes I to IV; A, ATP synthase; C, cytochrome c; PDH, pyruvate dehydrogenase complex; U, ubiquinone.

membrane, electron transport in the electron-transport chain creates an electrochemical gradient, called proton-motive force (Δp), or pmf, across the inner mitochondrial membrane. The proton-motive force consists of its chemical component − concentration gradient of protons, expressed as ΔpH, and an electric component − gradient of electric charge resulting in a membrane potential (ΔΨ). The mitochondrial matrix becomes depleted in protons, basic in pH, and negatively charged relative to the intermembrane space. Proton-motive force is a form of free energy, which can be utilized by cells to carry out work or to synthesize ATP. Prokaryotes use pmf to drive the rotation of their flagella during locomotion. Active transport of ions across biological membranes is also heavily dependent on this form of free energy. Protons reenter the mitochondrial matrix through the ATP synthase (abbreviated form ATPase, sometimes referred to as complex V), which catalyzes the reversible synthesis of ATP from ADP and Pi (Figure 3). This process of the flow of electrons from NADH and FADH2 to oxygen generates a proton gradient, which is then used to drive ATP synthesis from ADP and Pi, is called oxidative phosphorylation − it is ATP production powered by a proton-motive force generated through an electron-transport chain. The idea that ATP synthesis in mitochondria might entail ion gradient generation with the gradient powering ATP synthesis stems from Peter Mitchell (Mitchell 1961) and was called the chemiosmotic

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hypothesis. The hypothesis that the electron-transport chain and ATPase are biochemically separate systems linked only by a proton-motive force was controversial at first, but it turned out to be correct in all salient aspects, for which Mitchell received the 1978 Nobel Prize in Chemistry. The importance of chemiosmosis for understanding energy metabolism cannot be overstated. The electron-transport chain varies to some extent across eukaryotes. Many yeasts lack complex I, for example, and some eukaryotes have alternative versions of complex I or an alternative terminal oxidase (AOX) (Saari et al. 2019). An alternative complex I that does not pump protons occurs for example in Plasmodium (Biagini et al. 2006). An alternative terminal oxidase (AOX) that accepts electrons from UQ and then donating them to oxygen without pumping protons occurs for example in Trypanosoma brucei (Atteia et al. 2004; Degli Esposti et al. 2019). These alternative complexes are mainly involved in maintaining redox balance and not in the production of ATP. A consequence of the structure of the ATP synthase, namely the number of ring c-subunits in its membrane-bound Fo portion, which can vary from 8 to 15 subunits (Stewart et al. 2013), is that the synthesis of one ATP requires from 2.7 to 5 protons traversing the membrane, depending on the organism (Watt 2010). The number of protons translocated through the ATP synthase necessary to generate one ATP is called the H+/ ATP ratio (Nicholls and Ferguson 1992). The structure and function of rotor–stator ATP synthases allow for reversible function (Stewart et al. 2013). In the reverse direction, ATP hydrolysis powers pumping of protons (or, for instance, sodium ions), which is an indispensable function of ATP synthases in free-living prokaryotes in providing energy for metabolite import (Harold 1986) or for providing energy to render membrane-bound enzymatic redox reactions exergonic (Westphal et al. 2018; Schoelmerich and Müller 2019). When the electrontransport chain cannot be used to generate a membrane potential, some eukaryotes use the ATP synthase acting in reverse, hydrolyzing ATP to pump protons, to generate the membrane potential necessary for the import of the numerous essential mitochondrial proteins into the organelle (Neupert and Herrmann 2007). Examples are petitepositive baker’s yeast (Saccharomyces cerevisiae) when grown anaerobically and the long-slender bloodstream stage of Trypanosoma brucei, which has no protontranslocating electron-transport chain (Nolan and Voorheis 1992; Clark-Walker 2003; Schnaufer et al. 2005). Proton-motive force allows microbes to interconvert ATP and membrane potential. It is integral to many (if not most) fermentations in prokaryotes (Buckel and Thauer 2013), and some fermentations in eukaryotes. Just as reversed flow of protons through the ATPase can occur, reversed flow of electrons between the complexes of the electron-transport chain can occur under some conditions. In mammalian mitochondria, reverse electron transport is not the rule and can be very hazardous. It is encountered in situations where electron flow from UQ in the mitochondrial membrane into complex I generates reactive oxygen species (ROS). Such situations can include pathologies such as ischemia reperfusion

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injury (Robb et al. 2018), ROS mediated signaling of the physiological state of the cell (Fernandez-Aguera et al. 2015), or generation of ROS in macrophages (Mills et al. 2016). From an evolutionary standpoint, electron-transport chains were in existence long before there was oxygen. Those electron-transport chains just used different electron acceptors, such as sulfur, generating H2S instead of H2O from O2. Such anaerobic respiration is very common among prokaryotes (Rabus et al. 2015). Many microbes use either O2 or other compounds such as nitrate (Kraft et al. 2011) or iron(III) (Weber et al. 2006) as the terminal electron acceptor in the electron-transport chain. They are called facultative anaerobes, the most familiar example being Escherichia coli, which performs what are called “mixed acid fermentations” (Förster and Gescher 2014), that are, in terms of end products, virtually indistinguishable from the spectrum of end products generated by eukaryotes (Martin 2007). Under anaerobic conditions, E. coli is capable of both fermentation and anaerobic respiration (Bettenbrock et al. 2014). It can use O2 for growth at nanomolar concentrations if only nonfermentable substrates (Stolper et al. 2010) are provided. Under normal substrate availability, its anaerobic energy metabolic pathways switch on at oxygen levels below 1−4 μM (Becker et al. 1996), corresponding to the Pasteur point, the oxygen concentration below which facultative anaerobes typically switch to anaerobic metabolism. This occurs at about 2 µM O2 in water, which is the O2 concentration in water aerated with an atmosphere containing about 1% of our present atmospheric levels, that is, 0.2% O2 instead of 21% O2 by volume. When confronted with the presence of oxygen, some microbes prefer aerobic respiration. An exception is the Crabtree effect in some yeasts, which we will discuss later. The terminal acceptor that is used in the electron-transport chain can influence the type of quinone that is used. The quinone of mitochondria is UQ, which has a high midpoint potential (Em′ = +110 mV). Some mitochondria of invertebrates and protists use rhodoquinone (RQ) in addition (Em′ = −63 mV) (Hoffmeister et al. 2004). In prokaryotes, menaquinone (MQ) (Em′ = −80 mV) is very common (Sakai et al. 2012), RQ is rare but occurs in generalist α-proteobacteria (Hiraishi and Hoshino 1984; Hiraishi 1988). Archaea use MQ or alternatives to quinones such as benzothiophenes or methanophenazine (Berry 2003). Prokaryotes use a wide variety of anaerobic respiratory pathways, more than 140 such reactions using exogenous terminal electron acceptors were outlined by Amend and Shock (2001). By contrast, alternative inorganic terminal acceptors in eukaryotes are generally rare (Tielens et al. 2002; Müller et al. 2012), current known examples include inorganic nitrogen compounds. Nitrate (NO3−) is the terminal electron acceptor in denitrifying foraminifera, protists highly abundant in marine ecosystems (PinaOchoa et al. 2010), in several fungal species (Kobayashi et al. 1996; Watsuji et al. 2003), in diatoms (Kamp et al. 2011), and also among ciliates (Finlay et al. 1983). Anaerobic respiration of nitrite (NO2−) has been reported in mitochondria isolated from roots of two plant species (Stoimenova et al. 2007) and in several fungal species

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(Watsuji et al. 2003). Anaerobic reduction of elemental sulfur (S8) has been reported in a single eukaryotic species − fungus Fusarium oxysporum (Abe et al. 2007). A rather widespread anaerobic “respiratory” pathway that occurs both in prokaryotes and in eukaryotes is fumarate respiration (Tielens and van Hellemond 1998) in which fumarate serves as the terminal electron acceptor to yield succinate as the end product. The reason that respiratory is in quotation marks in the foregoing sentence is that fumarate is not an inorganic compound and not usually encountered in the environment, rather it is produced endogenously during metabolism, making fumarate respiration a fermentation, but a fermentation that involves an electron-transport chain. In mitochondria that perform fumarate respiration, a general pattern is observed. Phosphoenolpyruvate from glycolysis is first converted by phosphoenolpyruvate carboxykinase to oxaloacetate and reduced to malate (malate dehydrogenase) in the cytosol. During this reduction of oxaloacetate to malate, NADH produced by glycolysis is reoxidized, generating redox balance in the cytosol in the same way as lactate fermentation would. Malate is imported into the mitochondrion and metabolized by a set of pathways called malate dismutation (Tielens and van Hellemond 1998; Müller et al. 2012). As with fermentations, a portion of the malate is reduced and a portion is oxidized (dismutation is just another name for disproportionation). In the oxidative branch, malic enzyme catalyzes the oxidative decarboxylation of malate to pyruvate, which forms acetyl-CoA by traditional pyruvate dehydrogenase complex (PDH). In the so-called ASCT/SCS cycle, coenzyme A from acetyl-CoA is transferred by acetate:succinate CoA-transferase (ASCT) to succinate, giving rise to a highenergy succinyl-CoA intermediate. This is the substrate of succinyl-CoA synthetase (SCS), a Krebs cycle enzyme that both (i) catalyzes ATP synthesis by substrate-level phosphorylation and (ii) regenerates coenzyme A to be re-used by the PDH, and also (iii) provides succinate for the ASCT reaction. The oxidative end product of this part of the pathway is acetate, which is excreted to the environment. In the reducing branch, malate is reduced to succinate in two steps, which copy the Krebs cycle intermediates in reverse order. In the first step, fumarase catalyzes conversion of malate to fumarate, which is reduced to succinate in the second step. Reduction of fumarate is provided by a membrane-bound protein complex fumarate reductase, which accepts electrons from quinol − the reduced form of quinone. However, in contrast to the situation in aerobic electron-transport chains, this usually involves RQ because the UQ, playing role in aerobic respiration, does not have a sufficiently negative redox potential required to reduce fumarate (Tielens et al. 2002). Both fumarate reductase and RQ are parts of anaerobic electron-transport chains, which reduce RQ by electrons arriving from complex I of the electron-transport chain. Just like in aerobic respiration, complex I couples transport of electrons with pumping of protons into the intermembrane space of the mitochondrion and ATP is subsequently synthesized by ATP synthase (Tielens and van Hellemond 1998). With the mechanism of malate dismutation, malate is simultaneously the donor of electrons and, after its conversion to fumarate, also their terminal acceptor. After their

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extraction from malate in the oxidative branch of malate dismutation, electrons are directed to an anaerobic electron-transport chain through the reduced NADH cofactor and, in the reducing branch, eventually reduce fumarate to succinate by the membrane-bound fumarate reductase complex. Succinate is either released to the environment as an end product of energy metabolism or, depending on organism and growth conditions, it can be further utilized in the synthesis of extra ATP molecules through substrate-level phosphorylation in the propionate cycle with parallel propionate formation. Redox balance is maintained when acetate and propionate are excreted in the molar ratio of 1:2. In prokaryotes, it is common that fermentations entail the generation and use of ion gradients at the plasma membrane (Buckel and Thauer 2013). Microorganisms that live from small changes in free energy can, in principle, tap any exergonic reaction that is sufficient for translocation of even a single H+ (or Na+) ion across a membrane to support growth (Lim et al. 2014). The free energy change in an exergonic chemical reaction under physiological conditions that is needed to pump a proton across a membrane resides in the range of about –20 kJ/mol and is generally regarded as the biological energy quantum, or BEQ. This is the amount of free energy that must be released in the core bioenergetic reaction that the cell harnesses for pumping in order to synthesize ATP through chemiosmotic coupling. In some cellular configurations involving antiporters, exceptions to the “one proton rule” might exist (Müller and Hess 2017). For substrate-level phosphorylations, free energy changes of about –70 kJ/ mol in the main bioenergetic reaction are required, which is much greater than the free energy of hydrolysis of ATP (–31.5 kJ/mol) because biological energy conservation has an efficiency in the order of 40% (Thauer et al. 1977). Because malate dismutation is a fermentation, one could also call it fumarate fermentation , but the process as it occurs in eukaryotes is called fumarate respiration or malate dismutation in the literature, and there is no need to invent a new name here. Notably, malate dismutation is a fermentation that synthesizes ATP both by substrate-level phosphorylations and by using the proton gradient generated on the inner mitochondrial membrane and the ATP synthase. The overall energy yield from malate dismutation is about five ATP per glucose (four via substrate-level phosphorylation and about one additional ATP from proton pumping at complex I).

7 Using oxygen can be optional Oxygen is an option as a terminal electron acceptor. It offers more ATP per oxidized substrate than fermentations or anaerobic respiration. Living in its presence is, however, energetically 13 times more expensive than life without oxygen. Oxygen came late in evolution, the first electron-transport chains were anaerobic (Martin and Sousa 2016). When oxygen started accumulating in the Earth’s environments, it sparked the origin of five different kinds of O2 reductases (Degli Esposti et al. 2019), of which two are found in eukaryotes: complex IV and the alternative oxidase. When they arose, O2 reductases were integrated into preexisting anaerobic electrontransport chains. Many microbes welcomed the advent of oxygen, others remained anaerobes, and still others opted for a facultative lifestyle, either using O2 or growing anaerobically. Although O2 levels were low at the time when eukaryotes and mitochondria arose, there was at least some O2 available at the time of that symbiosis, because all O2 respiring mitochondria use the same machinery, essential components of which are still encoded in mitochondrial genomes (Allen 2015). From a facultatively anaerobic (or facultatively aerobic, which is the same) ancestral state, eukaryotes diversified into different lineages and underwent independent evolutionary trajectories. Some lost their electron-transport chains and specialized to anaerobic environments, some remained generalists who are able to survive with and without O2, one lineage (the plants) acquired a plastid, and still others retained the mitochondrial electron-transport chain but came to use it for functions in addition to ATP synthesis. In the following, we look at eukaryotes that live in oxic conditions and that possess mitochondria capable of respiration but that do not rely upon oxygen for the majority of their ATP synthesis.

7.1 An electron-transport chain with biosynthetic function Plasmodium falciparum is the infectious agent of malaria, every year it infects over 200 million humans and causes more than 400,000 deaths. It belongs to the phylum Apicomplexa and alternates between two hosts during its life cycle: female mosquitoes from the genus Anopheles and humans. Plasmodium parasites are unicellular eukaryotes that possess a single tubular mitochondrion whose biogenesis coordinates with the cell cycle. In the human bloodstream, Plasmodium multiplies inside erythrocytes as an obligate intracellular parasite. Mitochondrial DNA of Plasmodium species represents the smallest mitochondrial genome known, coding only for three proteins: a cytochrome b subunit of complex III and the subunits I and III of complex IV (Feagin et al. 1997). The remaining components of the electrontransport chain are encoded in the nucleus, such that all stages of P. falciparum have a fully functional electron-transport chain albeit with a bacterial-like type II https://doi.org/10.1515/9783110612417-008

7.2 The Crabtree effect: fermentation in the presence of O2

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NADH:ubiquinone oxidoreductase instead of the canonical complex I (Jacot et al. 2016; Goodman et al. 2017). However, despite living in an environment rich in glucose and oxygen, respiration contributes only marginally to the overall ATP formation, as oxygen consumption by the protist is minimal and electron-transport chain inhibitors hardly affect the overall ATP level in its cells. The main metabolic pathway of ATP synthesis in the asexual blood stages of Plasmodium is lactate fermentation using substratelevel phosphorylation (Jacot et al. 2016). What is then the purpose of the functional electron-transport chain in the mitochondria of the asexual blood stages of P. falciparum? Its sole known metabolic function is regeneration of ubiquinone (UQ), which is the electron-accepting cofactor for an enzyme essential for pyrimidine biosynthesis − dihydroorotate dehydrogenase (DHOD) (Painter et al. 2007; see also the following section). The mitochondrial electron-transport chain is completely expendable in a transgenic strain of P. falciparum, in which the native DHOD enzyme was replaced by a homolog from Saccharomyces cerevisiae, a yeast capable of aerobic or anaerobic growth. Yeast DHOD uses fumarate as its electron acceptor rather than the lipophilic UQ situated in the membrane. Thus, the asexual blood stages of P. falciparum utilize O2 to a limited extent in their mitochondrial electron-transport chain, yet not for bioenergetic purposes, but to regenerate oxidized UQ required by DHOD − an enzyme involved in pyrimidine biosynthesis. Most of the standard bioenergetic mitochondrial functions are not essential during the nonsexual blood stages. However, in contrast to earlier ideas, it is now clear that the bioenergetic function of the mitochondrion is also essential for the parasite when it moves from the red blood cell to the stages in the mosquito and thereafter in the liver stage upon infection of a new human host (Ke et al. 2015; Goodman et al. 2017).

7.2 The Crabtree effect: fermentation in the presence of O2 The Crabtree effect is the fungal version of “just say no” to O2. The budding yeast Saccharomyces cerevisiae has perfectly good functioning mitochondria, although they lack a proton-translocating NADH dehydrogenase and have an effective P/O ratio close to 1, giving them about 16 ATP per glucose oxidized to carbon dioxide instead of 30 in mammalian cells (Bakker et al. 2001). These yeast mitochondria support excellent growth also on nonfermentable substrates (Dejean et al. 2000). S. cerevisiae adjusts its metabolism to the conditions it encounters. When sugar is abundant, the yeast cells use fermentation as their major metabolic pathway and if sugar is scarce, they switch to an oxidative metabolism. During aerobic growth of yeast on glucose or fructose, only a minor fraction of the sugar substrate is completely oxidized to CO2 and H2O, whereas a major portion is partially oxidized to ethanol by alcohol fermentation. This is called aerobic fermentation or the Crabtree effect (De Deken 1966; Lin et al. 2013). Sometimes, two types of Crabtree effects are distinguished (Pronk et al. 1996),

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a long-term Crabtree effect (aerobic alcohol fermentation in the exponential growth phase of the culture regardless of whether saccharides are limited or present in excess), and a short-term Crabtree effect (short-term aerobic alcohol fermentation induced by culture transfer from sugar limitation to sugar excess. During evolution of ascomycetes, the ability to aerobically ferment simple saccharides to two-carbon organic compounds (ethanol and acetate), rather than their complete respiratory oxidation to CO2, arose in three independent lineages (Rhind et al. 2011; Rozpedowska et al. 2011; Hagman et al. 2013): the Saccharomyces cerevisiae lineage (which includes yeasts used in baking and brewing), the lineage leading to the budding yeast Dekkera bruxellensis, which diverged Saccharomyces about 200 million years ago, and the more anciently diverged fission yeasts including Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus. What evolutionary process allowed these three yeasts lineages to shun oxygendependent ATP synthesis in favor of O2-independent fermentation (Pfeiffer and Morley 2014)? It is hard to say what selection was involved, but one thing is certain, the mechanism did not involve lateral gene transfer, because no new genes entered these lineages to support these fermentations (the genes were present in their common ancestor). But the process did involve genome duplication, a leitmotif of eukaryotic genome evolution. Genome duplication gives rise to gene pairs (Wolfe and Shields 1997; Piskur and Langkjaer 2004). In budding yeast, several gene pairs are known in which one copy can be expressed under aerobic (normoxic) conditions while the other copy is expressed in hypoxic and/or anoxic conditions (Wolfe and Shields 1997). Gene pairs tracing to the whole genome duplication in the budding yeast lineage include CYC1/ CYC7 encoding cytochrome c, COX5A/COX5B coding for cytochrome c oxidase subunits, and paralogs for AAC2 (also called PET9) and AAC3 coding for mitochondrial ADP/ATP carriers (Langkjaer et al. 2003). AAC2 is expressed when yeast grows in the presence of oxygen and provides import of cytosolic ADP across the inner mitochondrial membrane into the matrix in exchange for mitochondrial ATP, synthesized by oxidative phosphorylation during aerobic respiration (Lawson and Douglas 1988). AAC3 is expressed in anaerobic conditions, where ATP is synthesized via fermentation in the cytosol. The function of the AAC3 carrier is to transfer cytosolic ATP to the mitochondrial matrix with simultaneous export of ADP from the mitochondrion, opposite to the aerobic condition (Drgon et al. 1991). The cytosol supplies the mitochondrion with ATP. The presence of a functional ADP/ATP carrier in the inner mitochondrial membrane is essential to anaerobic growth of S. cerevisiae yeast (Šubík et al. 1972; Kováč et al. 1977; Gbelská et al. 1983; Drgon et al. 1991). Under growth conditions in absence of oxygen, when yeast mitochondria do not synthesize ATP by oxidative phosphorylation, the energy demands of mitochondria have to be met by cytosolic ATP produced by substrate-level phosphorylation during fermentation. Inside anaerobically growing cells, the intramitochondrial ATP is utilized in multiple ways. ATP is hydrolyzed by the FoF1-ATP synthase, which pumps protons (H+) into the intermembrane space, generating pmf and maintaining membrane potential (ΔΨ; Visser et al. 1994).

7.2 The Crabtree effect: fermentation in the presence of O2

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The antiport ATPin (four negative charges) for ADPout (three negative charges) also contributes to membrane potential as in the case of Trypanosoma brucei (Loiseau et al. 2002). Since this transport is electrogenic (ATP4−/ADP3−), import of ATP and export of ADP generates the same membrane potential (ΔΨ) orientation as pumping of protons (negative in the matrix) (Visser et al. 1994). This ΔΨ is essential for protein import into the mitochondria (Gasser et al. 1982; Asai et al. 2004), as is the imported ATP itself (Wachter et al. 1994), which is needed by mitochondrial ATP-dependent chaperones in addition (Stuart et al. 1994; Kutejová et al. 1993). Imported ATP is also required for mitochondrial biosyntheses (Gbelská et al. 1983; Visser et al. 1994). In contrast to the facultative anaerobe S. cerevisiae, the strictly aerobic yeasts Kluyveromyces lactis, Schizosaccharomyces pombe, and Candida parapsilosis each have only one AAC gene, whose expression is, in all the three cases, suppressed in hypoxic cultivation conditions (Trézéguet et al. 1999; Neboháčová et al. 1999). It is possible that their inability to grow anaerobically relates to the lack of an ADP/ATP carrier for the import of cytosolic ATP from fermentations. The presence of an anaerobic form of ADP/ATP carrier is only one factor though. Another obligate aerobic yeast, Yarrowia lipolytica, encodes three AAC paralogs. Only the expression of YlAAC1 is suppressed during hypoxia, like the AAC genes in the strict aerobes K. lactis, S. pombe, and C. parapsilosis. However, even under hypoxia, YlAAC2 and YlAAC3 are expressed and the YlAAC3 paralog is slightly increased (Mentel et al. 2005). In Y. lipolytica, something other than the ADP/ATP carrier underlies its inability to grow in anaerobic conditions. Why should yeasts give up using O2 in aerobic environments? One theory has it that this relates to the origin of (sweet) angiosperm fruits, rich sources of energy and carbon. In this oxic ecological niche, there might be competition for fast growth, and the most successful species usually come from those three yeast lineages (Hagman et al. 2013). Fermentation provides ATP much faster than respiration, meaning more rapid reproduction, and more greedy consumption of resources for Crabtree-positive yeasts. The other advantage is the product of the fermentation − ethanol. Ethanol is toxic for most microorganisms (Piskur et al. 2006). On the other hand, some strains of S. cerevisiae and D. bruxellensis can tolerate up to 14% ethanol content (Rozpedowska et al. 2011). This lends itself to an interpretation of make-accumulate-consume, which evolved independently in D. bruxellensis and S. cerevisiae (Rozpedowska et al. 2011): rapid fermentation of saccharides to toxic ethanol that S. cerevisiae can subsequently utilize as a (nonfermentable) respiratory substrate. Budding yeast thus differs from the Crabtree-positive fission yeast Schizosaccharomyces pombe, which is incapable of using the produced ethanol as its sole carbon source because its so-called glyoxylate cycle is incomplete (Rhind et al. 2011). When glucose runs out, S. cerevisiae switches its metabolism from fermentation to respiration, referred to as the diauxic shift (diauxic: having two growth phases; see also the section on Fusarium oxysporum).

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It allows for respiratory ethanol degradation in the presence of oxygen (Pronk et al. 1996; Piskur et al. 2006), which entails downregulation (repression) of genes for aerobic respiration (Lin et al. 2013). Conspicuously detailed accounts of when and how this metabolic strategy originated are available for S. cerevisiae. A setting involving the origin of flowering plants in the late Triassic (Smith et al. 2010) has been proposed, with fruit production leading to freely available mono- and oligosaccharides in nature (Piskur and Langkjaer 2004), coinciding with a whole genome duplication in the yeast lineage (Wolfe and Shields 1997). For the evolution of the Crabtree effect, glucose repression and independence from oxygen for energy metabolism (Piskur and Langkjaer 2004) are important. In glucose repression, many genes required for respiratory sugar oxidation to CO2 and H2O, including the Krebs cycle, the electron-transport chain, and cytochromes (Piskur et al. 2006), are downregulated in their expression. Most of the facultative fermentative yeasts require small amounts of oxygen for growth even during alcoholic fermentation (Visser et al. 1990). In anaerobic conditions, ADH is induced in S. kluyveri (Gojkovic et al. 2004), as is a cytosolic fumarate dependent DHOD for pyrimidine biosynthesis. We will encounter DHOD in the section dealing with the malaria parasite Plasmodium, which has a quinone dependent DHOD that requires a respiratory chain to reoxidize the quinol; having a fumarate dependent DHOD allows pyrimidine biosynthesis in the absence of a functioning respiratory chain. Cytosolic ADH in S. cerevisiae is coded by two genes: ADH1 and ADH2 (Piskur et al. 2006). ADH1 is expressed constitutively, and the ADH1 enzyme has a relatively high Km (Michaelis constant) for ethanol, a low affinity to the substrate. ADH2 is only induced at low intracellular saccharide concentrations and the ADH2 enzyme’s Km for ethanol is 10 times lower than that of the ADH1 (a tenfold higher substrate affinity). The gene duplication and subsequent specialization of the newly formed isoenzymes ADH1 and ADH2 are estimated to have occurred less than 80 million years ago, after separation of Kluyveromyces lactis and S. cerevisiae lineages (Thomson et al. 2005). The duplication providing the AAC3 paralog that supplied the mitochondrion with ATP during aerobic fermentation was a key innovation. Though it evolved in three different yeast lineages, members of the genus Saccharomyces weaponized the Crabtree effect with a strategy that could be summarized as: “hold your breath, hog the loot, poison the witnesses, burn the evidence.” Greedy, uncontrolled proliferation is reminiscent of voracious insect masses during outbreaks of the 17 year locus and is more reminiscent of pathogenic growth than of microbes in communities. Uncontrolled glycolytic proliferation is the topic of the next section.

7.3 The Warburg effect: aerobic glycolysis in cancer cells Energy metabolism in cancer cells progresses from mitochondrial respiration towards fermentation, although oxygen is present (Warburg 1956). This is called aerobic

7.3 The Warburg effect: aerobic glycolysis in cancer cells

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glycolysis or the Warburg effect after Otto Warburg, one of the pioneers of biochemistry (and Nobel laureate in Physiology in 1931) who observed that in normal tissues 90% of the ATP comes from mitochondria whereas in tumor sections, more than 50% of the energy supply stems from glycolysis. Warburg’s observations came at a time when no one had a clue as to how respiratory ATP synthesis in mitochondria works. The observation that cancers mainly proliferate on the basis of glycolysis still generally holds (Denko 2008), although it should be mentioned that newer studies have uncovered a role for oxidative phosphorylation in several cancer types (Ashton et al. 2018). Glycolytic glucose consumption in tumor cells continues even when O2 supply would suffice to completely cover the needs of aerobic respiration. Like everything having to do with cancer, this shift to glycolytic metabolism is not simple. It is associated with changes in the action of oncogenes and tumor suppressor genes operating within numerous cellular signaling pathways, accompanying the transformation of healthy cells into cancer cells (Levine and Puzio-Kuter 2010). Aerobic glycolysis is not an efficient method of releasing energy from the energy substrate, but it is fast and simple. Aerobic glycolysis and lactate fermentation cause cancer cells to consume substantially more glucose than differentiated cells of healthy tissue while locally producing large amounts of lactate (Levine and Puzio-Kuter 2010; Vander Heiden et al. 2009). In contrast to the Crabtree effect in yeasts, the Warburg effect is no way an evolutionary strategy, because cancer is not an organism that produces progeny and cancer cells do not give rise to new organisms. Cancer is a manifestation of dysfunctional cell growth regulation. There is a vast literature describing tumor progression as an evolutionary process. Cancer cells do change over time during the course of clonal growth as the disease progresses. Evolution operates however over geological timescales, while cancer leads to cell proliferation within the body that generates no progeny surviving beyond the duration of a human lifespan. Fortunately, for almost all kinds of cancer with which humans can become afflicted, there are now very effective treatments. This book is about evolution of anaerobic energy metabolism in eukaryotes, not about cancer, but there is one protein that connects the two in a very straightforward manner: the hypoxia-inducible factor.

8 The hypoxia-inducible factor (HIF) What is hypoxia-inducible factor (HIF)? HIF is the main and most important O2-sensing system in animal cells, its discovery and characterization was awarded the Nobel Prize in Physiology or Medicine in 2019. HIF is a protein, a transcription factor that plays a key role in the response of cells to oxygen levels. The HIF pathway is present and operational in all animal lineages (Rytkönen and Storz 2011) tracing back to placozoa. The O2 levels at which HIF sensing elicits a response differs across lineages. Caenorhabditis elegans and other worms that live in soil live at low oxygen levels. Given a choice, Caenorhabditis prefers oxygen levels between 7 and 14% O2, only below 1% does Caenorhabditis accumulate HIF as a hypoxia warning signal (Branicky and Schafer 2008). Humans, by contrast, require O2 levels above about 14% for good brain function and good decision-making (Niedermeier et al. 2017); below 5–6% O2, corresponding to the O2 partial pressure at an altitude of about 8,000 m (the “death zone” feared by alpine climbers) human survival is almost impossible (Proffitt 2005), and over 300 humans have not survived the attempt to climb Mount Everest, either due to lack of oxygen to poor decisions, or both. HIF is suspected to be involved in human acclimatization to high altitude (Schuler et al. 2005), but pinpointing its role is not simple and genetic screens for signs of selection on HIF genes in high-altitude humans living in Tibet and the Andes have been equivocal (Bigham and Lee 2014). Note that one would not expect to see signs of selection in HIF if the protein of lowland inhabitants (like Reinhold Messner, the first recorded human to climb Mount Everest without an oxygen tank) does its job (gene activation via DNA binding) just as efficiently as the HIF of Tibet highland inhabitants. This is cause for reflection. The interested reader is directed to pages 192–210 of Hochachka and Somero (2002), who provide an outstanding survey of studies on physiological differences between human inhabitants of highlands (e.g., Tibet) and lowlands. The differences are many, obviously, and significant but they are not genetic, they are metabolic and physiological, and HIF plays a central role in those studies. What do we mean? Let us do a Gedankenexperiment. Let us conduct a thorough survey of physiological capabilities (heart rate, lung capacity, blood volume, red blood cell count, body mass index, etc.) of Olympic athletes worldwide compared to desk job employees of the same age from the same countries as the control group in each case. If we do that, we will find very real differences that are massively significant, but not due to genetic differences. Those differences will correlate with physical training and they will reflect the physiological responses of which the human body is capable. The chances that they will identify specific genes that increase the risk of having a desk job are negligible. There is currently a great deal of literature appearing on human genomes based on ancient DNA, conveying narratives that ancient humans had to interbreed (introgression) to acquire specific kinds of genes required in order to be better adapted to hypoxia at high altitude. (Note that the theory explains spread and not origin of alleles.) If those ancient adaptations were physiological (Hochachka and Somero 2002), mediated by https://doi.org/10.1515/9783110612417-009

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HIF and similar hypoxia response mechanisms (Semenza 2012), then all ancient humans had to do was find enough water, food, and good shelter at high altitudes, using the genes for physiological response that they already had. Put another way, humans might not have to acquire new genes or alleles to climb Mount Everest. How does HIF work? HIFs sense O2 (Figure 4) through a mechanism involving prolyl hydroxylases (PHD) (Semenza 2012; Samanta and Semenza 2018), the same family of enzymes that hydroxylate collagen, a connection to which we will return in Part III. HIFs are obligate heterodimers that consist of oxygen-regulated HIF-α subunit and a stable HIF-β subunit. HIF-α and -β themselves do not need to be induced, they are constitutively expressed but can be induced (Semenza 2012; Schuler et al. 2005). HIF-α subunits heterodimerize with the constantly expressed HIF-β subunits and when this dimer accumulates, it binds to hypoxia response elements, which results in increased transcription of genes for low oxygen response. The key to HIF activation is that under normal O2 levels HIF does not accumulate. That is because HIF-α is a good substrate for PHDs that hydroxylate proline residues in HIF-α in a reaction that requires O2 as a substrate. Hydroxylated HIF-α binds a protein called von Hippel–Lindau (VHL), which in turn recruits an ubiquitin protein ligase that attaches ubiquitin to the HIF-VHL complex. Ubiquitination is a signal that designates the complex for degradation in the proteasome (a large protein digesting complex). Thus, when O2 is around, HIF-α is degraded and the heterodimer is not formed, and hypoxia induced genes are, therefore, not activated because the activator is degraded in the presence of oxygen by a hydroxylation mechanism that directly involves O2 as a substrate (Maxwell et al. 1999; Fandrey et al. 2006). When O2 is lacking, HIF-α is not hydroxylated, because there is no O2 for the hydroxylation reaction, thus the HIF-α-β dimer accumulates and when it accumulates, it activates genes to elicit the physiological responses (Semenza 2012; Samanta and Semenza 2018). What does HIF do? The main physiological response to HIF in animal cells is a shift in energy metabolism from respiratory ATP synthesis in mitochondria to glycolytic ATP synthesis by diverting glycolytic flux at pyruvate through activating the transcription of many genes (Semenza 2012). The main reactions are as follows. Under hypoxic conditions, HIF activates genes encoding glucose transporters and the enzymes of the glycolytic pathway, thereby increasing the flux of glucose to pyruvate and glycolytic ATP synthesis in HIF-activated cells. At the same time, HIF increases transcription of pyruvate dehydrogenase kinase, which inactivates (via phosphorylation) PDH, the mitochondrial enzyme that converts pyruvate to acetylCoA for the Krebs cycle. HIF also activates transcription of LDH, which provides glycolytic redox balance by converting pyruvate to lactate. Furthermore, HIF activates transcription of two nuclear-encoded mitochondrial proteins called BNIP3 and BNIP3L, which induce mitochondrial-selective autophagy, also called mitophagy (Semenza 2012), a process in which cells digest their own mitochondria (Palikaras et al. 2015). HIF can also downregulate mitochondrial biogenesis, leading to lower numbers of mitochondria per cell (Denko 2008). HIF accumulation thus leads to

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HIF (hypoxia-inducible factor) Oxygen absent: HIF-1α is not degraded, transcriptional activation

Oxygen present: HIF-1α is hydroxylated and degraded

HIF-1α

PHD

HIF-1α O2

HIF-1β Cytosol

Proline hydroxylation

OH OH

HIF-1α

Nucleus VHL Ubiquitination OH

HIF-1α

Gene activation HIF-1α

HIF-1β

OH

Ub

OH OH

Ub

Ub Ub

Ub Ub

Ub Degradation in the proteasome

HRE (HIF-responsible element) Depending on the species and tissue ‒ Increased fermentation ‒ Reduced carbon flux to respiration ‒ Vascularization ‒ Erythropoietin induction

Figure 4: Oxygen-dependent regulation of HIF-1α (after Hammarlund et al. 2018). In normoxic conditions, the HIF-1α protein is recognized by prolyl hydroxylases (PHD), which hydroxylate prolines (–OH) in HIF-1α. Hydroxylated HIF-1α is recognized by the von Hippel–Lindau protein (VHL) and ubiquitinated (Ub) for degradation by the proteasome. In hypoxic conditions, HIF-1α and HIF-1β are translocated to the nucleus, where they form a heterodimer and bind to the hypoxia-responsive element (HRE) and then function as a transcription factor activating the expression of hypoxia-regulated genes.

shutdown of the Krebs cycle via inhibition of PDH, degradation of mitochondria, and induces fermentative pyruvate reduction to lactate. The role of HIF goes beyond energy metabolism. In mammals, embryogenesis occurs at O2 concentrations of 1–5%, rather than 21%, and HIF mediates the morphogenic role of O2 in various developmental systems (Dunwoodie 2009). There are also O2dependent proteins that hydroxylate the methylene group of asparaginyl residues in HIF-α; the hydroxylase is called factor inhibiting HIF (FIH), and it intervenes with the interaction between HIF-α with the coactivator p300, thereby impairing HIF transcriptional activity. The enzymatic mechanism of FIH and HIF PHDs is the same: one oxygen

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atom in O2 introduces a hydroxyl group into a methylene group of the substrate amino acid side chain while the other oxygen atom cleaves 2-oxoglutarate into succinate and CO2 (Elkins et al. 2003). The observation that the HIF-sensing system is conserved in terms of components and function back to the metazoan common ancestor clearly indicates that the first animals arose in environments where they were confronted with low oxygen and that the response to low oxygen has remained indispensable to the present. The Nobel prize in Physiology or Medicine 2019 was awarded jointly to William Kaelin Jr., Sir Peter Ratcliffe and Gregg Semenza for their discoveries of how cells sense and adapt to oxygen availability. They identified molecular machinery that regulates the activity of genes in response to varying levels of oxygen. Gregg Semenza studied the EPO gene and how it is regulated by varying oxygen levels. Sir Peter Ratcliffe also studied O2-dependent regulation of the EPO gene, and both research groups found that the oxygen sensing mechanism was present in virtually all tissues, not only in the kidney cells where EPO is normally produced. William Kaelin and Sir Peter Ratcliffe independently discovered that under normal oxygen levels, hydroxyl groups are added at two specific positions in HIF-1α (see Figure 4). Why is HIF so crucial for cancer? There are many investigations of the role of HIF in cancer (Denko 2008; Semenza 2012). As solid tumors grow, they tend to cut themselves off from the supply of nutrients for growth, including in particular glucose and O2, because these nutrients are supplied in blood vessels and are hence not adequately supplied by arterial blood if the tumors grow large enough to impair arterial blood flow. A crucial physiological role of HIF at high altitude is that it induces angiogenesis, the process of new capillary formation. This is an essential part of the acclimatization process, compensating low oxygen supply in air with an increased supply of (low oxygen) blood in tissues. This angiogenesis promoting activity of HIF is problematic in tumor tissue as it induces vascularization (the provision of blood vessels to a tissue), increasing blood supply and nutrient supply to the tumor (Krock et al. 2011; Semenza 2012; Masoud and Li 2015). This happens because the vascular endothelial growth factor, which regulates vascularization, is one of the genes that is activated by the HIF pathway (Hashimoto and Shibasaki 2015). There is nothing simple about cancer, but HIF is involved in many aspects of the growth of cells in tumors (Samanta and Semenza 2018) and that HIF is directly involved in stimulation of aerobic glycolysis is a result at many levels of tumor growth. Parallels and analogies between the energy metabolism of cancer cells and the energy metabolism of Crabtree-positive yeasts are evident (Ruckenstuhl et al. 2009; DiazRuiz et al. 2011). The energy metabolism of cancer cells has been added to the potential targets of antitumor therapy due to its specific differences from the energy metabolism of differentiated cells in healthy tissues of organisms. Are cancer cells aided by other energy metabolic pathways (Tomitsuka et al. 2010)? Cultivation of human cancer cells in hypoxic conditions with limited glucose supply leads to some ATP synthesis from fumarate respiration (Sakai et al. 2012). Glutamine also plays an important role in the oxygen-independent metabolism of cancer cells (Cluntun et al. 2017).

9 O2 dependent fermentations in trypanosomes Trypanosoma brucei causes sleeping sickness in humans (and nagana pest in cattle). It belongs to the larger group of protists called trypanosomatids, which in turn belong to the eukaryotic supergroup Excavata (Burki 2014). They are characterized by three unusual features: (i) Their cells have a single mitochondrion, which has a specific structure called the kinetoplast localized near the base of the flagellum. The kinetoplast is linked to the flagellum by filaments and comprises a nucleoprotein complex containing the mitochondrial DNA (mtDNA), also known as kinetoplast DNA (kDNA). In contrast to other mtDNAs, kDNA is a vast network of concatenated circular DNA molecules of two types − several thousands of minicircular DNAs and tens of maxicircular DNAs form single kDNA network packaged into a disk structure, the kinetoplast (Liu et al. 2005). The origin of kDNA occurred in successive steps from the traditional circular mtDNA, it gave the name to the Kinetoplastida (Lukes et al. 2002). (ii) In trypanosome mitochondria, most mRNA molecules undergo significant posttranscriptional modification during RNA editing, which includes insertion of hundreds and removal of dozens of uridylates (Stuart et al. 2005). Such edited mRNA is translated into fully functional proteins (Horváth et al. 2000). (iii) The major part of glycolysis is, in contrast with other eukaryotes, unusually localized inside glycosomes, which represent a metabolically specialized form of peroxisomes (Pieuchot and Jedd 2012) (Figures 5 and 6). The energy metabolism of all trypanosomatids studied to date is O2 dependent (Tielens and van Hellemond 2009). Why, beyond general interest, do they deserve treatment in a work focused on anaerobic energy metabolism of eukaryotes? There are two reasons. First, although they are unable to survive in the absence of oxygen, their energy metabolism is mainly fermentative. During oxidative degradation of organic compounds, they use several enzymes and metabolic pathways that are shared with anaerobic eukaryotes, which will feature later in the detailed description of (facultative) anaerobic mitochondrial metabolism in assorted eukaryotic species (Tielens and van Hellemond 2009). Second, although they are strictly aerobic and use oxygen as terminal electron acceptor, some species do not utilize, in all life cycle stages, a cytochrome-containing proton-pumping electron-transport chain and hence do not produce ATP by oxidative phosphorylation (OXPHOS). In such conditions, oxygen is utilized by a plant-like alternative oxidase (AOX) to maintain redox equilibrium (something we will also encounter in Trichomonads). The paradigm for energy metabolism in trypanosomes is the parasitic protist Trypanosoma brucei. Like many other parasitic organisms, the life cycle of T. brucei requires two hosts. It resides in infected mammalian hosts in the blood trypomastigote form, causing disease, being transmitted through bites of tsetse fly as vector. Inside the insect midgut, T. brucei undergoes transformation to the procyclic trypomastigote form, accompanied by a radical change of its energy metabolism. Throughout their life https://doi.org/10.1515/9783110612417-010

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Figure 5: Compartmentalized energy metabolism in bloodstream forms of T. brucei. Although mitochondria consume O2 as the terminal electron acceptor in the blood form stage, it is used solely for redox balance, not for ATP synthesis (Hannaert et al. 2003). O2 is reduced by the AOX to reoxidize UQH2 produced by the glycerol-3-phosphate shuttle, which regenerates NAD+ for glycolysis. Neither GPDH, nor AOX pump protons, hence no energy is conserved. On the contrary, the mitochondrial ATP synthase works in reverse, hydrolyzing glycolytic ATP imported from the cytosol to pump protons into the intermembrane space and generate a Δp. Abbreviations: A, ATP synthase; AOX, alternative oxidase; DHAP, dihydroxyacetone phosphate; G3P, glycerol3-phosphate; GAP, glyceraldehyde-3-phosphate; GPDH, glycerol-3-phosphate dehydrogenase; PGA, 3-phosphoglycerate; U, ubiquinone (photograph by Rob Koelewijn, Erasmus University Medical Center, Rotterdam, Netherlands).

cycle, T. brucei mitochondria are dependent on the presence of oxygen (compare Figures 5 and 6), but they are not always used for ATP synthesis (Bringaud et al. 2015). Then, what are the specific differences between the energy metabolic pathways of the procyclic and blood forms of T. brucei?

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Figure 6: Energy metabolism across three compartments in procyclic T. brucei. In contrast to the bloodstream form, pyruvate is further processed in the cytosol, mitochondria, and glycosomes. Cells of T. brucei do not reduce pyruvate to lactate (van Weelden et al. 2005). Part of the pyruvate is converted to alanine. The electron-transport chain of procyclic T. brucei generates a proton

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In the slender blood form of T. brucei, glycolysis is the sole metabolic pathway contributing to ATP synthesis. Glucose is only oxidized partially to an unusual and, here, virtually exclusive end product of energy metabolism − pyruvate. Although mitochondria of the blood form of T. brucei are functional, they do not synthesize ATP (Hannaert et al. 2003). The unusual role of mitochondria in the bloodstream form is redox balance (Figure 5) via a metabolic link between mitochondria and glycosomes. Since the end product of fermentative metabolism is pyruvate, reduced NADH cofactors produced in glycolysis are not reoxidized to NAD+ by the production of lactate or ethanol. Instead, trypanosomes use the same glycerol-3-phosphate shuttle present in mammalian cells to donate the electrons to a mitochondrial respiratory chain. In this shuttle, dihydroxyacetone phosphate is reduced by a soluble glycerol-3-phosphate dehydrogenase (GPDH) to glycerol-3-phosphate while the reduced NADH cofactor is oxidized to NAD+. Glycerol-3-phosphate acts as an electron shuttle from glycosomes to mitochondria, where it is reoxidized by a membrane associated GPDH (Opperdoes et al. 1977). The reduced GPDH transfers the electrons to ubiquinone (UQ). At that point, the trypanosomal pathway deviates from the mammalian one, as UQ now passes the electrons not to complex III, but to an AOX, catalyzing the formation of H2O out of O2 (Clarkson et al. 1989). Neither GPDH nor AOX couple the electron transport from NADH to O2 with proton pumping across the inner mitochondrial membrane into the intermembrane space, so the electron-transport chain of this composition does not generate proton-motive force for ATP synthesis. On the contrary, mitochondria of the bloodstream form of T. brucei, import ATP from the cytosol and use free energy released from its hydrolysis to ADP and Pi to generate proton-motive force (Nolan and Voorheis 1992; Schnaufer et al. 2005), which is essential for protein import and organelle biogenesis (Hewitt et al. 2014). In the procyclic insect form, the main end products are acetate and succinate, involving longer pathways in both glycosomes and mitochondria, while maintaining metabolic interconnections of the two organelles (Figure 6). Glycosomal metabolism becomes extended by phosphoenolpyruvate import from the cytosol. Phosphoenolpyruvate carboxykinase (ATP dependent), MDH, and a soluble FRD plus reoxidation of two NADH from glycolysis generate one mole of succinate

Figure 6 (continued) motive force (Δp), ATP is synthesized by oxidative phosphorylation. The branched electrontransport chain also includes an alternative NADH dehydrogenase insensitive to rotenone (Coustou et al. 2005). Abbreviations: I−IV, electron-transport chain complexes I to IV; A, ATP synthase; ALT, alanine aminotransferase; AN, alternative NADH dehydrogenase (rotenone-insensitive); AOX, alternative oxidase; ASCT, acetate:succinate CoA-transferase (protein subfamily IA); ATE, acetylCoA thioesterase; C, cytochrome c; CS, citrate synthase; FH, fumarase; GPDH, glycerol-3-phosphate dehydrogenase; KDH, α-ketoglutarate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; PDH, pyruvate dehydrogenase complex; PEP-CK, phosphoenolpyruvate carboxykinase (ATP dependent); PK, pyruvate kinase; SCS, succinyl-CoA synthetase; sFR, fumarate reductase (soluble); U, ubiquinone.

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(Besteiro et al. 2002). In the procyclic form of T. brucei, pyruvate is no longer excreted, but it is metabolized in mitochondria, where the Krebs cycle does not function as a cycle (van Weelden et al. 2003; van Weelden et al. 2005). Accordingly, even during the procyclic phase of its life cycle, T. brucei does not oxidize pyruvate to CO2, but performs only partial oxidations (despite the presence of O2). Acetyl-CoA from the PDH reaction is converted to acetate by two routes. The main pathway is via the ASCT/SCS cycle consisting of two enzymes. acetate:succinate CoA-transferase (of subfamily IA (Tielens et al. 2010); abbreviated as ASCT IA) transfers coenzyme A from acetyl-CoA to succinate. The energy of the thioester bond in succinyl-CoA is stored in ATP via substrate-level phosphorylation by succinyl-CoA synthetase (SCS), a typical Krebs cycle enzyme. SCS regenerates succinate, closing the cycle (Figure 6). In addition, T. brucei also has an alternative pathway for conversion of acetyl-CoA to acetate, making ASCT nonessential for the protist (Rivière et al. 2004). The alternative pathway of acetate synthesis from acetyl-CoA is catalyzed by acetyl-CoA thioesterase but does not conserve energy as ATP and the ASCT/SCS cycle play a key role in ATP production (Millerioux et al. 2012; Bringaud et al. 2015). Succinate in T. brucei mitochondrial fermentation is produced by two metabolic pathways. The first occurs in the glycosome and starts from malate (Figure 6) and entails the soluble, NADH dependent fumarate reductase that is not homologous to membrane-bound fumarate reductases (Coustou et al. 2005). The other pathway occurs in the mitochondrion and uses two Krebs cycle enzymes and allows procyclic T. brucei to utilize some amino acids such as proline as substrates for ATP synthesis (Figure 6). The electron-transport chain is branched: in addition to the typical complexes I to IV, it also incorporates AOX, GPDH, and an alternative, rotenoneinsensitive NADH dehydrogenase (Figure 6). In contrast to mitochondria of the bloodstream form, this electron-transport chain generates a proton-motive force (Δp) for ATP synthesis (Opperdoes and Michels 2008). These examples serve to make the point that oxygen, despite being a strong electron acceptor, that enables complete oxidation of substrates and OXPHOS, is often ignored by eukaryotes in their energy metabolism. Why shun such a good energy source? One reason is that, as mentioned at the outset of this book, O2 can improve energy yield from glucose or amino acids several fold, but life with O2 is energetically 13 times more expensive than without it, because O2 tends to oxidize organic compounds in general. O2 is certainly not required for fast growth, because fermenting forms of the same species generally grow faster than respiring forms (see the Crabtree effect and human tumor cells), or they produce ATP faster (see sprinters vs. long distance runners). “Yes but their resource use is not as efficient!” one might contend, yet if the fermenters have already consumed all the goods without O2, what use is having fuel efficiency with no fuel, other than to scavenge the products (acetate, propionate, succinate, ethanol, and lactate) that fermentation leaves behind? Also, cells that reduce O2 in their electron-transport chain are constantly confronted with managing the reactive oxygen species (ROS) that unavoidably result from that

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reaction, and ROS are very potent toxins for cells (Murphy 2009; Auten and Davis 2009; Sies et al. 2017), adding an element of deadly risk to the nonnegotiable necessity of maintaining redox balance (Allen and Raven 1996; Allen 2015). Even some multicellular animals switch to anaerobic mitochondrial energy metabolism in adult stages of their life cycle, even though their environment harbors O2 (Barrett 1991). These examples also show that eukaryotes that do not use O2 when it is present use enzymes that also occur in eukaryotic anaerobes. That means that there is no evolutionary process of de novo enzyme invention or acquisition underlying the origin of O2-independent lifestyles in eukaryotes, it is the result of ecological specialization (Müller et al. 2012). That makes sense because as we saw in Figure 1, eukaryotes arose and diversified during a billion years of the Earth’s history in which O2 levels were globally very low, as we will discuss in more detail in Part III.

10 Anaerobic mitochondria Mitochondria have a central position in the biology of eukaryotic cells (Lane 2005; van der Giezen 2013). In terms of energy metabolism, they are just as important for anaerobes as they are for aerobes (Shiflett and Johnson 2010; Müller et al. 2012). Most textbooks still depict mitochondria as a double membrane-bound organelle with cristae localized in the matrix, consuming oxygen and generating ATP through oxidative phosphorylation coupled with aerobic respiration, which satisfies energy demands of the eukaryotic cell after being exported to the cytosol by the ADP/ATP carrier. However, the term “mitochondria” refers to a broader spectrum of homologous (similar by virtue of common ancestry) organelles, which were once believed to have separate evolutionary origins. A simple functional classification of these organelles into five types is presented in Figure 7. The specialization of the organelles into strictly aerobic or strictly anaerobic types has occurred many times independently in evolution. As such, the classification in Figure 7 does not represent a phylogeny, it represents a classification. The best known mitochondria are aerobic mitochondria (class 1) functionally corresponding to the mammalian archetype. Recall that the oldest fossil mammals are only about 150 million years old (Sweetman et al. 2017), and that mammals arose from vertebrates that had been living on land for about 250 million years before they gave rise to mammals. Since the emergence of the first tetrapods, all land-dwelling vertebrates have required oxygen. At the time that animals evolved feet for land locomotion 395 million years ago (Niedzwiedzki et al. 2010), they irreversibly committed to O2 dependence in their evolutionary trajectory. It is not surprising that land vertebrate animals have an O2 dependent lifestyle and O2 dependent mitochondria. Here an observation is in order. There is something about the temporal order in which we learn things that affects the way we view evolution. Glycolysis is the first pathway we learned in school (or college), we therefore tend to think that it is also the most ancient pathway and that other pathways can be derived from it, which is not true. There was not a big pile of glucose or glucose-6-phosphate on the primordial Earth 4 billion years ago from which glycolysis arose; the first cells were autotrophs (Schönheit et al. 2016). Similarly, O2-specialized aerobic mitochondria (rat liver mitochondria) are the first kind of mitochondria we ever learned about in school or college, therefore it is perhaps natural to think that they represent the ancestral state from which all other mitochondria are derived, which is of course also not true. It is in particular not true because strictly aerobic environments did not exist anywhere on the Earth at the time that eukaryotes and mitochondria arose (Zimorski et al. 2019; Degli Esposti et al. 2019). Modern hypoxic habitats harbor eukaryotes whose mitochondria produce ATP but use terminal electron acceptors other than O2. The mitochondria that possess an electron-transport chain but do not use O2 as the terminal acceptor and do not https://doi.org/10.1515/9783110612417-011

10 Anaerobic mitochondria

ElectronATP transport production chain

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Figure 7: Functional classification of mitochondria. The classification is that of Müller et al. (2012). Aerobic mitochondria (class 1), also found in humans, use oxygen as their only terminal electron acceptor. Anaerobic mitochondria (class 2) are capable of utilizing (also) terminal electron acceptors other than oxygen, such as endogenously produced fumarate. Hydrogen-producing mitochondria (class 3), also known as hydrogenosomes with a genome, are an anaerobic form of mitochondria, which produce hydrogen gas and have an electron-transport chain. On the other hand, while the classic hydrogenosomes (class 4) also use protons as terminal electron acceptors, they have neither their own genome nor residues of an electron-transport chain. The most reduced mitochondrial forms are mitosomes (class 5), which do not participate in ATP synthesis.

produce H2 are called anaerobic mitochondria (class 2). If the mitochondria still have a functional electron-transport chain, even though in reduced form, and if they employ hydrogenases so that they can use protons as terminal acceptors of electrons, they are called H2-producing mitochondria (class 3). Because all mitochondria that possess an electron-transport chain still possess their own genetic material (Allen 2015), mitochondria in class III are sometimes called hydrogenosomes with a genome (Akhmanova et al. 1998), an intermediate form between mitochondria and hydrogenosomes (Boxma et al. 2005; de Graaf et al. 2011). Classic hydrogenosomes (class 4), originally discovered in trichomonads (Lindmark and Müller 1973), lack an electrontransport chain and as such they lack their own genetic material (Allen 2015), and their morphology has been reduced to the extent of a double membrane-bounded organelle lacking cristae in the matrix (van der Giezen 2009). Without selective pressure represented by the requirement of maintaining redox balance by respiration, the loss of electron-transport chain complexes could have occurred multiple times in independent eukaryotic evolutionary lineages with subsequent reduction of mitochondrial cristae and morphology of hydrogenosomes (Martin and Müller 1998).

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The most reduced form of mitochondria are mitosomes (class 5), which were discovered in 1999 in Entamoeba histolytica (Tovar et al. 1999; Mai et al. 1999). They have lost the mitochondrial genome, cristae, and also ATP synthesis. In addition to the mitosomes in these gastrointestinal parasitic protozoa, they have been found so far within intracellular parasites, which are sometimes localized in the vicinity of host mitochondria, stealing the host’s ATP products through bacterial-type nucleotide transporter localized within the parasite’s plasma membrane. Mitosomes contain enzymes involved in the synthesis of FeS clusters (Tovar et al. 2003; Burki 2016; Freibert et al. 2017). A highly reduced eukaryote, Monocercomonoides exilis, has recently been described which apparently lacks organelles of mitochondrial origin (OMOs) altogether (Karnkowska et al. 2016; Karnkowska et al. 2019). However, these flagellated protozoa branches within a eukaryotic group that possesses mitochondria, which shows that the lack of mitochondria in M. exilis, if the organelle is truly lacking, is a secondarily derived trait. There is no obvious reason why the only known conserved function of mitosomes, FeS cluster synthesis, should be localized in mitosomes (Tovar et al. 2003; Lill 2009; Nývltová et al. 2013; Freibert et al. 2017). FeS clusters are required for some enzymes of eukaryotic energy metabolism, including pyruvate:ferredoxin oxidoreductase and Fe–Fe hydrogenase. The different forms of mitochondria have been designated in different ways, including “mitochondria-like organelles,” (MLOs), though hydrogenosomes are not really like classical mitochondria in the functional sense and they are mitochondria in the evolutionary sense. The term “mitochondria-related organelles” (MROs) has also been proposed, but they are not related to mitochondria; they are mitochondria in the evolutionary sense. We and others (Krafsur and Jones 1967; Lopez-Garcia and Moreira 1999; Horner and Hirt 2004; van der Giezen 2009; PérezBrocal et al. 2010; Klute et al. 2011) have used the collective term “OMO, organelles of mitochondrial origin,” which is accurate in our view. The term amitochondriate eukaryotes was used in the 1980s and 1990s to designate those anaerobic eukaryotes such as species of Trichomonas, Giardia, and Entamoeba that lacked electron-transport chains, cytochromes, and quinones (Müller 1988; Müller 1992). Since then, it has been shown that the protists originally classified as amitochondriate were not truly amitochondriate as they all had organelles of mitochondrial origin − some form of mitochondria.

11 Mitochondria with and without oxygen Typically, when we think of mitochondria, we think of our own (human) textbooktype organelles, aerobic mitochondria with a Krebs cycle, β-oxidation of fatty acids, urea cycle, pyruvate dehydrogenase, and oxidative phosphorylation. Mitochondria also serve as calcium stores. They replicate and transcribe their own mitochondrial DNA, translate mitochondrial mRNA. Their activities require transport of metabolites across both mitochondrial membranes. Gasses like oxygen and carbon dioxide can diffuse across both membranes (Brandolin et al. 1996). For most ions, however, the mitochondrial membranes are impermeable. Transport across the outer membrane occurs via a nonspecific voltage-dependent anion channel, mitochondrial porin (Mannella et al. 1992). The inner mitochondrial membrane selectively transports metabolites by specific membrane proteins that are embedded within the phospholipid bilayer and form the mitochondrial carrier family (MCF) (Palmieri et al. 2011). One of the most important importers in mitochondria, and one of the most recent to be identified, the mitochondrial pyruvate carrier, does not belong to the MCF family, though (Herzig et al. 2012). In aerobes, oxidative phosphorylation in mitochondria provides the bulk of the cell’s ATP. The end products are water and carbon dioxide (Figure 3). ATP is exported from the mitochondrion in exchange for ADP by the ADP/ATP carrier, the prototype of the mitochondrial transporter family. Complete oxidation of glucose to CO2 and H2O using the Krebs cycle and electron-transport chain provides about 30 moles of ATP per glucose (Rich 2010). In all mammals and for almost all multicellular eukaryotes that live on land and above the soil line, the picture of mitochondrial function in energy metabolism is the same (some parasites are multicellular animals and live above the soil line but have anaerobic mitochondria). Non-parasitic, land-dwelling animals are oxygen addicts and have been for 400 million years. Recall that in some eukaryotic aerobes, the lack of O2 can induce mitophagy (Semenza 2012), that is, the cell starts digesting its own mitochondria. This is also part of the hypoxia-inducible factor (HIF) response in humans. In yeast, there is a clear increase in mitophagy during the shift from respiratory to fermentative growth (Bhatia-Kiššová and Camougrand 2010). Because of that connection, the term “mitochondria without oxygen” in the title of this section opens up a vast literature on the relationship of O2 to the redox state of mitochondria, reactive oxygen species (ROS) and ROS signaling, and the removal of damaged mitochondria by internal digestion in lysosomes, that is, the autophagy of mitochondria, or mitophagy. We cannot go into that literature here, we just note that there is an abundance of it, and the same mitophagy response processes occur in plants (Broda et al. 2018), indicating an ancient nature of the process, though underpinned by different enzymes as those in animals and fungi. The deeper evolutionary significance of

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mitophagy is still somewhat of a puzzle, but that it relates to redox balance, oxygen metabolism, and the nutritional state of the cell is certain. The situation regarding “mitochondria without oxygen” and the biology of aerobic mitochondria, like our own, that experience undersupply of O2 becomes even more complicated, however, because if O2-dependent mitochondria in a strict aerobe do not have enough O2 as terminal acceptor, the quinone pool in the mitochondrial membrane becomes very reduced, becoming effectively a charged battery in search of a place to unload an excess of electrons. This happens, for example, when the flow of blood to the mammalian heart is restricted because, for example, the arteries that supply oxygenated blood to heart muscle become clogged. Of course, that state of hypoxia leads to heart muscle damage via many mechanisms that we cannot get into here. When such a physiological state arises, one would think that the best remedy is to immediately supply as much O2 as possible, but what happens upon resupply of O2 is that the hypoxic mitochondrial membrane is so charged with electrons that it runs backwards (reverse electron transport), unloading electrons onto O2 at a specific site of complex I (instead of complex IV), generating the superoxide radical O2•– and a cascade of ROS. This is likely to be the main mechanism of reperfusion injury following resupply of O2 to the heart in the wake of ischemia. It was once thought to be a purely pathological reaction, but it is now thought to also be a major mechanism contributing to the O2-dependent physiological sensing of the redox state mitochondrial quinone pool (Robb et al. 2018). Sies et al. (2017) present a list of 12 point sources of superoxide radicals O2•– and H2O2 in mitochondria, of which complex I seems to be the major source in human tissues. Anaerobic mitochondria do not have such problems because they function without O2 in their natural physiological state. They are structurally typical mitochondria with cristae but in terms of metabolism, they can use compounds other than oxygen as terminal electron acceptors (Figure 7). This depends on environmental conditions or developmental stages of the organism in question. In anaerobic mitochondria of animals, the terminal electron acceptors are generated endogenously (malate dismutation) with CO2, acetate, propionate, and sometimes succinate being the end products. The end products of malate dismutation are excreted to the environment in free-living invertebrates or to the host in the case of parasites. In anaerobic mitochondria, malate dismutation involves an electron-transport chain, which, in contrast to that in aerobic mitochondria, uses rhodoquinone as the electron transporting quinone due to its strong electron donor potential, expressed by the redox potential value being lower than that of ubiquinone (Tielens et al. 2002; Sakai et al. 2012). Although the majority of the described eukaryotic species with anaerobic mitochondria use endogenously produced terminal electron acceptors, some are also able to reduce exogenous electron acceptors obtained from the environment. An example of such acceptors is nitrate (NO−3 ) participating in denitrification processes (Risgaard-Petersen et al. 2006) and the ability to reduce elemental sulfur (S8), as reported in the fungus Fusarium oxysporum (Abe et al. 2007). However, nonoxygen

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terminal electron acceptors obtained from the environment are still considered a rarity among eukaryotes. In contrast to ~30 ATP per glucose in humans, or ~18 ATP per glucose in yeast, malate dismutation (acetate, propionate and CO2) in anaerobic mitochondria yields about five ATP per glucose, four via substrate-level phosphorylation (two ATP from glycolysis, two ATP from acetate and propionate), and ~ one ATP generated by oxidative phosphorylation coupled with the anaerobic branch of the electron-transport chain (Atteia et al. 2013). The branch forms proton-motive force by the proton-pumping activity of complex I and that is subsequently conserved by the mitochondrial ATP synthase (~ one ATP per glucose), though without assistance from oxygen.

12 Hydrogenosomes and H2-producing mitochondria An alternative manifestation of mitochondria is the hydrogenosome, named for H2 that is produced as an end product of their fermentative energy metabolism (Lindmark and Müller 1973; Müller 1993). Typical hydrogenosomes have neither cytochromes nor quinones, lack an inner-membrane bound electron-transport chain and lack their own genome accordingly. They were first discovered in 1973, in the unicellular anaerobic livestock parasite Tritrichomonas foetus (Lindmark and Müller 1973). The T. foetus hydrogenosome and the organelle from another trichomonad representative, the human parasite Trichomonas vaginalis, which causes sexually transmitted urogenital tract infections in humans (Müller 1988), became a paradigm for energy metabolism studies of hydrogenosomes. With time, hydrogenosomes have been found in taxonomically diverse protist species adapted for life in anoxic environments, including ciliates (Yarlett et al. 1982), anaerobic fungi (Yarlett et al. 1986), and free-living amoeboflagellate species Psalteriomonas lanterna (Broers et al. 1990). Hydrogenosomes have only been found in protists. A report of hydrogenosomelike compartments in small marine animals, Loriciferans (Danovaro et al. 2010), raised controversy. The tiny animals (