Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes

Summary: Major insights into the phylogenetic distribution, biochemistry, and evolutionary significance of organelles involved in ATP synthesis (energy metabolism) in eukaryotes that thrive in anaerobic environments for all or part of their life cycles have accrued in recent years. All known eukaryotic groups possess an organelle of mitochondrial origin, mapping the origin of mitochondria to the eukaryotic common ancestor, and genome sequence data are rapidly accumulating for eukaryotes that possess anaerobic mitochondria, hydrogenosomes, or mitosomes. Here we review the available biochemical data on the enzymes and pathways that eukaryotes use in anaerobic energy metabolism and summarize the metabolic end products that they generate in their anaerobic habitats, focusing on the biochemical roles that their mitochondria play in anaerobic ATP synthesis. We present metabolic maps of compartmentalized energy metabolism for 16 well-studied species. There are currently no enzymes of core anaerobic energy metabolism that are specific to any of the six eukaryotic supergroup lineages; genes present in one supergroup are also found in at least one other supergroup. The gene distribution across lineages thus reflects the presence of anaerobic energy metabolism in the eukaryote common ancestor and differential loss during the specialization of some lineages to oxic niches, just as oxphos capabilities have been differentially lost in specialization to anoxic niches and the parasitic life-style. Some facultative anaerobes have retained both aerobic and anaerobic pathways. Diversified eukaryotic lineages have retained the same enzymes of anaerobic ATP synthesis, in line with geochemical data indicating low environmental oxygen levels while eukaryotes arose and diversified.     

Energy metabolism among eukaryotic anaerobes in light of Proterozoic ocean chemistry

Recent years have witnessed major upheavals in views about early eukaryotic evolution. One very significant finding was that mitochondria, including hydrogenosomes and the newly discovered mitosomes, are just as ubiquitous and defining among eukaryotes as the nucleus itself. A second important advance concerns the readjustment, still in progress, about phylogenetic relationships among eukaryotic groups and the roughly six new eukaryotic supergroups that are currently at the focus of much attention. From the standpoint of energy metabolism (the biochemical means through which eukaryotes gain their ATP, thereby enabling any and all evolution of other traits), understanding of mitochondria among eukaryotic anaerobes has improved. The mainstream formulations of endosymbiotic theory did not predict the ubiquity of mitochondria among anaerobic eukaryotes, while an alternative hypothesis that specifically addressed the evolutionary origin of energy metabolism among eukaryotic anaerobes did. Those developments in biology have been paralleled by a similar upheaval in the Earth sciences regarding views about the prevalence of oxygen in the oceans during the Proterozoic (the time from ca 2.5 to 0.6 Ga ago). The new model of Proterozoic ocean chemistry indicates that the oceans were anoxic and sulphidic during most of the Proterozoic. Its proponents suggest the underlying geochemical mechanism to entail the weathering of continental sulphides by atmospheric oxygen to sulphate, which was carried into the oceans as sulphate, fueling marine sulphate reducers (anaerobic, hydrogen sulphide-producing prokaryotes) on a global scale. Taken together, these two mutually compatible developments in biology and geology underscore the evolutionary significance of oxygen-independent ATP-generating pathways in mitochondria, including those of various metazoan groups, as a watermark of the environments within which eukaryotes arose and diversified into their major lineages.     

Endosymbiosis and the design of eukaryotic electron transport

The bioenergetic organelles of eukaryotic cells, mitochondria and chloroplasts, are derived from endosymbiotic bacteria. Their electron transport chains (ETCs) resemble those of free-living bacteria, but were tailored for energy transformation within the host cell. Parallel evolutionary processes in mitochondria and chloroplasts include reductive as well as expansive events: On one hand, bacterial complexes were lost in eukaryotes with a concomitant loss of metabolic flexibility. On the other hand, new subunits have been added to the remaining bacterial complexes, new complexes have been introduced, and elaborate folding patterns of the thylakoid and mitochondrial inner membranes have emerged. Some bacterial pathways were reinvented independently by eukaryotes, such as parallel routes for quinol oxidation or the use of various anaerobic electron acceptors. Multicellular organization and ontogenetic cycles in eukaryotes gave rise to further modifications of the bioenergetic organelles. Besides mitochondria and chloroplasts, eukaryotes have ETCs in other membranes, such as the plasma membrane (PM) redox system, or the cytochrome P450 (CYP) system. These systems have fewer complexes and simpler branching patterns than those in energy-transforming organelles, and they are often adapted to non-bioenergetic functions such as detoxification or cellular defense.     

Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS

Pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS) is an essential enzyme in the assembly of FeS clusters in bacteria as well as in the mitochondria of eukaryotes. Although FeS proteins are particularly important for the energy metabolism of amitochondrial anaerobic eukaryotes, there is no information about FeS cluster formation in these organisms. We identified and sequenced two IscS homologs of Trichomonas vaginalis (TviscS-1 and TviscS-2) and one of Giardia intestinalis (GiiscS). TviscS-1, TviscS-2, and GiiscS possess the typical conserved regions implicated in cysteine desulfurase activity. N-termini of TviscS-1 and TviscS-2 possess eight amino acid extensions, which resemble the N-terminal presequences that target proteins to hydrogenosomes in trichomonads. No presequence was evident in GiiscS from Giardia, an organism that apparently lacks hydrogenosmes or mitochondria. Phylogenetic analysis showed a close relationship among all eukaryotic IscS genes including those of amitochondriates. IscS of proteobacteria formed a sister group to the eukaryotic clade, suggesting that isc-related genes were present in the proteobacterial endosymbiotic ancestor of mitochondria and hydrogenosomes. NifS genes of nitrogen-fixing bacteria, which are IscS homologs required for specific formation of FeS clusters in nitrogenase, formed a more distant group. The phylogeny indicates the presence of a common mechanism for FeS cluster formation in mitochondriates as well as in amitochondriate eukaryotes. Furthermore, the analyses support a common origin of Trichomonas hydrogenosomes and mitochondria, as well as secondary loss of mitochondrion/hydrogenosome-like organelles in Giardia.     

Hydrogenosomes: convergent adaptations of mitochondria to anaerobic environments

Hydrogenosomes are membrane-bound organelles that compartmentalise the final steps of energy metabolism in a number of anaerobic eukaryotes. They produce hydrogen and ATP. Here we will review the data, which are relevant for the questions: how did the hydrogenosomes originate, and what was their ancestor? Notably, there is strong evidence that hydrogenosomes evolved several times as adaptations to anaerobic environments. Most likely, hydrogenosomes and mitochondria share a common ancestor, but an unequivocal proof for this hypothesis is difficult because hydrogenosomes lack an organelle genome - with one remarkable exception (Nyctotherus ovalis). In particular, the diversity of extant hydrogenosomes hampers a straightforward analysis of their origins. Nevertheless, it is conceivable to postulate that the common ancestor of mitochondria and hydrogenosomes was a facultative anaerobic organelle that participated in the early radiation of unicellular eukaryotes. Consequently, it is reasonable to assume that both, hydrogenosomes and mitochondria are evolutionary adaptations to anaerobic or aerobic environments, respectively.     

The missing link between hydrogenosomes and mitochondria

Mitochondria typically respire oxygen and possess a small DNA genome. But among various groups of oxygen-shunning eukaryotes, typical mitochondria are often lacking, organelles called hydrogenosomes being found instead. Like mitochondria, hydrogenosomes are surrounded by a double-membrane, produce ATP and sometimes even have cristae. In contrast to mitochondria, hydrogenosomes produce molecular hydrogen through fermentations, lack cytochromes and usually lack DNA. Hydrogenosomes do not fit into the conceptual mold cast by the classical endosymbiont hypothesis about the nature of mitochondria. Accordingly, ideas about their evolutionary origins have focussed on the differences between the two organelles instead of their commonalities. Are hydrogenosomes fundamentally different from mitochondria, the result of a different endosymbiosis? Or are our concepts about the mitochondrial archetype simply too narrow? A new report has uncovered DNA in the hydrogenosomes of anaerobic ciliates. The sequences show that these hydrogenosomes are, without a doubt, mitochondria in the evolutionary sense, even though they differ from typical mitochondria in various biochemical properties. The new findings are a benchmark for our understanding of hydrogenosome origins.     

The cellular machineries responsible for the division of endosymbiotic organelles

Chloroplasts (plastids) and mitochondria evolved from endosymbiotic bacteria. These organelles perform vital functions in photosynthetic eukaryotes, such as harvesting and converting energy for use in biological processes. Consistent with their evolutionary origins, plastids and mitochondria proliferate by the binary fission of pre-existing organelles. Here, I review the structures and functions of the supramolecular machineries driving plastid and mitochondrial division, which were discovered and first studied in the primitive red alga Cyanidioschyzon merolae. In the past decade, intact division machineries have been isolated from plastids and mitochondria and examined to investigate their underlying structure and molecular mechanisms. A series of studies has elucidated how these division machineries assemble and transform during the fission of these organelles, and which of the component proteins generate the motive force for their contraction. Plastid- and mitochondrial-division machineries have important similarities in their structures and mechanisms despite sharing no component proteins, implying that these division machineries evolved in parallel. The establishment of these division machineries might have enabled the host eukaryotic ancestor to permanently retain these endosymbiotic organelles by regulating their binary fission and the equal distribution of resources to daughter cells. These findings provide key insights into the establishment of endosymbiotic organelles and have opened new avenues of research into their evolution and mechanisms of proliferation.     

Suggested mitochondrial ancestry of non-mitochondrial ATP/ADP

One of the major evolutionary events that transformed endosymbiotic bacterium into mitochondrion was an acquisition of ATP/ADP carrier in order to supply the host with respiration-derived ATP. Along with mitochondrial carrier, unrelated carrier is known which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic alpha-Proteobacteria. This non-mitochondrial ATP/ADP carrier was recently described in rickettsia-like endosymbionts - a group of obligate intracellular bacteria, classified with the order Rickettsiales, which have diverged after free-living alpha-Proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on the non-mitochondrial carrier were reanalysed in the present work using both DNA and protein sequences, and various methods including Bayesian analysis. The data presented are consistent with classic endosymbiont theory for the origin of mitochondria and also suggest that even last but one common ancestor of rickettsiae and organelles may have been an endosymbiotic bacterium in which ATP/ADP carrier has first originated.     

Diversity and reductive evolution of mitochondria among microbial eukaryotes

All extant eukaryotes are now considered to possess mitochondria in one form or another. Many parasites or anaerobic protists have highly reduced versions of mitochondria, which have generally lost their genome and the capacity to generate ATP through oxidative phosphorylation. These organelles have been called hydrogenosomes, when they make hydrogen, or remnant mitochondria or mitosomes when their functions were cryptic. More recently, organelles with features blurring the distinction between mitochondria, hydrogenosomes and mitosomes have been identified. These organelles have retained a mitochondrial genome and include the mitochondrial-like organelle of Blastocystis and the hydrogenosome of the anaerobic ciliate Nyctotherus. Studying eukaryotic diversity from the perspective of their mitochondrial variants has yielded important insights into eukaryote molecular cell biology and evolution. These investigations are contributing to understanding the essential functions of mitochondria, defined in the broadest sense, and the limits to which reductive evolution can proceed while maintaining a viable organelle.     

Parallels in genome evolution in mitochondria and bacterial symbionts

Mitochondria, the energy-producing organelles of the eukaryotic cell, originate from an endosymbiotic alpha-proteobacterium. These organelles are believed to have arisen only once in evolutionary history, but despite their common ancestry, mitochondrial DNAs vary extensively throughout eukaryotes in genome architecture and gene content. New insights into early mitochondrial genome evolution come from the investigation of primitive mitochondriate eukaryotes, as well as the comparison between mitochondria and intracellular bacterial symbionts.     

Phylogenetic positions of several amitochondriate protozoa-Evidence from phylogenetic analysis of DNA topoisomerase II

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their 'lacking mitochondria' and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of 'long-branch attraction' appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be polyphyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

Phylogenetic positions of several amitochondriate protozoa

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their ‘lacking mitochondria’ and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of ‘long-branch attraction’ appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplo-monads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be poly-phyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

Evolution of macromolecular import pathways in mitochondria,hydrogenosomes and mitosomes

All eukaryotes require mitochondria for survival and growth. The origin of mitochondria can be traced down to a single endosymbiotic event between two probably prokaryotic organisms. Subsequent evolution has left mitochondria a collection of heterogeneous organelle variants. Most of these variants have retained their own genome and translation system. In hydrogenosomes and mitosomes, however, the entire genome was lost. All types of mitochondria import most of their proteome from the cytosol, irrespective of whether they have a genome or not. Moreover, in most eukaryotes, a variable number of tRNAs that are required for mitochondrial translation are also imported. Thus, import of macromolecules, both proteins and tRNA, is essential for mitochondrial biogenesis. Here, we review what is known about the evolutionary history of the two processes using a recently revised eukaryotic phylogeny as a framework. We discuss how the processes of protein import and tRNA import relate to each other in an evolutionary context.     

Hydrogenosomes, mitochondria and early eukaryotic evolution

Available data suggest that unusual organelles called hydrogenosomes, that make ATP and hydrogen, and which are found in diverse anaerobic eukaryotes, were once mitochondria. The evolutionary origins of the enzymes used to make hydrogen, pyruvate:ferredoxin oxidoreductase (PFO) and hydrogenase, are unresolved, but it seems likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobes, these proteins are found in diverse eukaryotic cells, including our own, where they are targeted to different cell compartments. Organelles related to mitochondria and hydrogenosomes have now been found in species of anaerobic and parasitic protozoa that were previously thought to have separated from other eukaryotes before the mitochondrial endosymbiosis. Thus it is possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, bearing testimony to the important role that the mitochondrial endosymbiosis has played in eukaryotic evolution. It remains to be seen if members of this family of organelles share a common function essential to the eukaryotic cell, that provides the underlying selection pressure for organelle retention under different living conditions.     

Presence of a member of the mitochondrial carrier family in hydrogenosomes: conservation of membrane-targeting pathways between hydrogenosomes and mitochondria

A number of microaerophilic eukaryotes lack mitochondria but possess another organelle involved in energy metabolism, the hydrogenosome. Limited phylogenetic analyses of nuclear genes support a common origin for these two organelles. We have identified a protein of the mitochondrial carrier family in the hydrogenosome of Trichomonas vaginalis and have shown that this protein, Hmp31, is phylogenetically related to the mitochondrial ADP-ATP carrier (AAC). We demonstrate that the hydrogenosomal AAC can be targeted to the inner membrane of mitochondria isolated from Saccharomyces cerevisiae through the Tim9-Tim10 import pathway used for the assembly of mitochondrial carrier proteins. Conversely, yeast mitochondrial AAC can be targeted into the membranes of hydrogenosomes. The hydrogenosomal AAC contains a cleavable, N-terminal presequence; however, this sequence is not necessary for targeting the protein to the organelle. These data indicate that the membrane-targeting signal(s) for hydrogenosomal AAC is internal, similar to that found for mitochondrial carrier proteins. Our findings indicate that the membrane carriers and membrane protein-targeting machinery of hydrogenosomes and mitochondria have a common evolutionary origin. Together, they provide strong evidence that a single endosymbiont evolved into a progenitor organelle in early eukaryotic cells that ultimately give rise to these two distinct organelles and support the hydrogen hypothesis for the origin of the eukaryotic cell.     

Analysis of two genomes from the mitochondrion-like organelle of the intestinal parasite Blastocystis: complete sequences, gene content, and genome organization

Acquisition of mitochondria by the ancestor of all living eukaryotes represented a crucial milestone in the evolution of the eukaryotic cell. Nevertheless, a number of anaerobic unicellular eukaryotes have secondarily discarded certain mitochondrial features, leading to modified organelles such as hydrogenosomes and mitosomes via degenerative evolution. These mitochondrion-derived organelles have lost many of the typical characteristics of aerobic mitochondria, including certain metabolic pathways, morphological traits, and, in most cases, the organellar genome. So far, the evolutionary pathway leading from aerobic mitochondria to anaerobic degenerate organelles has remained unclear due to the lack of examples representing intermediate stages. The human parasitic stramenopile Blastocystis is a rare example of an anaerobic eukaryote with organelles that have retained some mitochondrial characteristics, including a genome, whereas they lack others, such as cytochromes. Here we report the sequence and comparative analysis of the organellar genome from two different Blastocystis isolates as well as a comparison to other genomes from stramenopile mitochondria. Analysis of the characteristics displayed by the unique Blastocystis organelle genome gives us an insight into the initial evolutionary steps that may have led from mitochondria to hydrogenosomes and mitosomes.     

Biochemical and evolutionary aspects of anaerobically functioning mitochondria

Mitochondria are usually considered to be the powerhouses of the cell and to be responsible for the aerobic production of ATP. However, many eukaryotic organisms are known to possess anaerobically functioning mitochondria, which differ significantly from classical aerobically functioning mitochondria. Recently, functional and phylogenetic studies on some enzymes involved clearly indicated an unexpected evolutionary relationship between these anaerobically functioning mitochondria and the classical aerobic type. Mitochondria evolved by an endosymbiotic event between an anaerobically functioning archaebacterial host and an aerobic alpha-proteobacterium. However, true anaerobically functioning mitochondria, such as found in parasitic helminths and some lower marine organisms, most likely did not originate directly from the pluripotent ancestral mitochondrion, but arose later in evolution from the aerobic type of mitochondria after these were already adapted to an aerobic way of life by losing their anaerobic capacities. This review will focus on some biochemical and evolutionary aspects of these fermentative mitochondria, with special attention to fumarate reductase, the synthesis of the rhodoquinone involved, and the enzymes involved in acetate production (acetate : succinate CoA-transferase and succinyl CoA-synthetase).     

Sawyeria marylandensis (Heterolobosea) has a hydrogenosome with novel metabolic properties

Protists that live under low-oxygen conditions often lack conventional mitochondria and instead possess mitochondrion-related organelles (MROs) with distinct biochemical functions. Studies of mostly parasitic organisms have suggested that these organelles could be classified into two general types: hydrogenosomes and mitosomes. Hydrogenosomes, found in parabasalids, anaerobic chytrid fungi, and ciliates, metabolize pyruvate anaerobically to generate ATP, acetate, CO(2), and hydrogen gas, employing enzymes not typically associated with mitochondria. Mitosomes that have been studied have no apparent role in energy metabolism. Recent investigations of free-living anaerobic protists have revealed a diversity of MROs with a wider array of metabolic properties that defy a simple functional classification. Here we describe an expressed sequence tag (EST) survey and ultrastructural investigation of the anaerobic heteroloboseid amoeba Sawyeria marylandensis aimed at understanding the properties of its MROs. This organism expresses typical anaerobic energy metabolic enzymes, such as pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenase, and associated hydrogenase maturases with apparent organelle-targeting peptides, indicating that its MRO likely functions as a hydrogenosome. We also identified 38 genes encoding canonical mitochondrial proteins in S. marylandensis, many of which possess putative targeting peptides and are phylogenetically related to putative mitochondrial proteins of its heteroloboseid relative Naegleria gruberi. Several of these proteins, such as a branched-chain alpha keto acid dehydrogenase, likely function in pathways that have not been previously associated with the well-studied hydrogenosomes of parabasalids. Finally, morphological reconstructions based on transmission electron microscopy indicate that the S. marylandensis MROs form novel cup-like structures within the cells. Overall, these data suggest that Sawyeria marylandensis possesses a hydrogenosome of mitochondrial origin with a novel combination of biochemical and structural properties.     

Suggested mitochondrial ancestry of nonmitochondrial ATP/ADP carrier

One of the major evolutionary events that transformed an endosymbiotic bacterium into a mitochondrion was the acquisition of the ATP/ADP carrier (AAC) in order to supply the host with respiration-derived ATP. Along with the mitochondrial carrier, an unrelated carrier is known, which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic α-proteobacteria. This nonmitochondrial carrier was recently described in rickettsia-like endosymbionts (RLE), a group of obligate intracellular bacteria classified with the order Rickettsiales, which have diverged after free-living α-proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on nonmitochondrial AAC were re-analyzed in the present work, using both DNA and protein sequences and various methods including Bayesian analysis. The data presented are consistent with the classic endosymbiont theory for the origin of mitochondria and suggest that even the last but one common ancestor of rickettsiae and organelles was an endosymbiotic bacterium, in which AAC first originated.     

Mitochondria as we don't know them

Biochemistry textbooks depict mitochondria as oxygen-dependent organelles, but many mitochondria can produce ATP without using any oxygen. In fact, several other types of mitochondria exist and they occur in highly diverse groups of eukaryotes - protists as well as metazoans - and possess an often overlooked diversity of pathways to deal with the electrons resulting from carbohydrate oxidation. These anaerobically functioning mitochondria produce ATP with the help of proton-pumping electron transport, but they do not need oxygen to do so. Recent advances in understanding of mitochondrial biochemistry provide many surprises and furthermore, give insights into the evolutionary history of ATP-producing organelles.