Single Eubacterial Origin of Eukaryotic Sulfide:Quinone Oxidoreductase, a Mitochondrial Enzyme Conserved from the Early Evolution of Eukaryotes During Anoxic and Sulfidic Times

Ursula Theissen*, Meike Hoffmeister*, Manfred Grieshaber{dagger} and William Martin*,

* Institute of Botany III
{dagger} Institut für Zoophysiologie, University of Düsseldorf, Düsseldorf, Germany

Correspondence: E-mail: w.martin{at}uni-duesseldorf.de.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Mitochondria occur as aerobic, facultatively anaerobic, and, in the case of hydrogenosomes, strictly anaerobic forms. This physiological diversity of mitochondrial oxygen requirement is paralleled by that of free-living {alpha}-proteobacteria, the group of eubacteria from which mitochondria arose, many of which are facultative anaerobes. Although ATP synthesis in mitochondria usually involves the oxidation of reduced carbon compounds, many {alpha}-proteobacteria and some mitochondria are known to use sulfide (H2S) as an electron donor for the respiratory chain and its associated ATP synthesis. In many eubacteria, the oxidation of sulfide involves the enzyme sulfide:quinone oxidoreductase (SQR). Nuclear-encoded homologs of SQR are found in several eukaryotic genomes. Here we show that eukaryotic SQR genes characterized to date can be traced to a single acquisition from a eubacterial donor in the common ancestor of animals and fungi. Yet, SQR is not a well-conserved protein, and our analyses suggest that the SQR gene has furthermore undergone some lateral transfer among prokaryotes during evolution, leaving the precise eubacterial lineage from which eukaryotes obtained their SQR difficult to discern with phylogenetic methods. Newer geochemical data and microfossil evidence indicate that major phases of early eukaryotic diversification occurred during a period of the Earth's history from 1 to 2 billion years before present in which the subsurface ocean waters contained almost no oxygen but contained high concentrations of sulfide, suggesting that the ability to deal with sulfide was essential for prokaryotes and eukaryotes during that time. Notwithstanding poor resolution in deep SQR phylogeny and lack of a specifically {alpha}-protebacterial branch for the eukaryotic enzyme on the basis of current lineage sampling, a single eubacterial origin of eukaryotic SQR and the evident need of ancient eukaryotes to deal with sulfide, a process today germane to mitochondrial quinone reduction, are compatible with the view that eukaryotic SQR was an acquisition from the mitochondrial endosymbiont.

Key Words: sulfide-quinone reductase • endosymbiosis • hydrogenosomes • electron transport chain • anaerobiosis


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
ATP synthesis in most mitochondria involves the generation of a proton gradient with the help of the electron transport chain through respiratory complexes I to IV. Yet, numerous exceptions to that rule occur among anaerobic mitochondria, which can bypass complex IV or use alternative terminal acceptors other than O2, such as nitrate or fumarate (Tielens et al. 2002). Further exceptions are hydrogenosomes, anaerobic mitochondria that generate ATP through substrate level phosphorylation without the help of a proton gradient (Martin and Müller 1998). In all hydrogenosomes and in most mitochondria, ATP synthesis involves the use of reduced carbon compounds as the electron donor in one or a series of redox reactions. Yet some mitochondria can synthesize ATP chemolithotrophically, using proton gradients that are generated with the help of electrons taken not from carbon compounds, but rather from sulfide (H2S, HS-, and S2-), which is abundant in many anaerobic environments, such as marine sediments (Völkel, Hauschild, and Grieshaber 1995). In mitochondria of marine invertebrates from such environments, electrons from sulfide can be transferred to quinones as their entry point into the mitochondrial respiratory chain for ATP synthesis (Doeller et al. 1999; Doeller, Grieshaber, and Kraus 2001).

The terminal acceptor for electrons stemming from sulfide in mitochondria depends upon the sulfide concentration itself. This is because sulfide is a strong inhibitor of oxygen respiration (National Research Council 1979; Grieshaber and Völkel 1998). At sulfide concentrations below approximately 20 µM, electrons from sulfide can be donated to O2 as the terminal acceptor. Sulfide concentrations in the range of 10 to 50 µM inhibit the electron transfer from cytochrome c to complex IV (Bagarinao and Vetter 1990; Grieshaber and Völkel 1998). At higher sulfide concentrations, mitochondrial complex IV is blocked and the electrons are donated to alternative, yet unknown, acceptors, possibly involving a similar alternative oxidase as is found in plants (Völkel and Grieshaber 1996a; Parrino, Kraus, and Doeller 2000). Since sulfide concentrations can reach approximately 20 mM in environments inhabited by marine invertebrates (Fenchel and Riedel 1970; Völkel, Hauschild, and Grieshaber 1995), and since sulfide is a potent toxin, both the energy-producing and detoxifying functions of sulfide oxidation are essential to mitochondrial function in these organisms.

In eubacteria, sulfide oxidation is commonly catalyzed by the flavoprotein, sulfide:quinone oxidoreductase (SQR) (also called sulfide quinone reductase). The biochemistry of eubacterial SQR has been characterized in some detail (Reinartz et al. 1998; Griesbeck, Hauska, and Schütz 2000). Eubacterial SQR catalyzes the reaction H2S + Ubiquinone {longleftrightarrow} [S±0] + UbiquinoneH2 (Griesbeck et al. 2002). The enzyme has been purified and cloned from Rhodobacter capsulatus (Schütz et al. 1997) and Oscillatoria limnetica (Arieli et al. 1994) and biochemically characterized in Chlorobium limicola (Shahak et al. 1992), Rhodobacter capsulatus (Shahak et al. 1994), Paracoccus denitrificans (Schütz et al. 1998), Allochromatium vinosum (Reinartz et al. 1998), and Aquifex aeolicus (Nübel et al. 2000) (reviewed in Griesbeck, Hauska, and Schütz 2000). Bacterial SQR is a single polypeptide with an apparent molecular mass of 48 to 55 kDa, is possibly active as a dimer, is membrane associated, belongs to the glutathione reductase family of flavoproteins, and is inhibited by quinone analogs at micromolar or nanomolar concentrations (Arieli et al. 1994; Schütz et al. 1997; Griesbeck et al. 2002). Rhodobacter SQR was shown to reside in the periplasm (Schütz et al. 1997). In Chlorobium and Rhodobacter, electrons from sulfide enter into the electron transport chain of anaerobic photosynthesis through SQR (Griesbeck, Hauska, and Schütz 2000; Griesbeck et al. 2002). In the nonphotosynthetic {alpha}-proteobacterium Paracoccus denitrificans (Schütz et al. 1998) and in Aquifex aeolicus (Nübel et al. 2000), SQR introduces electrons from sulfide into the respiratory chain (reviewed in Griesbeck, Hauska, and Schütz 2000).

Mitochondrial SQR activity has been indirectly measured in many organisms through the mitochodrion-dependent formation of thiosulfate from sulfide, for example, in the annelids Heteromastus filiformis and Arenicola marina as well as in the molluscs Solemya reidi and Geukensia demissa (Oeschger and Visman 1994; reviewed in Grieshaber and Völkel 1998), but has not been purified to homogeneity from any multicellular eukaryote. A functional mitochondrial SQR was, however, recently cloned and characterized from the ascomycete Schizosaccharomyces pombe (Vande Weghe and Ow 1999). S. pombe SQR showed marked sequence similarity to the SQR purified and extensively characterized at the biochemical level from the {alpha}-proteobacterium Rhodobacter capsulatus (Schütz et al. 1997) and was furthermore shown to be imported into and functional in S. pombe mitochondria (Vande Weghe and Ow 1999). Mitochondrial SQR activity has been most extensively studied in marine invertebrates that inhabit sulfide-rich intertidal sediments, most notably the annelid lugworm Arenicola marina and the ribbed mussel Geukensia demissa (Völkel and Grieshaber 1996a; Parrino, Kraus, and Doeller 2000). Both in those organisms (Doeller et al. 1999; Doeller, Grieshaber, and Kraus 2001; Völkel and Grieshaber 1996b) and in recent biochemical studies of SQR from chicken mitochondria (Yong and Searcy 2001) it was shown that mitochondrial sulfide consumption was coupled to ATP synthesis. Since chickens do not inhabit sulfide-rich environments, the role of SQR in their mitochondria is probably not ATP production, but may involve detoxification. The primary oxidation product of sulfide produced by mitochondrial SQR is still not known with certainty. The two-electron reaction would yield either elemental sulfur (S±0) or sulfanes (HSSnH) as the primary oxidation product. The four-electron reaction would yield thiosulfate (S2O32-), which is the most commonly detected oxidation product (O'Brien and Vetter 1990; Völkel and Grieshaber 1992; Johns et al. 1997). Recent results by Yong and Searcy (2001) suggest that sulfanes might be produced during mitochondrial sulfide oxidation, but sulfanes have still not been directly detected.

The ability of mitochondria to perform sulfide oxidation for ATP synthesis raises the question as to the evolutionary origin of eukaryotic SQR genes, particularly from the standpoint of endosymbiotic theory (recently reviewed in Martin et al. 2001). In general, there are four simple possibilities for the origin of mitochondrial sulfide oxidation in eukaryotes. (1) The host that acquired the mitochondrion could have possessed an SQR enzyme that was retargeted to the mitochondrion to become functional there; in this case eukaryotic SQR should be related to archaebacterial SQR because the DNA replication (Tye 2000), translation (Lecompte et al. 2002), transcription (Reeve 2003), and chromatin-packaging systems (Reeve 2003) of eukaryotes are specifically related to their archaebacterial homologs. (2) The mitochondrial symbiont could have possessed the SQR enzyme, whereby the gene must have been transferred to the host's chromosomes, since SQR is not encoded in any mitochondrial DNA; in this case eukaryotic SQR should reveal a single eubacterial origin. (3) Neither host nor symbiont may have possessed SQR, and the SQR gene could have been acquired through horizontal gene transfer in organisms that inhabit sulfidic environments; in this case eukaryotic SQR should reveal multiple origins from diverse prokaryotic donors. (4) Eukaryotic SQR is an invention specific to the eukaryotic lineage; in this case eukaryotic SQR should be unrelated to prokaryotic SQR. The phylogenetic distribution of SQR-related enzymes among various genomes has been previously studied (Vande Weghe and Ow 1999; Griesbeck, Hauska, and Schütz 2000), but the phylogeny of SQR itself has not.

SQR was almost certainly an essential and possibly ubiquitous enzyme during the phase of eukaryotic evolution from 2 to 1 billion years ago, since newer geochemical evidence indicates that the Earth's ocean waters were anoxic and very sulfidic during that time (Canfield 1998; Shen, Buick, and Canfield 2001; Anbar and Knoll 2002), findings that underscore the evolutionary importance of anaerobic biochemistry in both ancient and modern eukaryotes (Tielens et al. 2002; Embley et al. 2003). Here, we report the occurrence of SQR and SQR-related enzymes among genomes of eubacteria, archaebacteria, and eukaryotes and examine the phylogeny of SQR, with particular attention to the evolutionary origin of the eukaryotic nuclear genes for mitochondrial sulfide:quinone oxidoreductase.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Database searching was performed using the sequences for the functionally characterized SQR from S. pombe (Vande Weghe and Ow 1999) and from Rb. capsulatus (Schütz et al. 1997) as queries against GenBank and against unfinished microbial genomes listed by the Institute for Genome Research (http://tigrblast.tigr.org/ufmg/). Sequence handling, data formatting, and alignment were performed with programs of the GCG Package version 10.3 (Genetics Computer Group, Madison, Wis.) and with ClustalW (Thompson, Higgins, and Gibson 1994). Alignments were reinspected and manually adjusted. For phylogenetic analyses, programs of the PHYLIP package (Felsenstein 1998), the MOLPHY package (Adachi and Hasegawa 1996), and Puzzle (Strimmer and von Haeseler 1996) were used. Uncorrected proportions of differences between sequences (p-distances) were calculated with ClustalW (Thompson, Higgins, and Gibson 1994). Logdet distances were calculated with the LDDist program available at the Web site http://artedi.ebc.uu.se/molev/software/LDDist.html. NeighborNet (Bryant and Moulton 2002) networks for representing the data were constructed with the software available at the Web site http://www.mcb.mcgill.ca/~bryant/NeighborNet/. For ProtML and Puzzle, the JTT-F matrix was used. Quartet-puzzling (QP) was employed using a gamma distribution and eight categories of rate heterogeneity.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Conservation and Fusions Among SQR-Related Proteins
Using Blast, SQR homologs were identified in genomes of eubacteria, archaebacteria, and eukaryotes and retrieved from the databases. Because of poor sequence conservation, the alignment of these sequences is challenging. For example, the functionally characterized SQR proteins from S. pombe and Rb. capsulatus share only 24% amino acid identity in the pairwise Needleman-Wunsch alignment. As a consequence, the automatic alignment programs ClustalW and Pileup align neither the FAD binding domains nor strictly conserved cysteine residues identified by Bronstein et al. (2000) and Griesbeck et al. (2002) in their analyses of SQR and related sequences from prokaryotes. Using those conserved domains as a guide, and using the terminology of Griesbeck et al. (2002), we manually identified the conserved regions corresponding to SQR fingerprint 2, containing Cys 159, and SQR fingerprint 5, containing Cys 353, as well as FAD binding domain III in all sequences shown in figure 1. These anchor points of sequence conservation were used for further manual refinement of the alignment. Residues Cys 159 and Cys 353, originally identified by Bronstein et al. (2000), refer to their positions in the SQR sequence from Rb. capsulatus (Griesbeck et al. 2002). The spacing of these conserved motifs is generally uniform throughout the alignment. Two further sequence motifs identified by Griesbeck et al. (2002) in the analysis of seven prokaryotic SQR homologs surrounding the residues Cys 127 and His 196 are not conserved across all 37 SQR homologs shown in figure 1 and hence are not shown. Patterns of sequence similarity visible in the alignment around Cys 159, Cys 353, and the FAD binding domain suggested the presence of three distinct groups of sequence diversity which we term groups I, II, and III. Although not thoroughly exhaustive with respect to all unfinished genomes, this database search provides a representative overview of sequence diversity for the SQR family, including all functionally characterized members. Across groups I, II, and III, amino acid identity in pairwise Needleman-Wunsch comparisons of unaligned sequences was approximately 25% on average. Of the sequences indicated in figure 1, only the homologs from Rb. capsulatus (Schütz et al. 1997), from S. pombe (Vande Weghe and Ow 1999), and from the cyanobacteria Aphanonthece and Oscillatoria (Bronstein et al. 2000) have been shown to represent active SQR enzymes.



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FIG. 1. Conserved regions from the alignment of SQR sequences (accession numbers given) corresponding to Cys 159, the FAD binding domain III, and Cys 353 using the numbering of Griesbeck et al. (2002). The residue number of the cysteines and the conserved glycine in FAD binding domain III (boldface type) in each sequence is given. Group designations (I, II, and III) refer to families of sequence similarity. Species names designated as SQR indicate that the sequence is known to encode SQR acitvity; those marked with an asterisk possess the N-terminal translational fusion indicated in figure 2. Gaps are indicated as dashes. Nostoc sp. PCC 7120 is synonymous with Anabaena PCC 7120

 
The partial sequence of an SQR homolog from Dictyostelium discoideum that contained the three strictly conserved motifs was assembled from unannotated sequence data (http://db.dictybase.org/) but lacked 125 N-terminal residues relative to the S. pombe sequence (fig. 1). One additional SQR homolog each was identified in the Caenorhabditis elegans (GenBank accession number NP_500688) and S. pombe (GenBank accession number T43278) genomes, but these sequences appeared to be C-terminally truncated and lacked the Cys 353 region. The Caenorhabditis homolog shown (GenBank accession number NP_502729) possesses a unique approximately 50–amino acid insertion at about position 230 that might represent a translated intron, but we were unable to identify possible intron donor and acceptor sites in the nucleotide sequence that would justify its removal. Searches among the genome sequence data for the anaerobic protists Giardia intestinalis and Entamoeba histolytica revealed no identifiable SQR homologs. No SQR homologs were detected among photosynthetic eukaryotes, although SQR detects glutathione reductase as a distant relative in several eukaryotic genomes in BLAST searches (data not shown).

The SQR homologs from Nitrosomonas europaea, Burkholderia fungorum, and Ralstonia metallidurans possess a well-conserved approximately 180–amino acid open reading frame (ORF) N-terminal to the SQR domain that is present as an independent ORF of unknown function in several prokaryotic genomes (fig. 2) but does not always co-occur with SQR. This N-terminal ORF contains one strictly conserved cysteine and an additional cysteine conserved in some sequences (fig. 2). Homologs of the N-terminal ORF from Agrobacterium, Sinorhizobium, Mesorhizobium, and Xylella are fused with yet another approximate 240–amino acid ORF of unknown function (fig. 3) that shares no similarity with SQR and that is annotated in several entries as a member of the metallo-ß-lactamase family by virtue of its similarity to the PFAM (Bateman et al. 2002) lactamase B family (PF00753; http://pfam.wustl.edu/), which includes several thiolesterases.



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FIG. 2. Conserved regions in an ORF N-terminally fused present to several SQR homologs (indicated with an asterisk next to the accession number; see also fig. 1) and present as an independent ORF in several genomes (no asterisk). Sequences marked with two asterisks possess at their C-terminus not SQR, but rather an unrelated ORF that is annotated in several genomes on the basis of weak sequence similarity as a ß-lactamase (see text). The strictly conserved cysteine is marked with "*," and the partially conserved cysteine is marked with "|." Their positions are indicated as in figure 1

 


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FIG. 3. Schematic overview of fusions involving SQR. The positions of conserved cysteine residues is indicated by dotted lines. (a) SQR as it occurs in most prokaryotes. (b) SQR as it occurs in S. pombe. T indicates the mitochondrial transit peptide identified (Vande Weghe and Ow 1999). (c) SQR as it occurs in sequences marked with an asterisk in figures 1 and 2. (d) Unfused ORF present in many prokaryotic genomes as indicated in figure 2. (e) Fusion of the ORF with an SQR-unrelated sequence as it occurs in sequences marked with two asterisks in figure 2

 
SQR Phylogeny
Phylogenetic analyses started with the complete alignment containing 36 OTUs (operational taxonomical units; sequences) and 715 positions, only seven of which were constant. Recalling that these sequences are highly divergent (<25% identity in many comparisons), and recalling that neither Pileup using the PAM250 matrix nor ClustalW using the Blosum matrices recovered the conserved cysteines (fig. 1) for all sequences, we started analyses with a NeighborNet network of protein logdet distances to obtain graphical representation of the data (fig. 4a). Neighbor-joining (NJ) (Saitou and Nei 1987) trees using the uncorrected proportion of amino acid differences (p-distance or Hamming distance) as a distance measure (NJP) gave a very similar picture. Although the p-distance is a rough measure, it has been shown to perform well in computer simulations when sequence divergence is high because it has a lower variance than correction procedures (Nei 1996; Nei and Kumar 2000). At the 95% bootstrap proportion (BP) level, the NJ tree of p-distances recovered all branches that protein logdet did plus only one additional branch—that joining Archaeoglobus and Magnetospirillum, which was found at BP = 93% using protein logdet.



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FIG. 4. Phylogenetic analyses of SQR sequences. (a) NeighborNet (NNet) network of protein logdet distances among SQR sequences. The NNet network depicts splits in the data as series of parallel lines, conflicting or non–tree-like signals for a given taxon or group of taxa can thus be represented. For example, in addition to the split linking Rhodospirillum and Pasteurella, there is also one linking Rhodospirillum and Sulfolobus tokadaii (a conflicting or non–tree-like signal). The splits joining members of group I and group II, respectively, are indicated. Rhodobacter is abbreviated as "Rb." (b) ProtML tree using the JTT-F matrix for sequences that pass the significance test for amino acid compositional equilibrium at P = 0.95. (c) ProtML tree using the JTT-F matrix for sequences from group II that pass the significance test for amino acid compositional equilibrium at P = 0.95. Bootstrap support for the monophyly of eukaryotic SQR in with various methods and for various subsets of the data is summarized in the box below the tree in (c) (see text). Branches supported at a BP >=95% with the method indicated are labeled with a dot; for the NNet graph, dots indicate splits with a BP >=95% in NJ trees of logdet distances. Eubacterial taxon designations are indicated for major recognized groups: {alpha}, ß, {gamma}, {delta}, {alpha}-proteobacteria, etc.; cy indicates cyanobacteria, and G+ indicates gram-positives. Sequence groups I, II, and III are indicated at the periphery of the tree in (a) and (b). Species names designated as SQR indicate that the sequence is known to encode SQR activity. Archaebacterial sequences are boxed. Eukaryotic sequences are indicated in bold. The scale bar indicates 0.1 substitution per site with the respective method

 
The groups I, II, and III identifiable in the alignment were recovered in the NJP topology, although the BP for group III was only 73% using NJP (48% using logdet). Local rearrangement using ProtML starting from the NJ tree of ML distances and using the JTT-F matrix strongly separated groups I, II, and III at BP greater than 95% each and furthermore grouped the eukaryotic sequences together (not shown) but with a low BP (59%). However, the amino acid composition of 14 sequences in the 36 OTU data deviated from the expectation at P = 0.95 as estimated with Puzzle: Thermosynechococcus, Magnetococcus, Rb. sphaeroides, P. syringae, P. aeruginosa, R. metallidurans, R. solanacearum, Staphylococcus, Rhodospirillum, Archaeoglobus, Desulfovibrio, Ferroplasma, S. tokodaii, and Thermoplasma. Removing these from the alignment (22 OTU data) and rechecking revealed that Burkholderia deviated, upon removal of which all remaining 21 sequences passed the significance test for amino acid composition. ProtML analysis of that data after excluding gapped sites (21 OTUs, 343 positions) produced the topology in figure 4b, which had a very low BP (< 50%) for the monophyly of the eukaryotic sequences, support for which increased to 86% when gapped sites were included (not shown).

However, the 21 OTU data (fig. 4b) was still highly divergent, with between group (I, II, and III) amino acid identity of sequences from the multiple alignment in the range of only 20%. To examine the possible monophyly of eukaryotic SQR more closely, we investigated the phylogeny of the 18 sequences belonging to group II in figure 4a. This data set (18 OTUs, 681 sites including gaps) was inspected for amino acid compositional bias, whereby P. aeruginosa, R. solanacearum, and Staphylococcus failed the frequency distribution test at P = 0.95. Removal of these sequences from the data and rechecking revealed that Rhodospirillum failed, leaving 14 OTUs, all of which passed the amino acid frequency distribution test. The resulting alignment (14 OTUs, 681 sites) contained better sequence conservation with 43 invariant sites and all aligned sequences being at least 30% identical in all comparisons, although this is still generally poor sequence conservation. Nonetheless, analysis with ML, NJ, and QP, also after exclusion of gapped positions (14 OTUs, 386 sites), provided good support for the monophyly of eukaryotic SQR (branch E in figure 4c) with all methods except MP (fig. 4c).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Sulfide:quinone oxidoreductase belongs to the larger family of disulfide oxidoreductase (DiSR) flavoproteins that includes glutathion reductases, the lipoamid dehydrogenase (E3) subunit of pyruvate dehydrogenase, thioredoxin reductase, and—importantly—flavocytochrome c (Fcc or FCC) (Schütz et al. 1997; Griesbeck et al. 2002). FCC is used by numerous prokaryotes for sulfide oxidation as an alternative to SQR (Schütz et al. 1997; Griesbeck et al. 2002). However, sequence comparisons revealed that all SQR sequences examined here are more similar to each other than they are to FCC or other members of the disulfide oxidoreductase flavoprotein family (fig. 5). For example, in Blast comparisons to GenBank, FCC members detect each other at E-values roughly less than 10-30 and usually share more than 25% sequence identity in pairwise comparisons, but FCC members detect SQR members at E-values roughly greater than 10-10, with which they usually share less than 20% sequence identity in pairwise comparisons, and vice versa. Other members of the DiSR family were detected at much lower similarity levels. This indicates that SQR and FCC are distinct but specifically related subfamilies within the larger family of DiSR flavoproteins. Notably, many of the prokaryotic genomes surveyed here encode both SQR and FCC homologs: Chlorobium (one SQR and two FCCs), Magnetospirillum, R. solanacaerum, R. metallidurans, Magnetococcus, Aquifex, Rb. sphaeroides, Paracoccus, Solfolobus, and Thermoplasma.



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FIG. 5. Schematic phylogeny depicting sequence similarity shared between SQR and FCC relative to other members of the DiSR family as estimated by Blast results and sequence identity in pairwise comparisons

 
Sequence Conservation and Motifs in SQR
Sequences such as SQR that share only about 20% identity in many pairwise comparisons pose a challenge to phylogenetic analysis. In such cases, phylogeny is strongly aided by information from three-dimensional structures (Schütz et al. 2000; Baymann et al. 2003). Crystal structures are available for several members of the DiSR flavoprotein family, including of FCC from Chromatium vinosum (Chen et al. 1994), but there are currently no three-dimensional structures available for SQR. In studies of ancient protein phylogeny or poorly conserved sequences, as with the present SQR data, the discrepancy between sequence conservation and structure conservation can become severe, as underscored by Rost (1997), who found that the majority of pairwise comparisons among protein sharing a common structure reveal only about 8% to 9% sequence identity. At low sequence identity, many assumptions of phylogenetic methods are almost certainly violated. For example, recent computer simulations showed that the fraction of sites determined to be neutral by the criterion of protein folding thermodynamics fluctuates in a manner that depends upon the randomly chosen neutral mutations accumulated by the sequence as it mutates through sequence space (Bastolla et al. 2002). Such findings suggest that at levels of sequence similarity where structural constraints can cause different fractions of sites in a protein to become neutral, current phylogenetic inference methods, which are founded in neutral theory, will reflect patterns of shared sequence similarity but will unlikely recover the true tree. Despite these problems, if one wishes to study the evolution of SQR, one has to work with the degree sequence conservation that SQR has to offer.

Based on spectroscopic and mutational analyses, Griesbeck et al. (2002) proposed a mechanism for the SQR that involved the participation of three cysteine residues, Cys 127, Cys 159, and Cys 353; however, they also noted that a catalytic mechanism involving only two cysteine residues instead of three would be compatible with the available data. We found that Cys 159 and Cys 353 are strictly conserved in SQR homologs investigated here, but we found no evidence for a conservation of Cys 127 outside of group I. Cysteine residues were lacking in several SQR sequences within 50 amino acids N-terminal of Cys 127 to Cys 159 (alignment available upon request) and also in the S. pombe SQR, the only sequence outside of group I that has been shown to be directly involved in sulfide oxidation.

Noting that Cys 127 is missing in S. pombe SQR, Griesbeck et al. (2002) pointed out that the Km values of roughly 2 mM each for sulfide and quinone measured for S. pombe SQR are 1000-fold higher than for the eubacterial enzymes. This low substrate affinity could, in principle, cast doubt on the functional identity of S. pombe SQR (HMT2, the product of the hmt2 gene) as a functional SQR enzyme. However, Vande Weghe and Ow (1999) showed (1) that isolated mitochondria from hmt2+ S. pombe cells could reduce exogenous quinones with sulfide, whereas hmt2- could not, (2) that hmt2+ S. pombe cells could oxidize endogenously produced sulfide, whereas hmt2- could not, (3) that HMT2 produced in E. coli is a flavoprotein, (4) that his-tagged HMT2 purified from E. coli reduces quinones in a sulfide-dependent manner in vitro, albeit with poor kinetic constants, and (5) that HMT2 resides in mitochondria. Thus, despite the high Km values measured for S. pombe HMT2 produced in E. coli (which lacks SQR), the brunt of evidence indicates that S. pombe HMT2 is an active mitochondrial SQR, but there remains the possibility that it requires an additional subunit or factor not required by group I SQR for full activity. This possibility and the lack of Cys 127 in several SQR sequences drew our attention to the ORF of unknown function translationally fused to the N-terminus of group I SQR from Nitrosomonas europaea, Burkholderia fungorum, and Ralstonia metallidurans. These possess a strictly conserved cysteine residue at position 40 of the Nitrosomonas sequence (fig. 2). Database searching revealed that this ORF is present in numerous eubacterial genomes and that Cys 40 is strictly conserved in all homologs, perhaps suggesting that it might be able to assume the function of Cys 127 in SQR from group II. However we could not identify this ORF in all genomes whose group II SQR lacks Cys 127, for example, Pasteurella multocida, leaving the question of whether two or three cysteine residues are involved in the SQR catalytic mechanism (Griesbeck et al. 2002) open from this standpoint.

SQR Sequence Diversity and Lateral Gene Transfer Among Prokaryotes
SQR homologs encompass three groups of sequence diversity that are nonuniformly distributed across eubacteria, archaebacteria, and eukaryotes (figs. 1 and 4a and b). Group I contains the functionally characterized eubacterial SQR enzymes from cyanobacteria and Rhodobacter capsulatus and furthermore contains only eubacterial homologs. Group III contains functionally uncharacterized SQR homologs detected in sequenced archaebacterial genomes in addition to eubacterial homologs from the sulfate reducer Desulfovibrio, from the anaerobic, photosynthetic, green sulfur bacterium Chlorobium, and from the {alpha}-proteobacterium Magnetospirillum. SQR sequences in group II (fig. 4a and b) comprise eubacterial and eukaryotic homologs.

Lateral gene transfer (LGT) exists among prokaryotes and has become a major issue in gene and genome evolution (Gogarten, Doolittle, and Lawrence 2002). The present analysis suggests that also SQR genes may have transferred among prokaryotes during evolution, judging from the interleaving of eubacterial and archae distribution of {alpha}-proteobacterial homologs (fig. 4a). However, when sequences possessing significant amino acid bias are removed from the data (fig. 4b), the degree of interleaving also decreases, suggesting that a phylogenetic argument for unrestricted LGT of SQR genes among prokaryotes cannot be made for these data. However, the presence of four robustly clustering cyanobacterial SQR genes in group I and the presence of a single cyanobacterial SQR in group II (Synechocystis) suggests that the Oscillatoria-type SQR (group I) might represent the endogenous cyanobacterial gene, whereas Synechocystis may have picked up its SQR from a proteobacterial donor (fig. 4c). The interleaving of ß-proteobacterial and {gamma}-proteobacterial homologs in figure 4c indicates further probable workings of LGT for SQR among prokaryotes.

A Single Origin of Eukaryotic SQR, a Eubacterial Relict from the Anoxic and Sulfidic Past
The present analyses provide evidence for a single eubacterial origin of eukaryotic SQR, indicating that eukaryotes sampled here acquired the gene for mitochondrial SQR once in evolution from a eubacterial donor (fig. 4c). The nature of that eubacterial donor is highly relevant to the issue of mitochondrial evolution and eukaryote origins. There are two simple possiblities: the donor of the SQR gene either was the ancestor of mitochondria or was not.

On the one hand, arguing meekly against the view that the SQR donor was the mitochondrial endosymbiont is a single finding, namely that eukaryotic SQR does not specifically branch with {alpha}-proteobacterial SQR. Indeed, it has recently been argued that any eukaryotic nuclear gene that is to be inferred to be of mitochondrial origin must be shown to branch specifically with {alpha}-proteobacterial homologs (Kurland and Andersson 2000; Canback, Andersson, and Kurland 2002). However, that view is probably too simplistic for several reasons. First, SQR is not a highly conserved protein, such that the early evolution of this eukaryotic gene as viewed from the perspective of phylogenetics may have simply been obscured by mutation. Sequence conservation in SQR permits one to trace the origins of the eukaryotic gene to eubacteria, but tracing it to any particular eubacterial lineage on the basis of 30% sequence identity is probably asking too much of phylogenetic inference methods. Such loss of phylogenetic signal among poorly conserved genes has been well documented in genome-wide phylogenies involving genes acquired from chloroplasts (Martin et al. 2002). Second, the overall pattern of sequence similarity in figure 4a suggests that contemporary {alpha}-proteobacteria may themselves have acquired their SQR genes from different sources, as evidenced by the presence of {alpha}-proteobacterial SQR homologs in group I (Rhodobacter), group II (Rhodospirillum, very close to a {gamma}-proteobacterial homolog), and group III (Magnetospirillum, branching among archaebacterial homologs). It is reasonable to assume that {alpha}-proteobacteria were undergoing LGT, also for SQR genes, at the time of mitochondrial origins and subsequently. Incorporating LGT into evolutionary thinking thus makes it difficult to pinpoint exactly which genes the ancestor of mitochondria possessed and/or contributed to eukaryotes on the basis of today's sequence comparisons. In other words, allowing for the existence of LGT during prokaryotic evolution (Gogarten, Doolittle, and Lawrence 2002), no single contemporary {alpha}-proteobacterium can be expected to contain exactly the same set of orthologous genes as the ancestral mitochondrial endosymbiont did (Rotte et al. 2001). Third, the current sampling of eubacterial lineages is currently quite sparse; in time, eubacterial homologs that are more closely related to eukaryotic SQR might be found. Thus, the lack of an {alpha}-proteobacterial branch for this poorly conserved and laterally transferred gene (SQR) does not constitute clear evidence against its mitochondrial origin.

On the other hand, several findings argue in favor of the view that the eukaryotic SQR gene was acquired from the ancestor of mitochondria. First, eukaryotic SQR functions in the mitochondrial membrane the same way that {alpha}-proteobacterial SQR functions in the eubacterial membrane, donating electrons from sulfide to quinones. Hence, a eubacterium with a diversified (facultatively) anaerobic electron transport chain would be the most likely SQR gene donor, for example one that could use fumarate as an electron acceptor. Eubacteria such as the {alpha}-proteobacterium Rhodospirillum rubrum, which possesses SQR, commonly use rhodoquinone (Okayama et al. 1968) alternatively to ubiquinone in their anaerobic electron transport chain, just like anaerobic mitochondria do today (Tielens et al. 2002). The inheritance by mitochondria of preexisting and functioning aerobic and anaerobic components in the same electron transport chain from a facultatively anaerobic ancestor of mitochondria that was perhaps similar in overall physiology to facultatively anaerobic {alpha}-proteobacteria, such as Rhodospirillum, Paracoccus denitrificans (John and Whatley 1975), or Rhodobacter, which possess SQR (Schütz et al. 1998; Griesbeck, Hauska, and Schütz 2000), seems much more likely than the piece-by-piece addition during eukaryotic evolution of anaerobic components (SQR, rhodoquinone, etc.) to an (hypothetical) ancestrally aerobic mitochondrial electron transport chain as envisaged by those who argue for an origin of mitochondria from strictly aerobic Rickettsia-like parasites (Kurland and Andersson 2000).

Second, the nuclear gene for SQR was apparently acquired once in eukaryotic evolution, not several times as would be predicted under models that envisage lateral gene acquisition from food bacteria as the major source of eubacterial, but apparently non–{alpha}-proteobacterial, genes in eukaryotes (Doolittle 1998). Such single acquisition with a phylogenetically unresolved eubacterial origin as seen for SQR is also observed for several other proteins involved in anaerobic ATP synthesis in eukaryotes, for example, [Fe]-hydrogenase (Horner, Foster, and Embley 2000; Horner et al. 2002), pyruvate:ferredoxin oxidoreductase (Horner, Hirt, and Embley 1999; Rotte et al. 2001; Embley et al. 2003), many glycolytic enzymes (Hannaert et al. 2000), and NADH oxidase (Nixon et al. 2002), not to mention many other eukaryotic proteins that are not involved directly in ATP synthesis, such as proteasome homologs HslV and HslU (Couvreur et al. 2002). Notably, [Fe]-hydrogenase and pyruvate:ferredoxin oxidoreductase possess several FeS clusters (Chabriere et al. 1999; Peters 1999), as do several proteins of the mitochondrial respiratory chain (Burger et al. 1996; Friedrich and Schiede 2000). Recent findings indicate that many proteins required for the assembly of FeS clusters are localized in mitochondria (Lill and Kispal 2000) and related organelles such as hydrogenosomes (Tachezy, Sanchez, and Müller 2001) and mitosomes (Katinka et al. 2001; Williams et al. 2002). The emerging monophyly of FeS cluster assembly in eukaryotes suggests that it was acquired en bloc from the ancestor of mitochondria (Huynen et al. 2001), as we suggest here for an SQR-containing mitochondrial respiratory chain.

Third, newer evidence suggests that during the period of Earth's history from 2 billion years ago to 1 billion years ago (2 to 1 Ga) SQR must have been very important, if not essential, for most, if not all, eukayrotes, at least the ones that inhabited the oceans. This is because the sulfur isotope record indicates that biological sulfate reduction, which produces sulfide, was highly active and globally widespread during that time (Canfield 1998; Shen, Buick, and Canfield 2001; Anbar and Knoll 2002). The consequence is that Earth's oceans subsurface water would have been both anoxic (without oxygen) and sulfidic (laden with sulfide) during that time. Anbar and Knoll (2002) discussed this anoxic, sulfidic marine environment in the context of low resulting copper and molybdenum concentrations, which they argued to have possibly impaired eukaryotic diversity, because these are important trace elements for eukaryotes. However, from the standpoint of our present findings, the more immediate problem posed by such environments for early eukaryotes would have been (1) ATP production without oxygen and (2) dealing with high concentrations of sulfide. Put another way, only osmotrophic eukaryotes such as fungi would have been limited by trace element availability—phagocytosing eukaryotes would have been able to obtain their trace elements from ingested prey, but all subsurface eukaryotes during the period from 1 to 2 Ga would have been confronted with high sulfide concentrations. SQR is the mechanism that contemporary eukaryotes use to deal with high sulfide concentrations today, both in terms of detoxification and in terms of utilizing sulfide for mitochondrial ATP synthesis (Grieshaber and Völkel 1998). It is therefore reasonable to assume that ancient eukaryotes dealt with sulfide the same way as contemporary eukaryotes do, namely with mitochondrial SQR. Hence, the sulfidic and anoxic phase of Earth's history revealed by the sulfur isotope record does not lead to the prediction of limited eukaryotic diversity during the period from 1 to 2 Ga as suggested by Anbar and Knoll (2002), rather it leads to the prediction that eukaryotes diversified during anaerobic times and therefore that they should have preserved abundant traces of that anaerobic past—which they have, particularly in their mitochondria and hydrogenosomes (Martin and Müller 1998; Tielens et al. 2002; Embley et al. 2003) and also in the form of mitochondrial SQR.

Unicellular eukaryotes are at least 1.5 Ga old (Javaux, Knoll, and Walter 2001) and multicellular red algae are at least 1.2 Ga old (Butterfield 2000), meaning that differentiation of eukaryotic lineages below the plant lineage occurred in an anoxic and sulfidic world. Thus, eukaryotes that today inhabit anoxic and sulfidic marine environments did not necessarily have to become especially adapted to such conditions, nor did they need to acquire SQR genes by lateral transfer to do so. Rather, it seems that they "grew up" in an anoxic and sulfidic world and that mitochondrial SQR is simply a relic retained from that phase of eukaryotic history, whereby it still fulfills those same essential functions in modern eukaryotes from sulfidic habitats. Newer data indicate the fungal-animal divergence to be among the deepest branches in the eukaryotic tree (Stechmann and Cavalier-Smith 2002), such that the animal and fungal lineages sampled here cover much of the depth but not the breadth of eukaryotic diversity. In eukaryotes from aerobic and/or nonsulfidic habitats, such as S. pombe, the SQR gene and activity have nonetheless been retained (Vande Weghe and Ow 1999), perhaps for detoxification functions, and the SQR gene has apparently been lost in many lineages, among them Arabidopsis and Saccharomyces. No SQR homologs have yet been sequenced from those eukaryotes in which mitochondrial SQR has been most extensively characterized at the biochemical level: marine invertebrates (Grieshaber and Völkel 1998; Doeller, Grieshaber, and Kraus 2001) and chicken (Yong and Searcy 2001). However, work on the marine invertebrates is ongoing. Clearly, our prediction is that SQR from these eukaryotes will share the same origin as S. pombe SQR.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank the Deutsche Forschungsgemeinschaft for financial support, Carmen Rotte for critical reading, and Günter Hauska for many helpful comments on the manuscript.


    Footnotes
 
Geoffrey McFadden, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 

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Accepted for publication May 8, 2003.