REVIEW ARTICLE Order within a mosaic distribution of mitochondrial c-type cytochrome biogenesis systems? James W A Allen1, Andrew P Jackson2, Daniel J Rigden3, Antony C Willis4, Stuart J Ferguson1 and Michael L Ginger5,6 Department of Biochemistry, University of Oxford, UK Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK School of Biological Sciences, University of Liverpool, UK MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, UK Sir William Dunn School of Pathology, University of Oxford, UK Department of Biological Sciences, Lancaster University, UK Keywords bioinformatics; Ccm system; cytochrome c; Diplonema papillatum; evolution; heme lyase; lateral gene transfer; mitochondria; post-translational modification; Trypanosoma Correspondence M Ginger, Department of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK Fax: +44 1524 593192 Tel: +44 1524 593922 E-mail: m.ginger@lancaster.ac.uk (Received February 2008, revised March 2008, accepted March 2008) doi:10.1111/j.1742-4658.2008.06380.x Mitochondrial cytochromes c and c1 are present in all eukaryotes that use oxygen as the terminal electron acceptor in the respiratory chain Maturation of c-type cytochromes requires covalent attachment of the heme cofactor to the protein, and there are at least five distinct biogenesis systems that catalyze this post-translational modification in different organisms and organelles In this study, we use biochemical data, comparative genomic and structural bioinformatics investigations to provide a holistic view of mitochondrial c-type cytochrome biogenesis and its evolution There are three pathways for mitochondrial c-type cytochrome maturation, only one of which is present in prokaryotes We analyze the evolutionary distribution of these biogenesis systems, which include the Ccm system (System I) and the enzyme heme lyase (System III) We conclude that heme lyase evolved once and, in many lineages, replaced the multicomponent Ccm system (present in the proto-mitochondrial endosymbiont), probably as a consequence of lateral gene transfer We find no evidence of a System III precursor in prokaryotes, and argue that System III is incompatible with multi-heme cytochromes common to bacteria, but absent from eukaryotes The evolution of the eukaryotic-specific protein heme lyase is strikingly unusual, given that this protein provides a function (thioether bond formation) that is also ubiquitous in prokaryotes The absence of any known c-type cytochrome biogenesis system from the sequenced genomes of various trypanosome species indicates the presence of a third distinct mitochondrial pathway Interestingly, this system attaches heme to mitochondrial cytochromes c that contain only one cysteine residue, rather than the usual two, within the heme-binding motif The isolation of single-cysteinecontaining mitochondrial cytochromes c from free-living kinetoplastids, Euglena and the marine flagellate Diplonema papillatum suggests that this unique form of heme attachment is restricted to, but conserved throughout, the protist phylum Euglenozoa Abbreviations ccm, cytochrome c maturation; EF-1a, elongation factor-1a; EST, expressed sequence tag; IMS, intermembrane space; KH test, Kishino– Hasegawa test; LGT, lateral gene transfer; ML, maximum likelihood; SOD, superoxide dismutase FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS 2385 Evolution of mitochondrial cytochrome c maturation J W A Allen et al Fig Structures of (A) heme (Fe-protoporphyrin IX) and (B) heme bound to a polypeptide chain as in a typical c-type cytochrome, in which the vinyl groups of the heme are saturated by the addition of cysteine thiols that occur in a Cys-Xxx-Xxx-Cys-His motif (only the sulfur atoms of the cysteines are shown), forming covalent bonds between heme and protein (C) Cartoon representation of heme attachment to protein in mitochondrial cytochrome c The porphyrin ring is shown in blue and the heme iron atom in brown The cysteines of the CXXCH motif form covalent bonds to the heme, and the histidine acts as a ligand to the heme iron atom via a nitrogen atom The sixth ligand to the iron atom is the sulfur of a methionine residue located distantly from the CXXCH motif in the primary structure of the protein In bacterial c-type cytochromes, histidine (rather than methionine) is often the sixth iron ligand, and there are examples with cysteine, an N-terminal amino group, asparagine, lysine or a vacant coordination site There are few restrictions on the nature of the Xxx-Xxx residues Multiple pathways for heme cysteine attachment The c-type cytochromes are characterized by the covalent attachment of heme to the apocytochrome through thioether (carbon–sulfur) bonds (Fig 1) Numerous examples of distinct c-type cytochromes have been described in Bacteria and, more recently, in some Archaea, where they typically function in electron transfer or at the catalytic sites of certain enzymes [2–9] {In agreement with the nomenclature proposed in [1], we refer to the three domains of life as Bacteria (formerly Eubacteria), Archaea (formerly Archaebacteria) and Eucarya (the eukaryotes) When using the expression ‘Bacteria’, we therefore refer to the domain; when using the term ‘bacteria’, we refer generically to nonarchaean prokaryotes} However, the best known examples from the c-type cytochrome family are mitochondrial cytochromes c and c1, which function as essential electron transfer components of the respiratory chain [7,8,10] The covalent attachment of two vinyl groups from the heme cofactor to the thiols in the CXXCH hemebinding motif of apocytochromes c is chemically far from facile (X is any amino acid, except cysteine), and there are multiple systems which catalyze this posttranslational modification in biology [2,4,11–15] Systems I and II are modular and widely distributed amongst bacteria [2,4,6,12,14]; they have been studied using a combination of genetic and biochemical approaches [2,4,14,16,17] System I is understood best 2386 in Escherichia coli, where it consists of eight dedicated essential proteins, named CcmA–H (Fig 2A), and a number of accessory proteins CcmA–H are all membrane anchored or integral membrane proteins, and collectively function in the periplasm The biogenesis of c-type cytochromes is a spatial and temporal problem; in bacteria, both heme and apoprotein are synthesized in the cytoplasm and must be transported to the periplasm, where heme attachment occurs The apocytochrome polypeptide is translocated by the general type II secretion (Sec) proteins [18] How heme is transported remains an intriguing mystery CcmA and CcmB are reminiscent of an ATPdependent (ABC-type) transporter, and CcmA has been shown to hydrolyze ATP [19] However, no transport substrate has yet been identified; heme has been proposed, but much evidence weighs against this possibility [19–22] A more recent hypothesis is that CcmA and CcmB are required to release heme from the heme chaperone CcmE by coupling the free energy gained from ATP hydrolysis [21] CcmE is a key player in the Ccm system; it binds heme covalently as an intermediate in the cytochrome c biogenesis pathway [23] This remarkable heme attachment occurs between a histidine residue and a heme vinyl group Heme attachment to CcmE is dependent on CcmC [24], an integral membrane protein with a number of interesting phenotypes arising from mutation in ccmC, some of which may be unrelated to c-type cytochrome biogenesis [25] CcmD is a very small ( 60 amino acids) integral membrane protein FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS J W A Allen et al Evolution of mitochondrial cytochrome c maturation Fig Cytochrome c biogenesis systems found in bacteria Each of these systems can mature a wide variety of c-type cytochromes, including those with multiple hemes (A) System I (the Ccm system) in Escherichia coli Some uncertainties are designated with ‘?’; for example, what, if anything, is transported by the ABC-type transporter CcmAB, and how is heme transported from its site of synthesis in the cytoplasm to the periplasm? DsbD has two thiols amongst its eight transmembrane helices which are believed to accept reducing equivalents from thioredoxin (TrxA) These thiols, in turn, pass on the reducing power to periplasmic C- and N-terminal domains From there, reductant passes to the c-type cytochrome biogenesis apparatus, tentatively by the route shown; CcmG has been shown in some schemes to be the electron acceptor from CcmH but, although there is experimental evidence for this order, more evidence indicates the arrangement shown in the figure DsbA is a strong, non-specific disulfide bond-oxidizing protein found in the periplasm of E coli Ultimately, the cysteine thiols of the apocytochrome CXXCH heme-binding motif become reduced to allow heme attachment Heme becomes covalently attached to the chaperone CcmE as an intermediate in the pathway The specific covalent attachment of heme to apocytochrome c is believed to involve CcmF and H (B) Cytochrome c biogenesis System II in a Gram-negative bacterium In some species, CcdA is replaced by the protein DsbD shown in Fig 2A CcdA and ResA provide a pathway by which reductant is transferred to the apocytochrome to reduce a disulfide bond in the CXXCH heme-binding motif ResB and ResC provide the covalent heme attachment function to produce the product holocytochrome c Heme delivery to the periplasm from the cytoplasm may also occur through the ResBC complex, but this is presently not certain Other names are in common use for ResA ⁄ B ⁄ C (ResA = CcsX = HCF164; ResB = CcsB = Ccs1; ResC = CcsA; and CcdA = CcsC) that mediates complex formation between CcmC and CcmE [26] CcmF and CcmH are implicated in the transfer of heme from holo-CcmE to apocytochrome c, including the covalent heme attachment step to produce the product holocytochrome E coli CcmH is a fusion protein which includes the proteins known as CcmH and CcmI in many bacteria CcmG is a thioredoxin-like protein [27] that forms part of an electron transfer chain Electrons are transferred from the cytoplasmic protein thioredoxin, via the multidomain membrane protein DsbD, to CcmG, and then to the apocytochrome to reduce a disulfide bond that forms between the cysteines of the apocytochrome CXXCH heme-binding motif; these thiols must be reduced for heme attachment to occur (reviewed in [2]) Such a reductive pathway is thought to be necessary in E coli, partly because the periplasm contains the strong, indiscriminate, disulfide-oxidizing protein DsbA System II (Fig 2B) is less well understood than System I at the molecular level, but it seems very likely to consist of four proteins [28] {Note: The nomencla- FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS 2387 Evolution of mitochondrial cytochrome c maturation J W A Allen et al ture for System II c-type cytochrome biogenesis proteins is somewhat inconsistent in the literature Here, we adopt the names used for the various biogenesis proteins found in Bacillus subtilis, i.e ResA (also called CcsX or HCF164), ResB (also called CcsB or Ccs1), ResC (also called CcsA) and CcdA (also called CcsC)} These include a thioredoxin-like protein called ResA (similar in structure to CcmG) [29] and CcdA, a functional analogue of DsbD, or DsbD itself (depending on the organism); together, these apparently form a pathway analogous to that observed in System I for reducing a disulfide bond in the apocytochrome CXXCH motif The heme attachment (and possibly heme delivery) function of System II is catalyzed by ResB and ResC Indeed, a fusion protein cloned from Helicobacter pylori containing elements of ResB and ResC was sufficient to mature c-type cytochromes when expressed in the periplasm of a ccm deletion strain of E coli [30] Several recent studies have provided insight into the flexible organization of prokaryotic c-type cytochrome biogenesis pathways For example, in the Archaea and some bacteria, a divergent System I has recently been described [3], and some bacteria contain components of both System I and System II [3,4,6] Although the presence of multiple cytochrome c biogenesis systems in a single bacterium might hint at possible redundancy, additional c-type cytochrome maturation components are sometimes required for heme attachment to specific substrates For example, in the e-proteobacterium Wolinella succinogenes, the ccsA1-encoded heme lyase is required for thioether bond formation to the remarkable CX15CH heme-binding motif of the multi-heme c-type cytochrome MccA [31] System III for cytochrome c maturation consists of a single primary component, the enzyme heme lyase, which is found only in the mitochondrial intermembrane space (IMS) of animals, fungi and some protists [11,32] {The kingdom Protista refers to those eukaryotes that cannot be classified as animals, plants or fungi: it includes protozoa and algae The protozoa [or ‘first (proto-) animals (zoa)’] are unicellular eukaryotes, which lack the chitinous cell wall found in fungi} At least in fungi, heme lyase is supplemented by the flavoprotein Cyc2, which is thought to provide reducing equivalents for the heme attachment process [33] The biochemical study of heme lyase has proved challenging, and the molecular details of its enzymology are still largely unclear Finally, a distinctive example of a biogenesis system that is required for the dedicated maturation of a particular substrate is provided by the recent description of System IV for cytochrome c maturation Heme is 2388 attached through a single thioether linkage to cytochromes b6 and b from the b6f and bc complexes of oxygenic phototrophs (cyanobacteria, plants, algae) and certain Bacillus species, respectively [34,35] The mechanism by which covalent heme attachment to Bacillus cytochrome b occurs is not yet known, but the identification of gene products from the green alga Chlamydomonas reinhardtii that restore cytochrome b6 formation in four ccb mutants constitutes the initial step in the characterization of System IV, which appears to be conserved in all oxygenic phototrophs [36] In species from the phylum Euglenozoa, which includes Euglena gracilis and the medically relevant trypanosomatids (Trypanosoma brucei, T cruzi and pathogenic Leishmania species), heme is uniquely attached to the mitochondrial c-type cytochromes by a single thioether bond within a F ⁄ AXXCH heme-binding motif [37–41] In an earlier study, we determined that, in the trypanosomatids, the occurrence of singlecysteine-containing mitochondrial cytochromes c and c1 correlates with the absence from both nuclear and mitochondrial genomes of genes encoding any component of the known c-type cytochrome maturation systems; we also provided experimental evidence that, for the single-cysteine-containing T brucei cytochrome c, spontaneous (i.e uncatalyzed) maturation is unlikely [41] These results indicate that at least one further pathway for cytochrome c maturation awaits discovery in the trypanosomatids In this article, we draw on the resources that are provided through the availability of numerous complete genome sequences and several ab initio modeling programs We consider in detail the evolutionary distribution of the machinery for mitochondrial cytochrome c assembly throughout the Eucarya, and the possible origins of heme lyase Although the origin of the exclusively eukaryotic heme lyase remains mysterious, replacement of a proto-mitochondrial System I pathway for c-type cytochrome maturation occurred multiple times during protist evolution With rare exceptions, these replacements probably occurred as a result of eukaryote-to-eukaryote lateral gene transfer (LGT) or endosymbiotic gene transfer of heme lyase We also approach defining the limits of the distribution of the single-cysteine heme-binding motif found in some mitochondrial cytochromes c Mapping character traits onto a consensus view of eukaryotic phylogeny The origin of the first eukaryotic cell has been debated for many years; during the 1980s and early 1990s, the FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS J W A Allen et al available experimental evidence was generally consistent with an evolutionary model (called the Archezoa theory), which posited two early phases to eukaryotic evolution: an ancestral phase, in which the hallmark features of the eukaryotic cytoskeleton, endomembrane system and nucleus were evolved, followed by the second critical phase, which saw the acquisition of the a-proteobacterial endosymbiont and the evolution of the proto-mitochondrion Although the results from some phylogenetic analyses conflicted with the model formulated by Cavalier-Smith (discussed in [42]), the Archezoa theory generally received robust support in phylogenetic trees derived from the analysis of small subunit rRNA or translation elongation factor proteins Grouped at the base of many of these trees were several eukaryotic lineages, including diplomonads (represented by Giardia), the parabasalids (represented by Trichomonas) and the Microsporidia [43,44] (and reviewed recently in [45,46]) The distinctive ultrastructure of these organisms suggested that they apparently possessed neither mitochondria nor other hallmark eukaryotic organelles, such as peroxisomes and golgi, and their status as Archezoa denoted that they were believed to be ancestrally without these organelles We now know that this is not the case; more recent phylogenetic treatments have resulted in the repositioning of at least some formerly basal or ‘primitive’ eukaryotes elsewhere within the eukaryotic tree [46–48] Furthermore, although the secondary loss of peroxisomes has occurred numerous times in evolution, the aforementioned organisms crucially retain mitochondria, golgi and other classically eukaryotic subcellular compartments that have merely been remodeled beyond obvious or easy recognition [49–53] Thus, there are no known examples of contemporary eukaryotes that lack double-membrane-bound organelles of mitochondrial descent; indeed, although difficult to prove, a popular current viewpoint is that the acquisition of the protomitochondrial endosymbiont could have been coincident with eukaryotic origins (see, for example, [47,54] for a further discussion) Although the position of the root for eukaryotic evolution remains a contentious issue – Cavalier-Smith has argued that the last common ancestor of all extant eukaryotes diverged with the unikont–bikont split (Fig 3) [55–57]; other results have suggested that it is still not possible to discount a previously long-standing view that the diplomonads and parabasalids belong to the earliest diverging eukaryotic lineage [46,47,58] – comparative interrogations of various morphological and molecular character traits, as well as phylogenies based on the analysis of multiple gene sets, have resulted in a seemingly robust resolution of eukaryotic Evolution of mitochondrial cytochrome c maturation diversity into six major groupings ([59] and reviewed in [46,47,60,61]) The framework provided by this resolution is increasingly being used to inform on the evolution of various fundamental aspects of eukaryotic biology, both within and between these major groupings [55–57,62–66] It is this consensus view of eukaryotic evolution on which the comparative analysis described below is based A phylogeny for mitochondrial c-type cytochrome maturation Using the complete or draft nuclear and mitochondrial genome sequences indicated in supplementary Doc S1, we mapped the distribution of mitochondrial cytochrome c maturation pathways onto a consensus view of eukaryotic phylogeny (Fig 3) Our aim was to assess whether there was any obvious order to the otherwise mosaic distribution of mitochondrial cytochrome c biogenesis machineries that has previously been hinted at [67,68] The presence of the Ccm system in higher plants and some unicellular eukaryotes [e.g the deeply divergent jakobid Reclinomonas americana, ciliates and the rhodophyte (red alga) Cyanidioschyzon merolae] has been described previously [69–74], whereas other eukaryotes, such as the animals, the chlorophyte green alga C reinhardtii and the malarial parasite Plasmodium falciparum (an apicomplexan) have heme lyase for maturation of mitochondrial cytochromes c [2,15,32,75–77] The mitochondrial genome sequences of various excavate, algal, plant and ciliate taxa very clearly point to the presence of System I within the a-proteobacterial endosymbiont from which mitochondria evolved [69,70,72,78,79] However, taking into account the generally robust support for relationships within and between the taxonomic groups shown in Fig 3, our comparative genomic analysis can be used to provide new insight into the evolution of mitochondrial cytochrome c maturation Observations that are key to the discussion that follows in subsequent sections are: (a) there is no evidence for the occurrence of heme lyase within the bikont supergroup Excavata; (b) in the unikonts, heme lyase is the only c-type cytochrome maturation system present; (c) there is a mosaic distribution of the Ccm system and heme lyase within the Chromoalveolata and Plantae; (d) wherever the multicomponent Ccm system is used for mitochondrial cytochrome c maturation, it is always partially encoded on the mitochondrial genome; this is perhaps unsurprising given that CcmC and CcmF are mitochondrial integral membrane proteins containing multiple predicted transmembrane helices Where a FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS 2389 Evolution of mitochondrial cytochrome c maturation J W A Allen et al Fig The phylogenetic distribution of the different pathways used for mitochondrial c-type cytochrome maturation in eukaryotes (A) Relationships within and between five of the six eukaryotic supergroups – no relevant data for c-type cytochrome maturation in the sixth supergroup, Rhizaria, are currently available The unikonts comprise the Amoebozoa (to which Dictyostelium discoideum and the human pathogen Entamoeba histolytica belong) and the Opisthokonts (the animals, fungi and various protozoa) The unikonts differ from the bikonts (which include the algae, land plants and many different protozoa) in that they possess (probably ancestrally [55]) only a single centriole (the barrelshaped structure from which flagellar basal bodies are derived and which, in many eukaryotes, is also involved in the organization of the mitotic spindle) The phylogeny reveals that, within some groups (e.g Viridiplantae), some species contain System I, whereas others contain System III; there were no examples of eukaryotes that contained multiple systems for the maturation of mitochondrial c-type cytochromes A more detailed overview of the distribution of mitochondrial c-type cytochrome maturation pathways in the Plantae is provided in (B) Lineages belonging to the Streptophyta are highlighted by the grey background The evolutionary relationships shown represent a consensus view of published data A complete list of species used to produce the phylogeny, including the databases searched, is provided in supplementary Doc S1 Species for which the identification of the mitochondrial c-type cytochrome biogenesis apparatus is based on the interrogation of a complete genome sequence are as follows: the choanoflagellate Monsiga brevicolis (System III); the amoebozoan Dictyostelium discoideum (System III); the chlorophyte green algae Chlamydomonas reinhardtii, Volvox carteri, Ostreococcus lucimarinus and Ostreococcus tauri (all System III); the red alga Cyanidioschyzon merolae; the ciliates Tetrahymena thermophila and Paramecium tetraurelia (System I); the Apicomplexans Plasmodium falciparum, Toxoplasma gondii and Theileria parva (System III); the dinoflagellate Perkinsus marinus (System III); the oomycetes Phytophthora ramorum and Phytophthora sojae and the diatoms Thalassiosira pseudonana and Phaeodactylum tricomutum (collectively belonging to a group known as the stramenopiles or chromists) (all System III); the Heterolobosean Naegleria gruberi (System I) Entamoeba histolytica (Amoebozoa), Encephalitozoon cuniculi (Microsporidia), Cryptosporidium parvum (Apicomplexa) and the diplomonad Giardia intestinalis all contain degenerate mitochondria known as mitosomes, and the parabasalid Trichomonas vaginalis possesses hydrogenosomes; such degenerate forms of mitochondria lack a respiratory chain and therefore not contain c-type cytochromes mitochondrial genome sequence is complemented by the availability of a complete or draft nuclear genome sequence for the same organism, the following always holds true: (i) if components of the Ccm system are encoded in the mitochondrial genome, further dedicated Ccm components are also encoded in the nuclear genome; (ii) there are no examples of eukaryotes possessing multiple systems for mitochondrial cyto2390 chrome c maturation Thus, even without the availability of a sequenced nuclear genome, the absence from a protozoan or algal mitochondrial genome of genes encoding Ccm components almost certainly provides a reliable indication that System I will not be used for the maturation of mitochondrial cytochromes c and c1 There are several green, red (rhodophyte) and chromist algae (belonging to the Chromalveolata), plus other FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS J W A Allen et al protozoan species (the amoebozoan Acanthamoeba castellanii), for which no nuclear genome sequence is available, but there is an accessible or annotated [79] mitochondrial genome sequence on which no component of the Ccm system is encoded Similarly, there is extensive sequence coverage for the uniquely organized (many small linear chromosomes of less than 8.3 kb in length) mitochondrial genome of the ichthyosporean Amoebidium parasiticum (belonging to the Opisthokonta); from the sequence released thus far, this genome also lacks genes encoding Ccm components [80] Assuming that none of these species contain mitochondrial c-type cytochromes with atypical hemebinding motifs, we suggest that it is likely that nuclearencoded heme lyase is used for the maturation of their mitochondrial cytochromes c and c1 We found a eukaryotic cytochrome c biogenesis System II only in those eukaryotes that contain chloroplasts (data not shown), and we assume that, in these cases, System II is used for the maturation of the chloroplast c-type cytochromes, given the ancestral relationship between chloroplasts and System II-containing cyanobacteria [81,82] Where System II was observed, a second c-type cytochrome biogenesis apparatus was always present and is presumed to be responsible for maturing the mitochondrial cytochromes c and c1 (e.g System I in Arabidopsis thaliana, System III in C reinhardtii and chromist algae) Similarly, genes encoding the four chloroplast proteins recently shown to be required for single-cysteine attachment to cytochrome b6 in Chlamydomonas – System IV for c-type cytochrome biogenesis – were also only present in phototrophic eukaryotes [36] The absence of heme lyase from the excavates, the possible origins of heme lyase and the molecular basis for the mosaic distribution of Systems I and III in chromalveolates and the Plantae are the critical issues upon which we focus in the remainder of this article Was heme lyase ever present in the Excavata? A number of important human pathogens, such as trypanosomes, Giardia and Trichomonas, as well as a diverse assortment of free-living protozoa, are included in the supergroup Excavata The validity of this classification was initially based on a number of shared morphological features, but has more recently received modest support from a variety of molecular phylogenies [59,83–85] Support for the monophyly of the Excavata is, however, equivocal [86]; indeed, the possibility that the earliest diverging eukaryote was an ancestor of diplomonads (Giardia) and parabasalids Evolution of mitochondrial cytochrome c maturation (Trichomonas) has not yet been entirely dismissed [46,58] Interestingly, if we accept the emerging evidence that groups the Excavata together, a deepbranching status for the supergroup can be inferred from a variety of character traits A prime example is the distinctive mitochondrial genome of the jakobid R americana which, in terms of both gene content and genome organization, more closely resembles an a-proteobacterial genome than any other mitochondrial genome that has presently been sequenced [70,87] Like Reclinomonas, some of the other excavates currently sampled (Naegleria and Malwimomonas) contain the Ccm system for cytochrome c maturation (Fig 3) Others (Trichomonas vaginalis and Giardia intestinalis) lack a capacity for respiration, and c-type cytochromes are accordingly absent from their degenerate mitochondria, making it impossible to assess which system for cytochrome c maturation would have been present in their last aerobic ancestors In trypanosomatids, cytochromes c and c1 are present, but there is no recognizable c-type cytochrome maturation system Thus, there is no evidence that heme lyase was ever present within the excavate supergroup The recently described absence [41] of any known cytochrome c biogenesis system from the various trypanosomatids represents a particularly intriguing scenario, as it correlates with the attachment of heme to single-cysteine XXXCH mitochondrial cytochromes in these organisms Such single cysteine cytochromes are also present in other kinetoplastids (the trypanosomatid family evolved from a kinetoplastid ancestor) and the euglenids Euglena gracilis and E viridis [37–41] All of these protists belong to the phylum Euglenozoa (Fig 4A), but, in addition to the euglenids and kinetoplastids, the Euglenozoa includes a third major taxonomic group, a family of mostly free-living marine flagellates known as the diplonemids Recent phylogenies suggest that the diplonemids are likely to be a sister group to the Kinetoplastida [88] Although there is no genome project for a diplonemid, we have used the relatively simple experiment of determining the type of mitochondrial cytochromes present (either CXXCH or XXXCH heme attachment) to look further at the evolution of cytochrome c biogenesis in the Excavata From a combination of spectroscopic methods and N-terminal sequencing (Fig 4), Diplonema papillatum unambiguously contains a single-cysteine c-type cytochrome (AGQCH heme-binding motif) Thus, all three major taxonomic groups of the Euglenozoa (diplonemids, kinetoplastids and euglenids) contain singlecysteine mitochondrial cytochromes c, and hence it is likely that they all contain the same, as yet unidentified, apparatus for maturation of cytochromes c, which FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS 2391 Evolution of mitochondrial cytochrome c maturation J W A Allen et al Fig Diplonema cytochrome c has only a single cysteine in its heme-binding motif (A) Probable evolutionary relationships within the phylum Euglenozoa, as suggested by taxon-rich small subunit rRNA phylogeny (B) Absorption spectrum of semi-purified D papillatum cytochrome c, recorded at 25 °C with the protein in 50 mM Tris ⁄ HCl (pH 8.0) containing a few grains of disodium dithionite to reduce the heme iron The protein was purified from a culture of D papillatum strain ATCC50162 by SP-Sepharose chromatography Absorption maxima were at 419.5, 523.5 and 554.0 nm Inset: reduced pyridine hemochrome spectrum of the same protein Pyridine hemochrome analysis was conducted according to Bartsch [133]: final concentrations of hydroxide and pyridine were 0.2 M and 30% (v ⁄ v), respectively, and a few grains of dithionite were added The a-band peak maximum at 553.0 nm (indicated by the vertical broken line) diagnostically indicates heme attachment to the polypeptide via one cysteine residue [37,39,41,133–135] Diplonema was cultured in artificial seawater as described previously [136], and subjected to detergent extraction [41] prior to isolation of cytochrome c (C) Sequence alignment of the N-terminal 40 amino acids of Diplonema cytochrome c, as determined by Edman degradation, and the N-terminal regions of cytochromes c from other organisms: Cf, Crithidia fasciculata; Dp, Diplonema papillatum; Eg, Euglena gracilis; iso, isoform; Sc, Saccharomyces cerevisiae; Tb, Trypanosoma brucei The c-type cytochrome heme-binding motif is highlighted in bold for each cytochrome Underlined residues denote differences between the major and minor isoforms of mitochondrial cytochrome c in D papillatum: Dpiso1 is the major form (75% of the total protein) and Dpiso2 is the minor form (25%) Cytochrome c as analyzed in (B) was further purified using a CM-Sepharose column before N-terminal sequencing Cysteine gives a blank (X) in the sequencing reaction unless appropriately alkylated [137]; thus X is what is expected and observed for cysteine covalently bound to a heme in a c-type cytochrome It is, however, clear that the first residue of the heme-binding motif of D papillatum cytochrome c is alanine not cysteine, and thus the cytochrome has a single cysteine heme-binding motif of the type found in other Euglenozoaons, rather than CXXCH as observed in typical mitochondrial cytochromes c is distinct from that found in any other organisms Analysis of all the available genome sequences and all publicly accessible expressed sequence tag (EST) collections (including ESTs for the excavates Malawimonas californiana, M jakobiformis and R americana) using blast reveals that, strikingly, single-cysteine attachment of heme to mitochondrial cyto2392 chrome c remains a characteristic that is unique to species from the phylum Euglenozoa Crucially, these analyses included the use of the draft nuclear genome sequence for Naegleria gruberi, an amoeboflagellate with an aerobic metabolism from the phylum Heterolobosea, the eukaryotes with the closest evolutionary relationship to the Euglenozoa [47,59,85] The FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS J W A Allen et al N gruberi mitochondrial cytochromes c and c1 contain CAQCH and CSACH motifs, respectively; these cytochromes are matured by cytochrome c biogenesis System I We postulated previously [41] that the acquisition of a novel mitochondrial cytochrome c biogenesis system in the Euglenozoa provided not only a driving force for the loss of a pre-existing maturation system, but also the evolutionary pressure to move from CXXCH to XXXCH cytochromes c If increased taxon sampling fails to detect the existence of an excavate heme lyase, this is likely to influence which of the models discussed below most parsimoniously explains the distribution of cytochrome c maturation systems shown in Fig Probing the origins of heme lyase Heme lyase has, since its discovery [15], remained a rather enigmatic enzyme: the origin of this eukaryoticspecific protein is obscure and little is known about the biochemistry of System III-dependent cytochrome c maturation [13] From the analysis shown in Fig 3, it is clear that, although animals and Dictyostelium each encode a single form of heme lyase, two isoforms of heme lyase are found in other eukaryotes At least in Saccharomyces cerevisiae, the presence of two lyases reflects the distinct substrate preferences of each enzyme: either cytochrome c or c1, respectively [15,32,75] In order to obtain an insight into the origin of heme lyase and to explore a molecular explanation for its evolutionary distribution, we performed a phylogenetic analysis, and also applied a number of bioinformatics tools that can be used to detect remote structural similarities between different proteins that are undetectable even by sensitive iterative database searches Assuming that the presence of multiple heme lyases always reflects, as it does in yeast, the deployment of one enzyme to catalyze the maturation of each mitochondrial c-type cytochrome, one aim with the phylogeny was to determine whether the transition from a single heme lyase with broad substrate specificity to dual enzymes, each with their own specificity for either cytochrome c or cytochrome c1 [15,32], was likely to have occurred just once or on a number of occasions With the exception of their N-termini, which were largely unique to each taxonomic group, heme lyase protein sequences were reliably aligned Following the omission of sequences corresponding to putative heme lyases from the choanoflagellate Monsiga brevicolis and the dinoflagellate Perkinsus marinus, a bootstrapped maximum likelihood (ML) phylogeny robustly resolved distinct heme lyase clades for the metazoan, fungal, Evolution of mitochondrial cytochrome c maturation algal and apicomplexan sequences These clades were supported by bootstrap values greater than 75 (Fig 5) However, the relationships between these clades were not robust, and therefore could not be resolved satisfactorily Clearly, the arrangement of the basal nodes towards the root of the phylogeny is crucial to an understanding of the evolution of the c–c1 heme lyase distinction, and the number of origins in particular However, all heme lyases from Apicomplexa clustered together with reasonable robustness (bootstrap value, 87), largely due to the distinct N-termini shared by these proteins The monophyly of all apicomplexan heme lyases points towards at least two origins of the c–c1 distinction amongst eukaryotes: one prior to the divergence of the fungi and one affecting the alveolates [the group that includes the ciliates, apicomplexans and dinoflagellates (Fig 3)] Further origins of the c–c1 distinction affecting diatoms (Thalassiosira pseudonana and Phaeodactylum tricomutum) and chlorophyte algae are possible, but increased taxon sampling is necessary to allow the resolution of these possibilities In the example of Dictyostelium, we cannot know whether the presence of a single heme lyase represents an ancestral state or the reverse transition of going from two distinct lyases to a single lyase of broader substrate specificity However, with regard to the opisthokonts, the presence of a single heme lyase in animals, but multiple lyases in the choanoflagellate Monsiga brevicolis (Fig 3) and the fungi, points either to multiple origins for the c–c1 dichotomy or a loss of a heme lyase isoform from animals with, presumably, relaxation of the substrate specificity To determine whether the monophyly of the apicomplexan sequences was an artifact introduced by the biased base composition common to apicomplexan genomes, a neighbor-joining phylogeny was estimated with logdet genetic distances [89], which correct for base composition imbalance Monophyly of apicomplexan sequences was still recovered after correction for base composition The result of the Kishino–Hasegawa (KH) test also corroborated the view that there have been multiple origins for the c–c1 distinction Here, to test whether the optimal topology obtained from the ML and Bayesian inference (BI) trees was significantly more likely than a ‘single-origin’ scenario, the likelihood score of an alternative tree, in which all c- and c1-type sequences were reciprocally monophyletic (i.e one simulating a single origin for the c–c1 distinction), was compared with the optimal ML estimate using a KH test [90] and phylip v3.65 [91] A significant reduction in likelihood score when this constraint was enforced demonstrated that a single origin of the c–c1 distinction could be rejected – the alternative ML FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS 2393 Evolution of mitochondrial cytochrome c maturation J W A Allen et al Fig An unrooted, maximum likelihood (ML) phylogeny of heme lyase protein sequences A WAG substitution matrix was applied with among-site rate heterogeneity described by a gamma distribution estimated from the data Branch lengths are measured in substitutions per site Non-parametric bootstrap values from the ML analysis over 50, and their corresponding posterior probabilities from the Bayesian analysis, are shown adjacent to the nodes An asterisk denotes bootstrap values > 95 and posterior probabilities of 1.00 Full details of the methods used for phylogeny construction and in the predictive modeling of heme lyase are provided in supplementary Doc S1 Clades are color coded by taxon: Fungi (red; c-type heme lyases are shaded lighter); Metazoa (yellow); Apicomplexa (blue; lighter and darker shading highlight distinct subclades); algal ⁄ stramenophile (green; lighter and darker shading highlight distinct subclades) tree topology had a likelihood score of )24248.9, which was significantly worse than the unconstrained, optimal tree topology (Dln L = )54.2, P < 0.001) To seek insight into the possible origin of System III for cytochrome c maturation, we used a variety of bioinformatics tools (as described in supplementary Doc S1) to search for protein families distantly related to heme lyase The application of these approaches served only to highlight further the enigmas that surround this fundamentally important enzyme; however, as cytochromes c and c1 are matured within the mitochondrial intermembrane space (IMS), two possible candidate proteins identified are nonetheless worthy of mention Thus, after the obvious match to the heme lyase domain itself, the first HHPRED result initially 2394 appeared interesting A small portion, 34 residues, of the heme lyase was matched to a region of a Pfam entry for the Erv1 ⁄ Alr family of IMS proteins involved in protein import into the IMS and export of mitochondrial Fe ⁄ S clusters into the cytoplasm [92–95] However, the heme lyase secondary structure prediction was not in good agreement with the four helical bundle architecture of the Erv1 ⁄ Alr sulfhydryl oxidase, and no other fold recognition method (below) flagged up this putative relationship The best 3D-Jury consensus fold recognition scores were obtained for the conserved domain of the human heme lyase but, at up to 45, did not reach the benchmark significance cut-off of 50 [96] Once again the matched protein, superoxide dismutase (SOD), was FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS J W A Allen et al imported into the mitochondrial IMS, and this time the match between the predicted heme lyase secondary structure and actual SOD secondary structure was reasonable However, SOD was not suggested as a good match when other heme lyase sequences were submitted, often being entirely absent from the list of top 3D-Jury hits, and the modeling of heme lyase based on the SOD structure required the deletion of a complete template helix Furthermore, when sequence conservation in the heme lyase family was mapped onto the model surface, conserved positions were distributed over most of the protein, in contrast with the clustering around binding and catalytic sites that would be expected Finally, programs for the ab initio modeling of small proteins are starting to provide useful predictions (for example [97]) We reasoned that a reliably ab initio predicted fold with a detectable similarity to known protein structures could therefore be indicative of a distant relationship Thus, rosetta was applied to the conserved domain of a Candida albicans heme lyase (accession code XP_722795.1 in the nr database [98]), chosen as, at 162 residues, it was the shortest in our set The top 10 clusters were processed and analyzed as described in supplementary Doc S1 In no case did dali discover any significant structural relationship between a model and a known structure Nor did profunc locate any matches to three-dimensional structural motifs, the presence of which could have increased confidence in the models From the bioinformatics analyses, therefore, it is clear that there is no strong evidence to support the existence of distant relationships between heme lyase and other proteins of known structure or function Although ab initio modeling is not yet a mature technology, the sequence and structure matching analyses represent the current state of the art Thus, this suggests that any relationship between heme lyase in the taxa sampled thus far and other characterized proteins must be exceedingly distant: the origin of the exclusively eukaryotic heme lyase therefore remains mysterious Eukaryote–eukaryote LGT events could readily account for the observed distribution of heme lyase Although several state-of-the-art predictive computational tools failed to shed any light on how heme lyase has evolved, two models can be invoked to explain the observed phylogenetic distribution of Systems I (Ccm system) and III (heme lyase) (Fig 3) The mitochondrial genome sequences from various excavate, algal, plant and ciliate taxa very clearly Evolution of mitochondrial cytochrome c maturation point to the presence of System I within the a-proteobacterial endosymbiont from which mitochondria evolved [69,70,72,78,79] System I is the only c-type cytochrome biogenesis apparatus identified to date in a-proteobacteria [6] Thus, was the eukaryotic-specific enzyme heme lyase also present in the last common ancestor of extant eukaryote taxa, or did heme lyase evolve in a single eukaryote following the divergence and radiation of the six eukaryotic supergroups (Excavata, Plantae, Chromalveolata, Rhizaria, Amoebozoa and Opisthokonts)? As sophisticated bioinformatics approaches have failed to detect any homology signature between heme lyase and any other known protein, we consider it highly unlikely that this enzyme, which is conserved between evolutionarily diverse taxa, has evolved independently on multiple occasions If heme lyase was present within a common ancestor of the unikont and bikont lineages, selective loss of either the partially mitochondrially encoded System I, or nuclear-encoded System III, would explain the observed phylogenetic distribution (model 1) Alternatively, if the origin of heme lyase postdates the divergence of the six eukaryotic supergroups, LGT of heme lyase on multiple occasions (model 2) provides the explanation for the phylogenetic distribution shown in Fig With respect to the LGT model (model 2), there are a number of other relevant points (a) The requirement in heme lyase-dependent cytochrome c maturation for a single obligatory protein component means that the System III pathway is a realistic candidate for lateral transfer (b) Given the widespread conservation of mitochondrial targeting sequences and protein import mechanisms [99–105], there is a high probability that, in any recipient lineage, the protein encoded by a heme lyase gene, laterally transferred from a eukaryotic donor, is targeted correctly into the mitochondrial IMS (c) Although it appears that many eukaryotes use distinct lyases for the maturation of cytochromes c and c1, respectively, the phylogenetic analysis shown in Fig suggests that the transition from using a single to two distinct isoforms of heme lyase has occurred multiple times, and, even in S cerevisiae, where distinct isoforms are present, the cytochrome c heme lyase can also mature cytochrome c1 [32] – thus, the use of distinct lyases for the maturation of each mitochondrial cytochrome only necessitates lateral transfer of a single gene, followed by a gene duplication (d) As the molecular components in the different c-type cytochrome maturation pathways (i.e Systems I and III) are completely nonhomologous, the case for LGT cannot be erroneously enhanced as a consequence of a phylogenetic artifact or the distribution of a misleading character trait, such FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS 2395 Evolution of mitochondrial cytochrome c maturation J W A Allen et al as amino acid insertions or deletions, within the sampled proteins (e.g as discussed in [46,106,107]) (e) No eukaryote analyzed thus far contains more than one system for mitochondrial cytochrome c maturation (f) Various phenomena, including the commonality of phagotrophic feeding modes, the independent acquisition or even replacement of algal plastids through secondary and tertiary endosymbiosis on multiple occasions [108–110], the variety of endosymbiotic associations seen in distantly related protozoa [111–115], and the ease with which stable transformation of many protists can be achieved, all support the likelihood that LGT, through a ‘you-are-what-you-eat’ gene ratchet model [116], is a significant process in the evolution of unicellular eukaryotes Classically, prokaryotic–eukaryotic LGT and, more recently, intertaxon eukaryote– eukaryote LGT have been invoked as critical factors in the metabolic adaptation of various protists – generally parasitic protozoa – to specific niche environments [117–122] However, there are also intriguing ‘punctate’ distributions for several nuclear-encoded genes that, at first glance, are unlikely to confer niche adaptation [e.g alanyl-tRNA synthetase and elongation factor-1a (EF-1a)-like GTPase, which is otherwise known as EFL], and LGT has been invoked as a possible explanation for these distributions [123–125] If LGT correctly explains the distribution of heme lyase within protists (including the green and red algae), the challenge is perhaps to also ask what selective advantage is provided by the lateral transfer of an alternative pathway for mitochondrial cytochrome c maturation A selective force for the evolution of System III for cytochrome c maturation? Many bacteria mature a wide range of c-type cytochromes with diverse functions and folds, and often with multiple (sometimes numerous) heme groups; these c-type cytochromes are matured using either biogenesis System I or System II [2,6] In contrast, heme lyase (System III) only has to mature the two mitochondrial c-type cytochromes c and c1, which are both monoheme proteins sharing essentially the same fold The available evidence suggests that the substrate specificities of heme lyases are limited to mitochondrial cytochromes c [126]; such strict specificity, in contrast with the wide variety of substrates matured by the modular biogenesis Systems I and II in bacteria, provides a plausible explanation for the absence, thus far, of a prokaryotic System III Moreover, the need to mature only the two similar mitochondrial cytochromes c would mean that the broad substrate speci2396 ficity possessed by System I, the ancestral system in mitochondria, was no longer required The derived and strict specificity of heme lyase for its mitochondrial cytochrome substrates provides a further argument in favor of a single evolutionary origin for this eukaryotic-specific c-type cytochrome maturation system Invoking biochemically significant LGT in the Plantae and endosymbiotic gene transfer of heme lyase Interestingly, a dichotomy between the use of heme lyase or the Ccm system is seen within the Plantae, and, in that regard, the results reported here extend the recently reported complex, mutually exclusive distribution of translation EF-1a and EFL in the green algae [127] Within chlorophyte green algae, there is evidence for the use of heme lyase only The placement of the scaly green flagellate Mesostigma viride within the Streptophyta (the groups highlighted by the grey background in Fig 3) is equivocal [128] However, if the absence of mitochondrially encoded Ccm components provides, as seems likely (see above), a reliable marker that heme lyase will be used for the maturation of conventional CXXCH-containing cytochromes c, there is evidence from the published mitochondrial genomes of three charophyte green algae [78,129,130] for the presence of both System III and System I in the algal group from which higher plants evolved ([78]; Fig 3) The phylogenetic distributions of System I versus System III and EF-1a versus EFL [127] are not identical Within red algae (Rhodophyta), although the Ccm system is found in the early diverging Cyanidiales, which live in extremely acidic (pH 1–2), high-salt environments, it is likely that species from other lineages (e.g potentially Chondrus crispus and Porphyra purpurea) contain heme lyase Phagotrophy is extremely rare within extant green and red algae, and in contrast with mixotrophic algae (i.e capable of photosynthesis and phagocytosis), with plastids of secondary or tertiary endosymbiotic origin, evidence of substantial LGT in the green or red algae is at best sparse – LGT has been invoked to explain the phylogeny of some shikimate pathway genes in the Plantae [131], but in a study of nuclear-encoded plastid genes in C reinhardtii no evidence of LGT was found [132] If the last common ancestor of glaucophytes, red algae and the Viridiplantae did not contain both the Ccm system and heme lyase, the survey presented here provides persuasive evidence for functionally significant LGT during algal evolution Of course, such speculation is only likely to be informed further by continued mapping of charac- FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS J W A Allen et al ter traits, such as the pathways used for c-type cytochrome maturation or the distribution of EF-1a and EFL, onto algal phylogenies Importantly, an insight into the extent of LGT during early algal evolution could have wider reaching implications for understanding the origins of LGT candidates in other eukaryotes For example, the likely widespread occurrence of heme lyase in green algae, including the early diverging chlorophytes Ostreococcus lucimarinus and O tauri, and plausibly its occurrence in red algae too, suggests that heme lyase is a candidate for endosymbiotic gene transfer rather than eukaryote-to-eukaryote LGT, within plastid-bearing chromalveolates, during the window of gene transfer from the nucleus of the endosymbiont to the host cell nucleus Conclusions and wider perspectives Obtaining a mechanistic understanding of how the chemically far from facile process of heme attachment to apocytochromes c is achieved by several very differently organized c-type cytochrome biogenesis machineries represents a formidable biochemical challenge, but one in which considerable progress is being made With regard to eukaryotes, the molecular diversity that is apparent in the organization of mitochondrial cytochrome c maturation contrasts with the strict co-occurrence of two c-type cytochrome biogenesis systems in apparently all chloroplasts and cyanobacteria In this article, we have sought to illustrate how a variety of predictive and comparative genomics approaches can be used to analyze the evolution of mitochondrial cytochrome c maturation With the release of more sequence data, the evolution of structural bioinformatics tools and a resolution of eukaryotic phylogeny, the hypotheses and models discussed here provide a useful framework which can be interrogated further in the years to come Acknowledgements This work was funded by grants from the Royal Society and the BBSRC (BB ⁄ C508118 ⁄ to S J F., M L G and J W A A., and BB ⁄ D019753 ⁄ to J W A A.) 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responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 2385–2402 ª 2008 The Authors Journal compilation ª 2008 FEBS ... divergence and radiation of the six eukaryotic supergroups (Excavata, Plantae, Chromalveolata, Rhizaria, Amoebozoa and Opisthokonts)? As sophisticated bioinformatics approaches have failed to detect any... the malarial parasite Plasmodium falciparum (an apicomplexan) have heme lyase for maturation of mitochondrial cytochromes c [2,15,32,75–77] The mitochondrial genome sequences of various excavate,... elongation factor 1alpha Proc Natl Acad Sci USA 101, 15380– 15385 124 Andersson JO, Sarchfield SW & Roger AJ (2005) Gene transfers from Nanoarchaeota to an ancestor of diplomonads and parabasalids