Maturation Of C-type Cytochromes Research Articles

Two disulfide-reducing pathways are required for the maturation of plastid c-type cytochromes in Chlamydomonas reinhardtii.

In plastids, conversion of light energy into ATP relies on cytochrome f, a key electron carrier with a heme covalently attached to a CXXCH motif. Covalent heme attachment requires reduction of the disulfide-bonded CXXCH by CCS5 and CCS4. CCS5 receives electrons from the oxidoreductase CCDA, while CCS4 is a protein of unknown function. In Chlamydomonas reinhardtii, loss of CCS4 or CCS5 yields a partial cytochrome f assembly defect. Here, we report that the ccs4ccs5 double mutant displays a synthetic photosynthetic defect characterized by a complete loss of holocytochrome f assembly. This defect is chemically corrected by reducing agents, confirming the placement of CCS4 and CCS5 in a reducing pathway. CCS4-like proteins occur in the green lineage, and we show that HCF153, a distant ortholog from Arabidopsis thaliana, can substitute for Chlamydomonas CCS4. Dominant suppressor mutations mapping to the CCS4 gene were identified in photosynthetic revertants of the ccs4ccs5 mutants. The suppressor mutations yield changes in the stroma-facing domain of CCS4 that restore holocytochrome f assembly above the residual levels detected in ccs5. Because the CCDA protein accumulation is decreased specifically in the ccs4 mutant, we hypothesize the suppressor mutations enhance the supply of reducing power through CCDA in the absence of CCS5. We discuss the operation of a CCS5-dependent and a CCS5-independent pathway controlling the redox status of the heme-binding cysteines of apocytochrome f.

Experimental evolution of Vibrio cholerae identifies hypervesiculation as a way to increase motility in the presence of polymyxin B.

Vibrio cholerae includes strains responsible for the cholera disease and is a natural inhabitant of aquatic environments. V. cholerae possesses a unique polar flagellum essential for motility, adhesion, and biofilm formation. In a previous study, we showed that motility and biofilm formation are altered in the presence of subinhibitory concentrations of polymyxin B in V. cholerae O1 and O139. In this study, we performed an experimental evolution to identify the genes restoring the motility in the presence of a subinhibitory concentration of polymyxin B. Mutations in five genes have been identified in three variants derived from two different parental strains A1552 and MO10: ihfA that encodes a subunit of the integration host factor (IHF), vacJ (mlaA) and mlaF, two genes belonging to the maintenance of the lipid asymmetry (Mla) pathway, dacB that encodes a penicillin-binding protein (PBP4) and involved in cell wall synthesis, and ccmH that encodes a c-type cytochrome maturation protein. We further demonstrated that the variants derived from MO10 containing mutations in vacJ, mlaF, and dacB secrete more and larger membrane vesicles that titer the polymyxin B, which increases the bacterial survival and is expected to limit its impact on the bacterial envelope and participate in the flagellum’s retention and motility.

An Archaea-specific c-type cytochrome maturation machinery is crucial for methanogenesis in Methanosarcina acetivorans

c-Type cytochromes (cyt c) are proteins that undergo post-translational modification to covalently bind heme, which allows them to facilitate redox reactions in electron transport chains across all domains of life. Genomic evidence suggests that cyt c are involved in electron transfer processes among the Archaea, especially in members that produce or consume the potent greenhouse gas methane. However, neither the maturation machinery for cyt c in Archaea nor their role in methane metabolism has ever been functionally characterized. Here, we have used CRISPR-Cas9 genome editing tools to map a distinct pathway for cyt c biogenesis in the model methanogenic archaeon Methanosarcina acetivorans, and have also identified substrate-specific functional roles for cyt c during methanogenesis. Although the cyt c maturation machinery from M. acetivorans is universally conserved in the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this machinery through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we demonstrate the convergent evolution of a novel Archaea-specific cyt c maturation machinery and its physiological role during methanogenesis, a process which contributes substantially to global methane emissions.

Identification of an extracytoplasmic function sigma factor that facilitates c-type cytochrome maturation and current generation under electrolyte-flow conditions in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 was cultured on electrodes in electrochemical flow cells (EFCs), and transcriptome profiles of electrode-attached cells grown under electrolyte-flow conditions were compared with those under static (nonflow) conditions. Results revealed that, along with genes related to c-type cytochrome maturation (e.g., dsbD), the SO_3096 gene encoding a putative extracytoplasmic function (ECF) sigma factor was significantly upregulated under electrolyte-flow conditions. Compared to wild-type MR-1 (WT), an SO_3096-deletion mutant (∆SO_3096) showed impaired biofilm formation and decreased current generation in EFCs, suggesting that SO_3096 plays critical roles in the interaction of MR-1 cells with electrodes under electrolyte-flow conditions. We also compared transcriptome profiles of WT and ∆SO_3096 grown in EFCs, confirming that many genes upregulated under the electrolyte-flow conditions, including dsbD, are regulated by SO_3096. LacZ reporter assays showed that transcription from a promoter upstream of dsbD is activated by SO_3096. Measurement of current generated by a dsbD-deletion mutant revealed that this gene is essential for the transfer of electrons to electrodes. These results indicate that the SO_3096 gene product facilitates c-type cytochrome maturation and current generation under electrolyte-flow conditions. The results also offer ecophysiological insights into how Shewanella regulates extracellular electron transfer to solid surfaces in the natural environment.

Maturation of Plastid c-type Cytochromes.

Cytochromes c are hemoproteins, with the prosthetic group covalently linked to the apoprotein, which function as electron carriers. A class of cytochromes c is defined by a CXXCH heme-binding motif where the cysteines form thioether bonds with the vinyl groups of heme. Plastids are known to contain up to three cytochromes c. The membrane-bound cytochrome f and soluble cytochrome c6 operate in photosynthesis while the activity of soluble cytochrome c6A remains unknown. Conversion of apo- to holocytochrome c occurs in the thylakoid lumen and requires the independent transport of apocytochrome and heme across the thylakoid membrane followed by the stereospecific attachment of ferroheme via thioether linkages. Attachment of heme to apoforms of plastid cytochromes c is dependent upon the products of the CCS (for cytochrome c synthesis) genes, first uncovered via genetic analysis of photosynthetic deficient mutants in the green alga Chlamydomonas reinhardtii. The CCS pathway also occurs in cyanobacteria and several bacteria. CcsA and CCS1, the signature components of the CCS pathway are polytopic membrane proteins proposed to operate in the delivery of heme from the stroma to the lumen, and also in the catalysis of the heme ligation reaction. CCDA, CCS4, and CCS5 are components of trans-thylakoid pathways that deliver reducing equivalents in order to maintain the heme-binding cysteines in a reduced form prior to thioether bond formation. While only four CCS components are needed in bacteria, at least eight components are required for plastid cytochrome c assembly, suggesting the biochemistry of thioether formation is more nuanced in the plastid system.

Shewanella oneidensis cytochrome c maturation component CcmI is essential for heme attachment at the non-canonical motif of nitrite reductase NrfA.

Shewanella oneidensis is renowned for its respiratory versatility, which is largely due to abundant c-type cytochromes. Maturation of these proteins depends on a Ccm system encoded by genes in an unusual chromosomal arrangement, but the detailed mechanism is not understood. In this study, we identify SO0265 as CcmI, an apocytochrome c chaperone that is important and essential for maturation of c-type cytochromes with the canonical heme binding motif(s) (HBM; CX(2)CH) and nitrite reductase NrfA carrying a non-canonical CX(2)CK motif respectively. We show that the N-terminal transmembrane segment of CcmI, CcmI-1, is sufficient for maturation of the former but the entire protein is required for maturation of the latter. Although S. oneidensis possesses a heme lyase, SirEFG, dedicated for non-canonical HBMs, it is specific for SirA, a sulfite reductase with a CX(15)CH motif. By presenting evidence that the periplasmic portion of CcmI, CcmI-2, interacts with NrfA, we suggest that CcmI also takes the role of Escherichia coli NrfG for chaperoning apo-NrfA for maturation at CX(2)CK. Moreover, intact CcmI is required for maturation of NrfA, presumably by ensuring that heme attachment at canonical HBMs occurs before apoprotein degradation.

Thiol/Disulfide System Plays a Crucial Role in Redox Protection in the Acidophilic Iron-Oxidizing Bacterium Leptospirillum ferriphilum

Thiol/disulfide systems are involved in the maintenance of the redox status of proteins and other molecules that contain thiol/disulfide groups. Leptospirillum ferriphilum DSM14647, an acidophilic bacterium that uses Fe2+ as electron donor, and withstands very high concentrations of iron and other redox active metals, is a good model to study how acidophiles preserve the thiol/disulfide balance. We studied the composition of thiol/disulfide systems and their role in the oxidative stress response in this extremophile bacterium. Bioinformatic analysis using genomic data and enzymatic assays using protein extracts from cells grown under oxidative stress revealed that the major thiol/disulfide system from L. ferriphilum are a cytoplasmic thioredoxin system (composed by thioredoxins Trx and thioredoxin reductase TR), periplasmic thiol oxidation system (DsbA/DsbB) and a c-type cytochrome maturation system (DsbD/DsbE). Upon exposure of L. ferriphilum to reactive oxygen species (ROS)-generating compounds, transcriptional activation of the genes encoding Trxs and the TR enzyme, which results in an increase of the corresponding activity, was observed. Altogether these data suggest that the thioredoxin-based thiol/disulfide system plays an important role in redox protection of L. ferriphilum favoring the survival of this microorganism under extreme environmental oxidative conditions.

Biochemical properties and catalytic domain structure of the CcmH protein from Escherichia coli

In the Gram-negative bacterium of Escherichia coli, eight genes organized as a ccm operon (ccmABCDEFGH) are involved in the maturation of c-type cytochromes. The proteins encoded by the last three genes ccmFGH are believed to form a lyase complex functioning in the reduction of apocytochrome c and haem attachment. Among them, CcmH is a membrane-associated protein; its N-terminus is a catalytic domain with the active CXXC motif and the C-terminus is predicted as a TPR-like domain with unknown function. By using SCAM (scanning cysteine accessibility mutagenesis) and Gaussia luciferase fusion assays, we provide experimental evidence for the entire topological structure of E. coli CcmH. The mature CcmH is a periplasm-resident oxidoreductase anchored to the inner membrane by two transmembrane segments. Both N- and C-terminal domains are located and function in the periplasmic compartment. Moreover, the N-terminal domain forms a monomer in solution, while the C-terminal domain is a compact fold with helical structures. The NMR solution structure of the catalytic domain in reduced form exhibits mainly a three-helix bundle, providing further information for the redox mechanism. The redox potential suggests that CcmH exhibits a strong reductase that may function in the last step of reduction of apocytochrome c for haem attachment.

Functional Characterization of the FoxE Iron Oxidoreductase from the Photoferrotroph Rhodobacter ferrooxidans SW2

Photoferrotrophy is presumed to be an ancient type of photosynthetic metabolism in which bacteria use the reducing power of ferrous iron to drive carbon fixation. In this work the putative iron oxidoreductase of the photoferrotroph Rhodobacter ferrooxidans SW2 was cloned, purified, and characterized for the first time. This protein, FoxE, was characterized using spectroscopic, thermodynamic, and kinetic techniques. It is a c-type cytochrome that forms a trimer or tetramer in solution; the two hemes of each monomer are hexacoordinated by histidine and methionine. The hemes have positive reduction potentials that allow downhill electron transfer from many geochemically relevant ferrous iron forms to the photosynthetic reaction center. The reduction potentials of the hemes are different and are cross-assigned to fast and slow kinetic phases of ferrous iron oxidation in vitro. Lower reactivity was observed at high pH and may contribute to prevent ferric iron precipitation inside or at the surface of the cell. These results help fill in the molecular details of a metabolic process that likely contributed to the deposition of precambrian banded iron formations, globally important sedimentary rocks that are found on every continent today.

The interplay between the disulfide bond formation pathway and cytochrome c maturation in Escherichia coli

Heme attachment to c-type cytochromes in bacteria requires cysteine thiols in the CXXCH motif of the protein. The involvement of the periplasmic disulfide generation system in this process remains unclear. We undertake a systematic evaluation of the role of DsbA and DsbD in cytochrome c biogenesis in Escherichia coli and show unequivocally that DsbA is not essential for holocytochrome production under aerobic or anaerobic conditions. We also prove that DsbD is important but not essential for maturation of c-type cytochromes. We discuss the findings in the context of a model in which heme attachment to, and oxidation of, the apocytochrome are competing processes.

CcmI Subunit of CcmFHI Heme Ligation Complex Functions as an Apocytochrome c Chaperone during c-Type Cytochrome Maturation

Cytochrome c maturation (Ccm) is a sophisticated post-translational process. It occurs after translocation of apocytochromes c to the p side of energy transducing membranes and forms stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of cysteines at their conserved heme binding sites. In many organisms this process involves up to 10 (CcmABCDEFGHI and CcdA) membrane proteins. One of these proteins is CcmI, which has an N-terminal membrane-embedded domain with two transmembrane helices and a large C-terminal periplasmic domain with protein-protein interaction motifs. Together with CcmF and CcmH, CcmI forms a multisubunit heme ligation complex. How the CcmFHI complex recognizes its apocytochrome c substrates remained unknown. In this study, using Rhodobacter capsulatus apocytochrome c(2) as a Ccm substrate, we demonstrate for the first time that CcmI binds apocytochrome c(2) but not holocytochrome c(2). Mainly the C-terminal portions of both CcmI and apocytochrome c(2) mediate this binding. Other physical interactions via the conserved structural elements in apocytochrome c(2), like the heme ligating cysteines or heme iron axial ligands, are less crucial. Furthermore, we show that the N-terminal domain of CcmI can also weakly bind apocytochrome c(2), but this interaction requires a free thiol group at apocytochrome c(2) heme binding site. We conclude that the CcmI subunit of the CcmFHI complex functions as an apocytochrome c chaperone during the Ccm process used by proteobacteria, archaea, mitochondria of plants and red algae.

Maturation of a eukaryotic cytochrome c in the cytoplasm of Escherichia coli without the assistance by a dedicated biogenesis apparatus

Maturation of c-type cytochromes involves the covalent and stereospecific enzymatic attachment of a heme b via thioether linkages to two conserved cysteines within apocytochromes. Horse cytochrome c is readily matured into its native holoform in the cytoplasm of E. coli when co-expressed with yeast cytochrome c heme lyase. Here we report the low yield formation of holocytochrome with covalently attached heme also in the absence of heme lyase. This is the first demonstration of in vivo maturation of a eukaryotic cytochrome c in a prokaryotic cytoplasm without the assistance by a dedicated enzymatic maturation system. The assembled cytochrome c can be oxidized by cytochrome c oxidase, indicating the formation of a functional protein. The absorption spectrum is typical of a low spin, six coordinated c-type heme. Nevertheless, minor spectral differences relative to the native cytochrome c, deviation of the midpoint reduction potential and slightly altered kinetic parameters of the interaction with cytochrome c oxidase emphasize the importance of cytochrome c heme lyase in folding cytochrome c into its native conformation.

Substrate specificity of three cytochromechaem lyase isoenzymes fromWolinella succinogenes: unconventional haemcbinding motifs are not sufficient for haemcattachment by NrfI and CcsA1

Bacterial c-type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX(2)CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Escherichia coli as host organism. Several reporter c-type cytochromes were employed including cytochrome c nitrite reductases (NrfA) from E. coli and Campylobacter jejuni that differ in their active-site HBMs (CX(2)CK or CX(2)CH). W. succinogenes CcsA2 was found to attach haem to standard CX(2)CH motifs in various cytochromes whereas other HBMs were not recognized. NrfI was able to attach haem c to the active-site CX(2)CK motif of both W. succinogenes and E. coli NrfA, but not to NrfA from C. jejuni. Different apo-cytochrome variants carrying the CX(15)CH motif, assumed to be recognized by CcsA1 during maturation of the octahaem cytochrome MccA, were not processed by CcsA1 in either W. succinogenes or E. coli. It is concluded that the dedicated CCHLs NrfI and CcsA1 attach haem to non-standard HBMs only in the presence of further, as yet uncharacterized structural features. Interestingly, it proved impossible to delete the ccsA2 gene from the W. succinogenes genome, a finding that is discussed in the light of the available genomic, proteomic and functional data on W. succinogenes c-type cytochromes.

Structure of a trypanosomatid mitochondrial cytochrome c with heme attached via only one thioether bond and implications for the substrate recognition requirements of heme lyase

The principal physiological role of mitochondrial cytochrome c is electron transfer during oxidative phosphorylation. c-Type cytochromes are almost always characterized by covalent attachment of heme to protein through two thioether bonds between the heme vinyl groups and the thiols of cysteine residues in a Cys-Xxx-Xxx-Cys-His motif. Uniquely, however, members of the evolutionarily divergent protist phylum Euglenozoa, which includes Trypanosoma and Leishmania species, have mitochondrial cytochromes c with heme attached through only one thioether bond [to an (A/F)XXCH motif]; the implications of this for the cytochrome structures are unclear. Here we present the 1.55 A resolution X-ray crystal structure of cytochrome c from the trypanosomatid Crithidia fasciculata. Despite the fundamental difference in heme attachment and in the cytochrome c biogenesis machinery of the Euglenozoa, the structure is remarkably similar to that of typical (CXXCH) mitochondrial cytochromes c, both in overall fold and, other than the missing thioether bond, in the details of the heme attachment. Notably, this similarity includes the stereochemistry of the covalent heme attachment to the protein. The structure has implications for the maturation of c-type cytochromes in the Euglenozoa; it also hints at a distinctive redox environment in the mitochondrial intermembrane space of trypanosomes. Surprisingly, Saccharomyces cerevisiae cytochrome c heme lyase (the yeast cytochrome c biogenesis system) cannot efficiently mature Trypanosoma brucei cytochrome c or a CXXCH variant when expressed in the cytoplasm of Escherichia coli, despite their great structural similarity to yeast cytochrome c, suggesting that heme lyase requires specific recognition features in the apocytochrome.

Probing the Heme-Binding Site of the Cytochrome c Maturation Protein CcmE

Maturation of c-type cytochromes in many bacterial species and plant mitochondria requires the participation of the heme chaperone CcmE that binds heme covalently via a His residue (H130 in Escherichia coli) before transferring it stereospecifically to the apo form of cytochromes c. Only the structure of the apo form of CcmE is known; the heme-binding site has been modeled on the surface of the protein in the vicinity of H130. We have determined the reduction potential of CcmE, which suggests that heme bound to CcmE is not as exposed to solvent as was initially thought. Alanine insertions in the vicinity of the heme-binding histidine (which we showed by NMR do not perturb the protein fold) strikingly abolish formation of both holo-CcmE and cytochrome c, whereas previously reported point mutations of residues adjacent to H130 gave only a partial attenuation. The heme iron coordinating residue Y134 proved to be strictly required for axial ligation of both ferrous and ferric heme. These results indicate the existence of a conformationally well-defined heme pocket that involves amino acids located in the proximity of H130. However, mutation of Y134 affected neither heme attachment to CcmE nor cytochrome c maturation, suggesting that heme binding and release from CcmE are hydrophobically driven and relatively indifferent to axial ligation.

Mechanism and Consequences of Anaerobic Respiration of Cobalt byShewanella oneidensisStrain MR-1

Bacteria from the genus Shewanella are the most diverse respiratory organisms studied to date and can utilize a variety of metals and metal(loid)s as terminal electron acceptors. These bacteria can potentially be used in bioremediation applications since the redox state of metals often influences both solubility and toxicity. Understanding molecular mechanisms by which metal transformations occur and the consequences of by-products that may be toxic to the organism and thus inhibitory to the overall process is significant to future applications for bioremediation. Here, we examine the ability of Shewanella oneidensis to catalyze the reduction of chelated cobalt. We describe an unexpected ramification of [Co(III)-EDTA](-) reduction by S. oneidensis: the formation of a toxic by-product. We found that [Co(II)-EDTA](2-), the product of [Co(III)-EDTA](-) respiration, inhibited the growth of S. oneidensis strain MR-1 and that this toxicity was partially abolished by the addition of MgSO(4). We demonstrate that [Co(III)-EDTA](-) reduction by S. oneidensis requires the Mtr extracellular respiratory pathway and associated pathways required to develop functional Mtr enzymes (the c-type cytochrome maturation pathway) and ensure proper localization (type II secretion). The Mtr pathway is known to be required for a variety of substrates, including some chelated and insoluble metals and organic compounds. Understanding the full substrate range for the Mtr pathway is crucial for developing S. oneidensis strains as a tool for bioremediation.

Proteins involved in maturation pathways of plant mitochondrial and plastid c-type cytochromes.

c-type cytochromes are characterized by the presence of two covalent bonds linking heme to apocytochrome and by the heme attachment motif in the apoprotein. Several molecular systems for the maturation of c-type cytochromes have evolved in different organisms. The best characterized are three of them: system I, system II and system III. Heme is synthesized in bacterial cytoplasm, in plastids, and in animal and fungal mitochondria. Therefore the maturation of bacterial and plastid c-type cytochromes involves the transport of heme and apocytochrome from the n-side to the p-side of the respective biological membranes and the formation of the covalent bond at the p-side. It should be underlined that the site of the c-type apocytochrome synthesis is also distinct from the site of its functioning. The aim of this review is to present the current state of knowledge concerning the structure and function of two systems - system I and system II - in the maturation of plant mitochondrial and plastid c-type cytochromes, respectively.

The Three Mitochondrial Encoded CcmF Proteins Form a Complex That Interacts with CCMH and c-Type Apocytochromes in Arabidopsis

Three reading frames called ccmF(N1), ccmF(N2), and ccmF(c) are found in the mitochondrial genome of Arabidopsis. These sequences are similar to regions of the bacterial gene ccmF involved in cytochrome c maturation. ccmF genes are always absent from animal and fungi genomes but are found in mitochondrial genomes of land plant and several evolutionary distant eukaryotes. In Arabidopsis, ccmF(N2) despite the absence of a classical initiation codon is not a pseudo gene. The 3 ccmF genes of Arabidopsis are expressed at the protein level. Their products are integral proteins of the mitochondrial inner membrane with in total 11 to 13 predicted transmembrane helices. The conserved WWD domain of CcmF(N2) is localized in the inter membrane space. The 3 CcmF proteins are all detected in a high molecular mass complex of 500 kDa by Blue Native PAGE. Direct interaction between CcmF(N2) and both CcmF(N1) and CcmF(C) is shown with the yeast two-hybrid split ubiquitin system, but no interaction is observed between CcmF(N1) and CcmF(C). Similarly, interaction is detected between CcmF(N2) and apocytochrome c but also with apocytochrome c(1). Finally, CcmF(N1) and CcmF(N2) both interact with CCMH previously shown to interact as well with cytochrome c. This strengthens the hypothesis that CcmF and CCMH make a complex that performs the assembly of heme with c-type apocytochromes in plant mitochondria.

Helix swapping leads to dimerization of the N-terminal domain of the c-type cytochrome maturation protein CcmH from Escherichia coli

In the process of cytochrome c maturation, heme groups are covalently attached to reduced cysteines of specific heme-binding motifs (CXXCH) in an apocytochrome c sequence. In Escherichia coli, the CcmH protein maintains apo-protein cysteines in a reduced state prior to heme attachment. We have purified and biophysically, as well as structurally characterized the soluble, N-terminal domain of E. coli CcmH that carries the functionally relevant LRCXXC-motif. In contrast to a recently presented structure of the homologous domain from Pseudomonas aeruginosa, the E. coli protein forms a tightly interlinked dimer by swapping its N-terminal helix between two monomers. We propose that an altered environment of the functional motif may help to discern between the two redox partners CcmG and apocytochrome c.

Order within a mosaic distribution of mitochondrial c‐type cytochrome biogenesis systems?

Mitochondrial cytochromes c and c(1) 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-cysteine-containing 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.