Covalent Attachment Of Heme Research Articles

Architecture of the Heme-translocating CcmABCD/E complex required for Cytochrome c maturation

Mono- and multiheme cytochromes c are post-translationally matured by the covalent attachment of heme. For this, Escherichia coli employs the most complex type of maturation machineries, the Ccm-system (for cytochrome cmaturation). It consists of two membrane protein complexes, one of which shuttles heme across the membrane to a mobile chaperone that then delivers the cofactor to the second complex, an apoprotein:heme lyase, for covalent attachment. Here we report cryo-electron microscopic structures of the heme translocation complex CcmABCD from E. coli, alone and bound to the heme chaperone CcmE. CcmABCD forms a heterooctameric complex centered around the ABC transporter CcmAB that does not by itself transport heme. Our data suggest that the complex flops a heme group from the inner to the outer leaflet at its CcmBC interfaces, driven by ATP hydrolysis at CcmA. A conserved heme-handling motif (WxWD) at the periplasmic side of CcmC rotates the heme by 90° for covalent attachment to the heme chaperone CcmE that we find interacting exclusively with the CcmB subunit.

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.

Characterization of the apicoplast-localized enzyme TgUroD in Toxoplasma gondii reveals a key role of the apicoplast in heme biosynthesis

Apicomplexan parasites such as Toxoplasma gondii possess an unusual heme biosynthesis pathway whose enzymes localize to the mitochondrion, cytosol, or apicoplast, a nonphotosynthetic plastid present in most apicomplexans. To characterize the involvement of the apicoplast in the T. gondii heme biosynthesis pathway, we investigated the role of the apicoplast-localized enzyme uroporphyrinogen III decarboxylase (TgUroD). We found that TgUroD knockdown impaired parasite proliferation, decreased free heme levels in the parasite, and decreased the abundance of heme-containing c-type cytochrome proteins in the parasite mitochondrion. We validated the effects of heme loss on mitochondrial cytochromes by knocking down cytochrome c/c1 heme lyase 1 (TgCCHL1), a mitochondrial enzyme that catalyzes the covalent attachment of heme to c-type cytochromes. TgCCHL1 depletion reduced parasite proliferation and decreased the abundance of c-type cytochromes. We further sought to characterize the overall importance of TgUroD and TgCCHL1 for both mitochondrial and general parasite metabolism. TgUroD depletion decreased cellular ATP levels, mitochondrial oxygen consumption, and extracellular acidification rates. By contrast, depletion of TgCCHL1 neither diminished ATP levels in the parasite nor impaired extracellular acidification rate, but resulted in specific defects in mitochondrial oxygen consumption. Together, our results indicate that the apicoplast has a key role in heme biology in T. gondii and is important for both mitochondrial and general parasite metabolism. Our study highlights the importance of heme and its synthesis in these parasites.

The CcmC–CcmE interaction during cytochrome c maturation by System I is driven by protein–protein and not protein–heme contacts

Cytochromes c are ubiquitous proteins, essential for life in most organisms. Their distinctive characteristic is the covalent attachment of heme to their polypeptide chain. This post-translational modification is performed by a dedicated protein system, which in many Gram-negative bacteria and plant mitochondria is a nine-protein apparatus (CcmA–I) called System I. Despite decades of study, mechanistic understanding of the protein–protein interactions in this highly complex maturation machinery is still lacking. Here, we focused on the interaction of CcmC, the protein that sources the heme cofactor, with CcmE, the pivotal component of System I responsible for the transfer of the heme to the apocytochrome. Using in silico analyses, we identified a putative interaction site between these two proteins (residues Asp47, Gln50, and Arg55 on CcmC; Arg73, Asp101, and Glu105 on CcmE), and we validated our findings by in vivo experiments in Escherichia coli. Moreover, employing NMR spectroscopy, we examined whether a heme-binding site on CcmE contributes to this interaction and found that CcmC and CcmE associate via protein–protein rather than protein–heme contacts. The combination of in vivo site-directed mutagenesis studies and high-resolution structural techniques enabled us to determine at the residue level the mechanism for the formation of one of the key protein complexes for cytochrome c maturation by System I.

CCS2, an Octatricopeptide-Repeat Protein, Is Required for Plastid Cytochrome c Assembly in the Green Alga Chlamydomonas reinhardtii

In bacteria and energy generating organelles, c-type cytochromes are a class of universal electron carriers with a heme cofactor covalently linked via one or two thioether bonds to a heme binding site. The covalent attachment of heme to apocytochromes is a catalyzed process, taking place via three evolutionarily distinct assembly pathways (Systems I, II, III). System II was discovered in the green alga Chlamydomonas reinhardtii through the genetic analysis of the ccs mutants (cytochrome csynthesis), which display a block in the apo- to holo- form conversion of cytochrome f and c6, the thylakoid lumen resident c-type cytochromes functioning in photosynthesis. Here we show that the gene corresponding to the CCS2 locus encodes a 1,719 amino acid polypeptide and identify the molecular lesions in the ccs2-1 to ccs2-5 alleles. The CCS2 protein displays seven degenerate amino acid repeats, which are variations of the octatricopeptide-repeat motif (OPR) recently recognized in several nuclear-encoded proteins controlling the maturation, stability, or translation of chloroplast transcripts. A plastid site of action for CCS2 is inferred from the finding that GFP fused to the first 100 amino acids of the algal protein localizes to chloroplasts in Nicotiana benthamiana. We discuss the possible functions of CCS2 in the heme attachment reaction.

Heme Trafficking by the Cytochrome C Biogenesis Pathways

Cytochromes function in electron transport chains to perform critical cellular functions, such as respiration and photosynthesis. Cytochromes c are unique due to their requirement for the covalent attachment of heme via two thioether bonds at a conserved CXXCH motif. Three pathways have been identified for cytochrome c maturation: System I (prokaryotes), System II (prokaryotes) and System III (eukaryotes). System I consists of 8 integral membrane proteins (CcmABCDEFGH), System II is comprised of 2 membrane proteins (CcsBA) and these pathways will be the focus of this presentation. Trafficking of heme from the site of its synthesis (cytoplasm) to the site of attachment to apocytochrome c (periplasm) is critical for cytochrome c biogenesis, yet little direct evidence of heme trafficking exists. The study of heme trafficking has proved elusive in this and most other systems due to tight cellular regulation, as well as the cytotoxic and amphipathic nature of heme. Here, we use key putative heme transporters in the prokaryotic pathways as model systems to develop a novel technique to covalently ‘trap’ heme during the trafficking process. First, the conserved WWD domain, which is predicted to interact with heme in the periplasmic space and is found in both System I and System II pathways, was used to develop the heme trapping technique. Heme was covalently trapped in the WWD domains of CcmC (System I) and CcsA (System II), providing the first direct evidence of how heme is trafficked to the periplasm in cytochrome c biogenesis. Currently, this trapping approach is being used on other integral membrane proteins in the pathway to capture heme intermediates and delineate the exact paths for trafficking in vivo. We envision that this approach will be applicable to other heme transporters and trafficking pathways from prokaryotes through eukaryotes.

Heme Trafficking and Modifications during System I Cytochrome c Biogenesis: Insights from Heme Redox Potentials of Ccm Proteins.

Cytochromes c require covalent attachment of heme via two thioether bonds at conserved CXXCH motifs, a process accomplished in prokaryotes by eight integral membrane proteins (CcmABCDEFGH), termed System I. Heme is trafficked from inside the cell to outside (via CcmABCD) and chaperoned (holoCcmE) to the cytochrome c synthetase (CcmF/H). Purification of key System I pathway intermediates allowed the determination of heme redox potentials. The data support a model whereby heme is oxidized to form holoCcmE and subsequently reduced by CcmF/H for thioether formation, with Fe(2+) being required for attachment to CXXCH. Results provide insight into mechanisms for the oxidation and reduction of heme in vivo.

Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site, Cys-XX-Cys-His

Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19.

The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE

Cytochrome c maturation (ccm) in many bacteria, archaea and plant mitochondria requires eight membrane proteins, CcmABCDEFGH, called system I. This pathway delivers and attaches haem covalently to two cysteines (of Cys-Xxx-Xxx-Cys-His) in the cytochrome c. All models propose that CcmFH facilitates covalent attachment of haem to the apocytochrome; namely, that it is the synthetase. However, holocytochrome c synthetase activity has not been directly demonstrated for CcmFH. We report formation of holocytochromes c by CcmFH and CcmG, a periplasmic thioredoxin, independent of CcmABCDE (we term this activity CcmFGH-only). Cytochrome c produced in the absence of CcmABCDE is indistinguishable from cytochrome c produced by the full system I, with a cleaved signal sequence and two covalent bonds to haem. We engineered increased cytochrome c production by CcmFGH-only, with yields approaching those from the full system I. Three conserved histidines in CcmF (TM-His1, TM-His2 and P-His1) are required for activity, as are the conserved cysteine pairs in CcmG and CcmH. Our findings establish that CcmFH is the system I holocytochrome c synthetase. Although we discuss why this engineering would likely not replace the need for CcmABCDE in nature, these results provide unique mechanistic and evolutionary insights into cytochrome c biosynthesis.

Interaction of HoloCcmE with CcmF in Heme Trafficking and Cytochrome c Biosynthesis

The periplasmic heme chaperone holoCcmE is essential for heme trafficking in the cytochrome c biosynthetic pathway in many bacteria, archaea, and plant mitochondria. This pathway, called system I, involves two steps: (i) formation and release of holoCcmE (by the ABC-transporter complex CcmABCD) and (ii) delivery of the heme in holoCcmE to the putative cytochrome c heme lyase complex, CcmFH. CcmFH is believed to facilitate the final covalent attachment of heme (from holoCcmE) to the apocytochrome c. Although most models for system I propose that holoCcmE delivers heme directly to CcmF, no interaction between holoCcmE and CcmF has been demonstrated. Here, a complex between holoCcmE and CcmF is “trapped”, purified, and characterized. HoloCcmE must be released from the ABC-transporter complex CcmABCD to interact with CcmF, and the holo-form of CcmE interacts with CcmF at levels at least 20-fold higher than apoCcmE. Two conserved histidines (here termed P-His1 and P-His2) in separate periplasmic loops in CcmF are required for interaction with holoCcmE, and evidence that P-His1 and P-His2 function as heme-binding ligands is presented. These results show that heme in holoCcmE is essential for complex formation with CcmF and that the heme of holoCcmE is coordinated by P-His1 and P-His2 within the WWD domain of CcmF. These features are strikingly similar to formation of the CcmC:heme:CcmE ternary complex [Richard-Fogal C, Kranz RG. The CcmC:heme:CcmE complex in heme trafficking and cytochrome c biosynthesis. J Mol Biol 2010;401:350–62] and suggest common mechanistic and structural aspects.

Perturbation of Cytochrome c Maturation Reveals Adaptability of the Respiratory Chain in Mycobacterium tuberculosis

ABSTRACTMycobacterium tuberculosis depends on aerobic respiration for growth and utilizes an aa3-type cytochrome c oxidase for terminal electron transfer. Cytochrome c maturation in bacteria requires covalent attachment of heme to apocytochrome c, which occurs outside the cytoplasmic membrane. We demonstrate that in M. tuberculosis the thioredoxin-like protein Rv3673c, which we named CcsX, is required for heme insertion in cytochrome c. Inactivation of CcsX resulted in loss of c-type heme absorbance, impaired growth and virulence of M. tuberculosis, and induced cytochrome bd oxidase. This suggests that the bioenergetically less efficient bd oxidase can compensate for deficient cytochrome c oxidase activity, highlighting the flexibility of the M. tuberculosis respiratory chain. A spontaneous mutation in the active site of vitamin K epoxide reductase (VKOR) suppressed phenotypes of the CcsX mutant and abrogated the activity of the disulfide bond-dependent alkaline phosphatase, which shows that VKOR is the major disulfide bond catalyzing protein in the periplasm of M. tuberculosis.

Structural and functional characterization of HP0377, a thioredoxin-fold protein fromHelicobacter pylori

Maturation of cytochrome c is carried out in the bacterial periplasm, where specialized thiol-disulfide oxidoreductases provide the correct reduction of oxidized apocytochrome c before covalent haem attachment. HP0377 from Helicobacter pylori is a thioredoxin-fold protein that has been implicated as a component of system II for cytochrome c assembly and shows limited sequence similarity to Escherichia coli DsbC, a disulfide-bond isomerase. To better understand the role of HP0377, its crystal structures have been determined in both reduced and partially oxidized states, which are highly similar to each other. Sedimentation-equilibrium experiments indicate that HP0377 is monomeric in solution. HP0377 adopts a thioredoxin fold but shows distinctive variations as in other thioredoxin-like bacterial periplasmic proteins. The active site of HP0377 closely resembles that of E. coli DsbC. A reductase assay suggests that HP0377 may play a role as a reductase in the biogenesis of holocytochrome c553 (HP1227). Binding experiments indicate that it can form a covalent complex with HP0518, a putative L,D-transpeptidase with a catalytic cysteine residue, via a disulfide bond. Furthermore, physicochemical properties of HP0377 and its R86A variant have been determined. These results suggest that HP0377 may perform multiple functions as a reductase in H. pylori.

Probing the biological significance of c‐heme attachment in cytochrome c

Heme is an iron‐containing porphyrin ring that some iron‐dependent bacteria are able to use as their iron source. c‐type heme is differentiated from other hemes (b, d, etc.) by covalent attachment of the heme to the protein chain via two thioether bonds to Cysteines. Covalent attachment of the heme group is energetically expensive, suggesting that there is a biological benefit to the attachment. One proposed advantage, supported by our studies, is that the covalent attachment stops iron‐dependent bacteria, such as Nontypable Haemophilus influenzae (NTHi), from stealing heme. In our study, we evaluated the role of c heme attachment by creating single‐ and double‐mutant cytochrome c proteins, which lacked one or both of the covalent attachments to the heme group, respectively. We then grew NTHi in the presence of wild‐type (two covalent heme attachments), single‐mutant (one covalent attachment) and double‐mutant (no covalent attachments) protein, as well as appropriate controls, to examine the effect of covalent heme attachment on NTHi growth. Results from our studies demonstrated that the covalent linkages do prevent NTHi from scavenging heme from wild‐type and single‐mutant cytochrome c, while non‐covalently attached heme (in double‐mutant cytochrome c) is readily scavenged and used as an iron source. This study was funded by the Rochester Institute of Technology.

Covalent Attachment of Heme to the Protein Moiety in an Insect E75 Nitric Oxide Sensor

We have recombinantly expressed and purified the ligand binding domains (LBDs) of four insect nuclear receptors of the E75 family. The Drosophila melanogaster and Bombyx mori nuclear receptors were purified as ferric hemoproteins with Soret maxima at 424 nm, whereas their ferrous forms had a Soret maximum at 425 nm that responds to (•)NO and CO binding. In contrast, the purified LBD of Oncopeltus fasciatus displayed a Soret maximum at 415 nm for the ferric protein that shifted to 425 nm in its ferrous state. Binding of (•)NO to the heme moiety of the D. melanogaster and B. mori E75 LBD resulted in the appearance of a peak at 385 nm, whereas this peak appeared at 416 nm in the case of the O. fasciatus hemoprotein, resembling the behavior displayed by its human homologue, Rev-erbβ. High-performance liquid chromatography analysis revealed that, unlike the D. melanogaster and B. mori counterparts, the heme group of O. fasciatus is covalently attached to the protein through the side chains of two amino acids. The high degree of sequence homology with O. fasciatus E75 led us to clone and express the LBD of Blattella germanica, which established that its spectral properties closely resemble those of O. fasciatus and that it also has the heme group covalently bound to the protein. Hence, (•)NO/CO regulation of the transcriptional activity of these nuclear receptors might be differently controlled among various insect species. In addition, covalent heme binding provides strong evidence that at least some of these nuclear receptors function as diatomic gas sensors rather than heme sensors. Finally, our findings expand the classes of hemoproteins in which the heme group is normally covalently attached to the polypeptide chain.

Electron self-exchange and self-amplified posttranslational modification in the hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002

Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539-552, 2011). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 10(2)-10(3) M(-1) s(-1) and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment.

The flavoprotein Cyc2p, a mitochondrial cytochrome c assembly factor, is a NAD(P)H‐dependent haem reductase

Cytochrome c assembly requires sulphydryls at the CXXCH haem binding site on the apoprotein and also chemical reduction of the haem co-factor. In yeast mitochondria, the cytochrome haem lyases (CCHL, CC(1) HL) and Cyc2p catalyse covalent haem attachment to apocytochromes c and c(1) . An in vivo indication that Cyc2p controls a reductive step in the haem attachment reaction is the finding that the requirement for its function can be bypassed by exogenous reductants. Although redox titrations of Cyc2p flavin (E(m) = -290 mV) indicate that reduction of a disulphide at the CXXCH site of apocytochrome c (E(m) = -265 mV) is a thermodynamically favourable reaction, Cyc2p does not act as an apocytochrome c or c(1) CXXCH disulphide reductase in vitro. In contrast, Cyc2p is able to catalyse the NAD(P)H-dependent reduction of hemin, an indication that the protein's role may be to control the redox state of the iron in the haem attachment reaction to apocytochromes c. Using two-hybrid analysis, we show that Cyc2p interacts with CCHL and also with apocytochromes c and c(1) . We postulate that Cyc2p, possibly in a complex with CCHL, reduces the haem iron prior to haem attachment to the apoforms of cytochrome c and c(1) .

Heme Ligand Identification and Redox Properties of the Cytochrome c Synthetase, CcmF

Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conserved transmembrane histidines (His261 or His491) impairs stoichiometric b-heme binding in CcmF and results in spectral perturbations in the remaining heme. Exogeneous imidazole is able to correct cytochrome c maturation for His261 and His491 substitutions with small side chains (Ala or Gly), suggesting that a "cavity" is formed in these CcmF mutants in which imidazole binds and acts as a functional ligand to the b-heme. The results of resonance Raman spectroscopy on wild-type CcmF are consistent with a hexacoordinate low-spin b-heme with at least one endogeneous axial His ligand. Analysis of purified recombinant CcmF proteins from diverse prokaryotes reveals that the b-heme in CcmF is widely conserved. We have also determined the reduction potential of the CcmF b-heme (E(m,7) = -147 mV). We discuss these results in the context of CcmF structure and functions as a heme reductase and cytochrome c synthetase.

Cytochrome c biogenesis System I

Cytochromes c are widespread respiratory proteins characterized by the covalent attachment of heme. The formation of c-type cytochromes requires, in all but a few exceptional cases, the formation of two thioether bonds between the two cysteine sulfurs in a –CXXCH– motif in the protein and the vinyl groups of heme. The vinyl groups of the heme are not particularly activated and therefore the addition reaction does not physiologically occur spontaneously in cells. There are several diverse post-translational modification systems for forming these bonds. Here, we describe the complex multiprotein cytochrome c maturation (Ccm) system (in Escherichia coli comprising the proteins CcmABCDEFGH), also called System I, that performs the heme attachment. System I is found in plant mitochondria, archaea and many Gram-negative bacteria; the systems found in other organisms and organelles are described elsewhere in this minireview series.

Composition and function of cytochrome c biogenesis System II

Organisms employ one of several different enzyme systems to mature cytochromes c. The biosynthetic process involves the periplasmic reduction of cysteine residues in the heme c attachment motif of the apocytochrome, transmembrane transport of heme b and stereospecific covalent heme attachment via thioether bonds. The biogenesis System II (or Ccs system) is employed by β-, δ- and ε-proteobacteria, Gram-positive bacteria, Aquificales and cyanobacteria, as well as by algal and plant chloroplasts. System II comprises four (sometimes only three) membrane-bound proteins: CcsA (or ResC) and CcsB (ResB) are the components of the cytochrome c synthase, whereas CcdA and CcsX (ResA) function in the generation of a reduced heme c attachment motif. Some ε-proteobacteria contain CcsBA fusion proteins constituting single polypeptide cytochrome c synthases especially amenable for functional studies. This minireview highlights the recent findings on the structure, function and specificity of individual System II components and outlines the future challenges that remain to our understanding of the fascinating post-translational protein maturation process in more detail.

The mitochondrial cytochrome c N-terminal region is critical for maturation by holocytochrome c synthase

The covalent attachment of heme to mitochondrial cytochrome c is catalysed by holocytochrome c synthase (HCCS, also called heme lyase). How HCCS functions and recognises the substrate apocytochrome is unknown. Here we have examined HCCS recognition of a chimeric substrate comprising a short mitochondrial cytochrome c N-terminal region with the C-terminal sequence, including the CXXCH heme-binding motif, of a bacterial cytochrome c that is not otherwise processed by HCCS. Heme attachment to the chimera demonstrates the importance of the N-terminal region of the cytochrome. A series of variants of a mitochondrial cytochrome c with amino acid replacements in the N-terminal region have narrowed down the specificity determinants, providing insight into HCCS substrate recognition.