Substrate recognition of holocytochrome c synthase: N-terminal region and CXXCH motif of mitochondrial cytochrome c.

Holocytochrome c synthase (HCCS) attaches heme covalently to mitochondrial respiratory cytochromes c. Little is known about the reaction of heme attachment to apocytochromes c by HCCS, although recently it has been established that the CXXCH motif and the N-terminus of the apocytochrome polypeptide are important protein-protein recognition motifs. Here, we explore further the important features of the N-terminal sequence and investigate what variations in the CXXCH residues are productively recognised by HCCS in its substrate.

Probing heme delivery processes in cytochrome c biogenesis System I.

Cytochromes c comprise a diverse and widespread family of proteins containing covalently bound heme that are central to the life of most organisms. In many bacteria and in certain mitochondria, the synthesis of cytochromes c is performed by a complex post-translational modification apparatus called System I (or cytochrome c maturation, Ccm, system). In Escherichia coli , there are eight maturation proteins, several of which are involved in heme handling, but the mechanism of heme transfer from one protein to the next is not known. Attachment of the heme to the apocytochrome occurs via a novel covalent bond to a histidine residue of the heme chaperone CcmE. The discovery of a variant maturation system (System I*) has provided a new tool for studying cytochrome c assembly because the variant CcmE functions via a cysteine residue in the place of the histidine of System I. In this work, we use site-directed mutagenesis on both maturation systems to probe the function of the individual component proteins as well as their concerted action in transferring heme to the cytochrome c substrate. The roles of CcmA, CcmC, CcmE, and CcmF in the heme delivery process are compared between Systems I and I*. We show that a previously proposed quinone-binding site on CcmF is not essential for either system. Significant differences in the heme chemistry involved in the formation of cytochromes c in the variant system add new pieces to the cytochrome c biogenesis puzzle.

Cytochrome c assembly.

Cytochromes c are central proteins in energy transduction processes by virtue of their functions in electron transfer in respiration and photosynthesis. They have heme covalently attached to a characteristic CXXCH motif via protein-catalyzed post-translational modification reactions. Several systems with diverse constituent proteins have been identified in different organisms and are required to perform the heme attachment and associated functions. The necessary steps are translocation of the apocytochrome polypeptide to the site of heme attachment, transport and provision of heme to the appropriate compartment, reduction and chaperoning of the apocytochrome, and finally, formation of the thioether bonds between heme and two cysteines in the cytochrome. Here we summarize the established classical models for these processes and present recent progress in our understanding of the individual steps within the different cytochrome c biogenesis systems.

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.

A pivotal heme-transfer reaction intermediate in cytochrome c biogenesis.

c-Type cytochromes are widespread proteins, fundamental for respiration or photosynthesis in most cells. They contain heme covalently bound to protein in a highly conserved, highly stereospecific post-translational modification. In many bacteria, mitochondria, and archaea this heme attachment is catalyzed by the cytochrome c maturation (Ccm) proteins. Here we identify and characterize a covalent, ternary complex between the heme chaperone CcmE, heme, and cytochrome c. Formation of the complex from holo-CcmE occurs in vivo and in vitro and involves the specific heme-binding residues of both CcmE and apocytochrome c. The enhancement and attenuation of the amounts of this complex correlates completely with known consequences of mutations in genes for other Ccm proteins. We propose the complex is a trapped catalytic intermediate in the cytochrome c biogenesis process, at the point of heme transfer from CcmE to the cytochrome, the key step in the maturation pathway.

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.

Mitochondrial cytochrome c synthase: CP motifs are not necessary for heme attachment to apocytochrome c.

The function of holocytochrome c synthase (HCCS, also called heme lyase) is to attach covalently the heme cofactor to cytochromes c in the mitochondria of animals, fungi and protozoa. Little is known about how the protein functions but CP motifs, commonly found in heme-binding proteins, are present. Here we examine holocytochrome c production by Saccharomyces cerevisiae HCCS in the Escherichia coli cytoplasm with emphasis on the conserved CP motifs long implicated in heme transfer by this enzyme. Unexpectedly, the two motifs, both towards the N-terminus, were not required for activity. Mutations in HCCS on the C-terminal side of the CP motifs, known to cause disease states in humans (microphthalmia with linear skin defects) abolished or drastically attenuated holocytochrome c production.

Oxidation state-dependent protein-protein interactions in disulfide cascades.

Bacterial growth and pathogenicity depend on the correct formation of disulfide bonds, a process controlled by the Dsb system in the periplasm of Gram-negative bacteria. Proteins with a thioredoxin fold play a central role in this process. A general feature of thiol-disulfide exchange reactions is the need to avoid a long lived product complex between protein partners. We use a multidisciplinary approach, involving NMR, x-ray crystallography, surface plasmon resonance, mutagenesis, and in vivo experiments, to investigate the interaction between the two soluble domains of the transmembrane reductant conductor DsbD. Our results show oxidation state-dependent affinities between these two domains. These observations have implications for the interactions of the ubiquitous thioredoxin-like proteins with their substrates, provide insight into the key role played by a unique redox partner with an immunoglobulin fold, and are of general importance for oxidative protein-folding pathways in all organisms.

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.

Metal and redox selectivity of protoporphyrin binding to the heme chaperone CcmE.

The interaction of heme with the heme chaperone CcmE is central to our understanding of cytochrome c maturation, a complex post-translational process involving at least eight proteins in many Gram-negative bacteria and plant mitochondria. We have shown previously that Escherichia coli CcmE can interact with heme non-covalently in vitro, before forming a novel covalent histidine-heme bond, in a redox-sensitive manner. The function of CcmE is to bind heme in the periplasm before transferring it to apocytochromes c. In the absence of structural information on the complex of CcmE and heme, we have further characterized it by examining the binding of the soluble domain of CcmE (CcmE') to protoporphyrins containing metals other than Fe, namely Zn-, Sn-, Co- and Mn-protoporphyrin (PPIX). CcmE' demonstrated no affinity for the Zn- or Sn-containing protoporphyrins and low affinity for Mn(ii)-PPIX. High-affinity, reversible binding was, however, observed for Co(iii)-PPIX, which was highly sensitive to oxidation state as demonstrated by release of the ligand from the chaperone on reduction; no binding to Co(ii)-PPIX was observed. The non-covalent complex of CcmE' and Co(iii)-PPIX was characterized by non-denaturing mass spectrometry. The implications of these observations for the in vivo function of CcmE are discussed.

Cytochrome c as an experimental model protein.

Mitochondrial cytochrome c is among the most intensively studied of all proteins. Initial interest was in its role in the respiratory chain and as a model for studies of protein structure, folding and electron transfer. The function of cytochrome c in signalling apoptosis has brought a new wave of research into the protein. Bacterial cytochromes c are more complex and varied in function. This review highlights some of these roles and expands on systems for producing holocytochrome c proteins.

c-Type cytochrome biogenesis can occur via a natural Ccm system lacking CcmH, CcmG, and the heme-binding histidine of CcmE.

The Ccm cytochrome c maturation System I catalyzes covalent attachment of heme to apocytochromes c in many bacterial species and some mitochondria. A covalent, but transient, bond between heme and a conserved histidine in CcmE along with an interaction between CcmH and the apocytochrome have been previously indicated as core aspects of the Ccm system. Here, we show that in the Ccm system from Desulfovibrio desulfuricans, no CcmH is required, and the holo-CcmE covalent bond occurs via a cysteine residue. These observations call for reconsideration of the accepted models of System I-mediated c-type cytochrome biogenesis.

Comparing the substrate specificities of cytochrome c biogenesis Systems I and II: bioenergetics.

c-Type cytochromes require specific post-translational protein systems, which vary in different organisms, for the characteristic covalent attachment of heme to the cytochrome polypeptide. Cytochrome c biogenesis System II, found in chloroplasts and many bacteria, comprises four subunits, two of which (ResB and ResC) are the minimal functional unit. The ycf5 gene from Helicobacter pylori encodes a fusion of ResB and ResC. Heterologous expression of ResBC in Escherichia coli lacking its own biogenesis machinery allowed us to investigate the substrate specificity of System II. ResBC is able to attach heme to monoheme c-type cytochromes c(550) from Paracoccus denitrificans and c(552) from Hydrogenobacter thermophilus, both normally matured by System I. The production of holocytochrome is enhanced by the addition of exogenous reductant. Single-cysteine variants of these cytochromes were not efficiently matured by System II, but System I was able to produce detectable amounts of AXXCH variants; this adds to evidence that there is no obligate requirement for a disulfide-bonded intermediate for the latter c-type cytochrome biogenesis system. In addition, System II was able to mature an AXXAH-containing variant into a b-type cytochrome, with implications for both heme supply to the periplasm and substrate recognition by System II.

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.

Control of periplasmic interdomain thiol:disulfide exchange in the transmembrane oxidoreductase DsbD.

The bacterial protein DsbD transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm. This reducing power is required for several essential pathways, including disulfide bond formation and cytochrome c maturation. DsbD includes a transmembrane domain (tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade. The final step in the cascade involves reduction of the Cys(103)-Cys(109) disulfide of nDsbD by Cys(461) of cDsbD. Here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmDsbD. We have found previously ( Mavridou, D. A., Stevens, J. M., Ferguson, S. J., & Redfield, C. (2007) J. Mol. Biol. 370, 643-658 ) that the attacking cysteine (Cys(461)) in isolated cDsbD has a high pK(a) value (10.5) that makes this thiol relatively unreactive toward the target disulfide in nDsbD. Here we show using NMR that active-site pK(a) values change significantly when cDsbD forms a complex with nDsbD. This modulation of pK(a) values is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in complex with nDsbD, the nucleophilicity of cDsbD increases permitting reductant transfer. The observation of significant changes in active-site pK(a) values upon complex formation has wider implications for understanding reactivity in thiol:disulfide oxidoreductases.

Avoidance of the cytochrome c biogenesis system by periplasmic CXXCH motifs.

The CXXCH motif is usually recognized in the bacterial periplasm as a haem attachment site in apocytochromes c. There is evidence that the Escherichia coli Ccm (cytochrome c maturation) system recognizes little more than the CXXCH sequence. A limited number of periplasmic proteins have this motif and yet are not c-type cytochromes. To explore how unwanted haem attachment to CXXCH might be avoided, and to determine whether haem attachment to the surface of a non-cytochrome protein would be possible, we converted the active-site CXXCK motif of a thioredoxin-like protein into CXXCH, the C-terminal domain of the transmembrane oxidoreductase DsbD (cDsbD). The E. coli Ccm system was found to catalyse haem attachment to a very small percentage of the resultant protein ( approximately 0.2%). We argue that cDsbD folds sufficiently rapidly that only a small fraction fails to avoid the Ccm system, in contrast with bona fide c-type cytochromes that only adopt their tertiary structure following haem attachment. We also demonstrate covalent haem attachment at a low level in vivo to the periplasmic disulfide isomerase DsbC, which contains a native CXXCH motif. These observations provide insight into substrate recognition by the Ccm system and expand our understanding of the requirements for covalent haem attachment to proteins. The possible evolutionary relationship between thioredoxins and c-type cytochromes is discussed.

Dispensable residues in the active site of the cytochrome c biogenesis protein CcmH.

CcmH functions in the assembly of c-type cytochromes in the Escherichia coli periplasm. The conserved cysteine pair in the N-terminal of its two membrane-anchored periplasmic domains is thought to reduce the CXXCH motif of cytochromes c. The recent structure of Pseudomonas aeruginosa CcmH identified conserved residues that might be functionally important. We replaced with alanine the active-site cysteines of E. coli CcmH, as well as R42, S54, R63, and tested the effects on cytochrome c production anaerobically and aerobically. Unexpectedly, replacement of the conserved non-cysteine active-site residues had little effect, whilst the cysteines were required under aerobic, but not anaerobic, conditions. We confirmed that removal of the C-terminal tetratricopeptide-like domain does not, surprisingly, abolish assembly of cytochromes c.

Cytochrome c assembly: a tale of ever increasing variation and mystery?

Formation of cytochromes c requires a deceptively simple post-translational modification, the formation of two thioether bonds (or rarely one) between the thiol groups of two cysteine residues found in a CXXCH motif (with some occasional variations) and the vinyl groups of heme. There are three partially characterised systems for facilitating this post-translational modification; within these systems there is also variation. In addition, there are clear indications for two other distinct systems. Here some of the current issues in understanding the systems are analysed.

Cytochrome c Biogenesis.

Escherichia coli employs several c-type cytochromes, which are found in the periplasm or on the periplasmic side of the cytoplasmic membrane; they are used for respiration under different growth conditions. All E. colic-type cytochromes are multiheme cytochromes; E. coli does not have a monoheme cytochrome c of the kind found in mitochondria. The attachment of heme to cytochromes c occurs in the periplasm, and so the apoprotein must be transported across the cytoplasmic membrane; this step is mediated by the Sec system, which transports unfolded proteins across the membrane. The protein CcmE has been found to bind heme covalently via a single bond and then transfer the heme to apocytochromes. It should be mentioned that far less complex systems for cytochrome c biogenesis exist in other organisms and that enterobacteria do not function as a representative model system for the process in general, although plant mitochondria use the Ccm system found in E. coli. The variety and distribution of cytochromes and their biogenesis systems reflect their significance and centrality in cellular bioenergetics, though the necessity for and origin of the diverse biogenesis systems are enigmatic.

Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins.

We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC (E. coli; cytochrome c biogenesis System I), CcmF (E. coli; cytochrome c biogenesis System I) and ResC (Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.