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.     

Physical interaction of CcmC with heme and the heme chaperone CcmE during cytochrome c maturation

Biogenesis of c-type cytochromes requires the covalent attachment of heme to the apoprotein. In Escherichia coli, this process involves eight membrane proteins encoded by the ccmABCDEFGH operon. CcmE binds heme covalently and transfers it to apocytochromes c in the presence of other Ccm proteins. CcmC is necessary and sufficient to incorporate heme into CcmE. Here, we report that the CcmC protein directly interacts with heme. We further show that CcmC co-immunoprecipitates with CcmE. CcmC contains two conserved histidines and a signature sequence, the so-called tryptophan-rich motif, which is the only element common to cytochrome c maturation proteins of bacteria, archae, plant mitochondria, and chloroplasts. We report that mutational changes of these motifs affecting the function of CcmC in cytochrome c maturation do not influence heme binding of CcmC. However, the mutants are defective in the CcmC-CcmE interaction, suggesting that these motifs are involved in the formation of a CcmC-CcmE complex. We propose that CcmC, CcmE, and heme interact directly with each other, establishing a periplasmic heme delivery pathway for cytochrome c maturation.     

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.     

Dynamic features of a heme delivery system for cytochrome C maturation

In Escherichia coli, heme is delivered to cytochrome c in a process involving eight proteins encoded by the ccmABCDEFGH operon. Heme is transferred to the periplasmic heme chaperone CcmE by CcmC and from there to apocytochrome c. The role of CcmC was investigated by random as well as site-directed mutagenesis. Important amino acids were all located in periplasmic domains of the CcmC protein that has six membrane-spanning helices. Besides the tryptophan-rich motif and two conserved histidines, new residues were identified as functionally important. Mutants G111S and H184Y had a clear defect in CcmC-CcmE interaction, did not transfer heme to CcmE, and lacked c-type cytochromes. Conversely, mutants D47N, R55P, and S176Y were affected neither in interaction with nor in delivery of heme to CcmE but produced less than 10% c-type cytochromes. A strain carrying a CcmCE fusion had a similar phenotype, suggesting that CcmC is important not only for heme transfer to CcmE but also for its delivery to cytochrome c. Co-immunoprecipitation of CcmC with CcmF was not detectable although CcmE co-precipitated individually with CcmC and CcmF. This contradicts the idea of CcmCEF supercomplex formation. Our results favor a model that predicts CcmE to shuttle between CcmC and CcmF for heme delivery.     

The interaction of covalently bound heme with the cytochrome c maturation protein CcmE

The heme chaperone CcmE is a novel protein that binds heme covalently via a histidine residue as part of its essential function in the process of cytochrome c biogenesis in many bacteria as well as plant mitochondria. In the continued absence of a structure of the holoform of CcmE, identification of the heme ligands is an important step in understanding the molecular function of this protein and the role of covalent heme binding to CcmE during the maturation of c-type cytochromes. In this work, we present spectroscopic data that provide insight into the ligation of the heme iron in the soluble domain of CcmE from Escherichia coli. Resonance Raman spectra demonstrated that one of the heme axial ligands is a histidine residue and that the other is likely to be Tyr134. In addition, the properties of the heme resonances of the holo-protein as compared with those of a form of CcmE with non-covalently bound heme provide evidence for the modification of one of the heme vinyl side chains by the protein, most likely the 2-vinyl group.     

Solution NMR structure, backbone dynamics, and heme-binding properties of a novel cytochrome c maturation protein CcmE from Desulfovibrio vulgaris

Cytochrome c maturation protein E, CcmE, plays an integral role in the transfer of heme to apocytochrome c in many prokaryotes and some mitochondria. A novel subclass featuring a heme-binding cysteine has been identified in archaea and some bacteria. Here we describe the solution NMR structure, backbone dynamics, and heme binding properties of the soluble C-terminal domain of Desulfovibrio vulgaris CcmE, dvCcmE'. The structure adopts a conserved β-barrel OB fold followed by an unstructured C-terminal tail encompassing the CxxxY heme-binding motif. Heme binding analyses of wild-type and mutant dvCcmE' demonstrate the absolute requirement of residue C127 for noncovalent heme binding in vitro.     

Dynamic ligation properties of the Escherichia coli heme chaperone CcmE to non-covalently bound heme

The cytochrome c maturation protein CcmE is an essential membrane-anchored heme chaperone involved in the post-translational covalent attachment of heme to c-type cytochromes in Gram-negative bacteria such as Escherichia coli. Previous in vitro studies have shown that CcmE can bind heme both covalently (via a histidine residue) and non-covalently. In this work we present results on the latter form of heme binding to a soluble form of CcmE. Examination of a number of site-directed mutants of E. coli CcmE by resonance Raman spectroscopy has identified ligands of the heme iron and provided insight into the initial steps of heme binding by CcmE before it binds the heme covalently. The heme binding histidine (His-130) appears to ligate the heme iron in the ferric oxidation state, but two other residues ligate the iron in the ferrous form, thereby freeing His-130 to undergo covalent attachment to a heme vinyl group. It appears that the heme ligation in the non-covalent form is different from that in the holo-form, suggesting that a change in ligation could act as a trigger for the formation of the covalent bond and showing the dynamic and oxidation state-sensitive ligation properties of CcmE.     

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.     

The membrane anchors of the heme chaperone CcmE and the periplasmic thioredoxin CcmG are functionally important

The cytochrome c maturation system of Escherichia coli contains two monotopic membrane proteins with periplasmic, functional domains, the heme chaperone CcmE and the thioredoxin CcmG. We show in a domain swap experiment that the membrane anchors of these proteins can be exchanged without drastic loss of function in cytochrome c maturation. By contrast, the soluble periplasmic forms produced with a cleavable OmpA signal sequence have low biological activity. Both the chimerical CcmE (CcmG'-'E) and the soluble periplasmic CcmE produce low levels of holo-CcmE and thus are impaired in their heme receiving capacity. Also, both forms of CcmE can be co-precipitated with CcmC, thus restricting the site of interaction of CcmE with CcmC to the C-terminal periplasmic domain. However, the low level of holo-CcmE formed in the chimera is transferred efficiently to cytochrome c, indicating that heme delivery from CcmE does not involve the membrane anchor.     

Interaction of heme with variants of the heme chaperone CcmE carrying active site mutations and a cleavable N-terminal His tag

Cytochrome c maturation in the periplasms of many bacteria requires the heme chaperone CcmE, which binds heme covalently both in vivo and in vitro via a histidine residue before transferring the heme to apocytochromes c. To investigate the mechanism and specificity of heme attachment to CcmE, we have mutated the conserved histidine 130 of a soluble C-terminally His-tagged version of CcmE (CcmEsol-C-His6) from Escherichia coli to alanine or cysteine. Remarkably, covalent bond formation with heme occurs with the protein carrying the cysteine mutation, and the process occurs both in vivo and in vitro. The yield of holo-H130C CcmEsol-C-His6 produced in vivo is low compared with the wild type. In vitro heme attachment occurs only under reducing conditions. We demonstrate the involvement of one of the heme vinyl groups and a side chain at residue 130 in the bond formation by showing that in vitro attachment does not occur either with the heme analogue mesoheme or when alanine is present at residue 130. These results have implications for the mechanism of heme attachment to the histidine of CcmE. In vitro, CcmEsol lacking a His tag binds 8-anilino-1-naphthalenesulphonate and heme, the latter both noncovalently and via a covalent bond from the histidine side chain, similarly to the tagged proteins, thus countering a recent proposal that the His tag causes the heme binding. However, the His tag does appear to enhance the rate of in vitro covalent heme binding and to affect the heme ligation in the ferric b-type cytochrome form.     

Loss of ATP hydrolysis activity by CcmAB results in loss of c-type cytochrome synthesis and incomplete processing of CcmE

The proteins CcmA and CcmB have long been known to be essential for cytochrome c maturation in Escherichia coli. We have purified a complex of these proteins, and found it to have ATP hydrolysis activity. CcmA, which has the features of a soluble ATP hydrolysis subunit, is found in a membrane-bound complex only when CcmB is present in the membrane. Mutation of the Walker A motif in CcmA(K40D) results in loss of the in vitro ATPase activity and in loss of cytochrome c biogenesis in vivo. The same mutation does not prevent covalent attachment of heme to the heme chaperone CcmE, but holo-CcmE is, for some unidentified reason, incompetent for heme transfer to an apocytochrome c or for release into the periplasm as a soluble variant. Addition of exogenous heme to heme-permeable E. coli with a ccmA deletion did not restore cytochrome c production. Our results suggest a role for CcmAB in the handling of heme by CcmE, which is chemically complex and involves an unusual histidine-heme covalent bond.     

Continued surprises in the cytochrome c biogenesis story

Cytochromes c covalently bind their heme prosthetic groups through thioether bonds between the vinyl groups of the heme and the thiols of a CXXCH motif within the protein. In Gram-negative bacteria, this process is catalyzed by the Ccm (cytochrome c maturation) proteins, also called System I. The Ccm proteins are found in the bacterial inner membrane, but some (CcmE, CcmG, CcmH, and CcmI) also have soluble functional domains on the periplasmic face of the membrane. Elucidation of the mechanisms involved in the transport and relay of heme and the apocytochrome from the bacterial cytosol into the periplasm, and their subsequent reaction, has proved challenging due to the fact that most of the proteins involved are membrane-associated, but recent progress in understanding some key components has thrown up some surprises. In this Review, we discuss advances in our understanding of this process arising from a substrate’s point of view and from recent structural information about individual components.     

CcmD is involved in complex formation between CcmC and the heme chaperone CcmE during cytochrome c maturation

CcmD is a small membrane protein involved in heme delivery to the heme chaperone CcmE during cytochrome c maturation. Here we show that it physically interacts with CcmE and CcmC, another essential component of the heme delivery system. We demonstrate the formation of a ternary complex consisting of CcmCDE. A deletion analysis of individual domains revealed that the central hydrophobic domain is essential for its function. Moreover, the C-terminal, cytoplasmic domain seems to require a net positive charge to be functional. Our topology analysis indicates that CcmD is an integral interfacial membrane protein with its N and C termini extruding into the cytoplasmic side of the membrane. Interactions of CcmD with either ferrochelatase, the last heme biosynthetic enzyme, or directly with heme were not detectable. We postulate a function for CcmD in protein-protein interaction or membrane protein assembly required for the heme delivery process.     

Functional characterization of the C-terminal domain of the cytochrome c maturation protein CcmE

CcmE is a heme chaperone involved in the periplasmic maturation of c-type cytochromes in many bacteria and plant mitochondria. It binds heme covalently and subsequently transfers it to the apo form of cytochromes c. To examine the role of the C-terminal domain of CcmE in the binding of heme, in vitro heme binding to the apo form of a truncated (immediately before Pro-136) version of the periplasmic domain of the heme chaperone from Escherichia coli was studied. Removal of the C-terminal domain dramatically altered the ligation of non-covalently bound heme in CcmE' (the soluble form lacking the membrane anchor) but only slightly affected its affinity for protoporphyrin IX and 8-anilino-1-naphthalenesulfonate. This finding has significant mechanistic implications for in vivo holo-CcmE formation and indicates that the C-terminal region is not required for the recruitment and docking of heme into its binding site but is likely to contain amino acid(s) involved in heme iron axial coordination. Removal of the C-domain significantly impaired in vivo heme binding to CcmE and conversion of apocytochrome to holoprotein by a similar factor, suggesting that the C-terminal domain of the chaperone is primarily involved in heme binding to CcmE rather than in heme transfer to the apo cytochrome.     

Biochemical and mutational characterization of the heme chaperone CcmE reveals a heme binding site

CcmE is a heme chaperone that binds heme transiently in the periplasm of Escherichia coli and delivers it to newly synthesized and exported c-type cytochromes. The chemical nature of the covalent bond between heme and H130 is not known. We have purified soluble histidine-tagged CcmE and present its spectroscopic characteristics in the visible range. Alanine scanning mutagenesis of conserved amino acids revealed that H130 is the only residue found to be strictly required for heme binding and delivery. Mutation of the hydrophobic amino acids F37, F103, L127, and Y134 to alanine affected CcmE more than mutation of charged and polar residues. Our data are in agreement with the recently solved nuclear magnetic resonance structure of apo-CcmE (PDB code 1LIZ) and suggest that heme is bound to a hydrophobic platform at the surface of the protein and then attached to H130 by a covalent bond. Replacement of H130 with cysteine led to the formation of a covalent bond between heme and C130 at a low level. However, the H130C mutant CcmE was not active in cytochrome c maturation. Isolation and characterization of the heme-binding peptides obtained after a tryptic digest of wild-type and H130C CcmE support the hypothesis that heme is bound covalently at a vinyl group.     

NMR structure of the heme chaperone CcmE reveals a novel functional motif

The concept of metal chaperones involves transient binding of metallic cofactors by specific proteins for delivery to enzymes in which they function. Metal chaperones thus provide a protective, as well as a transport, function. We report the first structure of a heme chaperone, CcmE, which comprises these two functions. We propose that the covalent attachment of heme to an exposed histidine occurs after heme binding at the surface of a rigid molecule with a flexible C-terminal domain. CcmE belongs to a family of proteins with a specific fold, which all share a function in delivery of specific molecular cargo.     

A bacterial cytochrome c heme lyase. CcmF forms a complex with the heme chaperone CcmE and CcmH but not with apocytochrome c.

Biogenesis of c-type cytochromes in Escherichia coli involves a number of membrane proteins (CcmA-H), which are required for the transfer of heme to the periplasmically located apocytochrome c. The pathway includes (i) covalent, transient binding of heme to the periplasmic domain of the heme chaperone CcmE; (ii) the subsequent release of heme; and (iii) transfer and covalent attachment of heme to apocytochrome c. Here, we report that CcmF is a key player in the late steps of cytochrome c maturation. We demonstrate that the conserved histidines His-173, His-261, His-303, and His-491 and the tryptophan-rich signature motif of the CcmF protein family are functionally required. Co-immunoprecipitation experiments revealed that CcmF interacts directly with the heme donor CcmE and with CcmH but not with apocytochrome c. We propose that CcmFH forms a bacterial heme lyase complex for the transfer of heme from CcmE to apocytochrome c.     

A variant System I for cytochrome c biogenesis in archaea and some bacteria has a novel CcmE and no CcmH

C-type cytochromes are characterized by post-translational covalent attachment of heme to thiols that occur in a Cys-Xxx-Xxx-Cys-His motif. Three distinct biogenesis systems are known for this heme attachment. Archaea are now shown to contain a significantly modified form of cytochrome c maturation System I (the Ccm system). The most notable adaptation relative to the well-studied apparatus from proteobacteria and plants is a novel form of the heme chaperone CcmE, lacking the highly conserved histidine that covalently binds heme and is essential for function in Escherichia coli. In most archaeal CcmEs this histidine, normally found in a His-Xxx-Xxx-Xxx-Tyr motif, is replaced by a cysteine residue that occurs in a Cys-Xxx-Xxx-Xxx-Tyr motif. The CcmEs from two halobacteria contain yet another form of CcmE, having HxxxHxxxH approximately corresponding in alignment to the H/CxxxY motif. The CxxxY-type of CcmE is, surprisingly, also found in some bacterial genomes (including Desulfovibrio species). All of the modified CcmEs cluster together in a phylogenetic tree, as do other Ccm proteins from the same organisms. Significantly, CcmH is absent from all of the complete archaeal genomes we have studied, and also from most of the bacterial genomes that have CxxxY-type CcmE.     

Structurally Mapping Endogenous Heme in the CcmCDE Membrane Complex for Cytochrome c Biogenesis

Although many putative heme transporters have been discovered, it has been challenging to prove that these proteins are directly involved with heme trafficking in vivo and to identify their heme binding domains. The prokaryotic pathways for cytochrome c biogenesis, Systems I and II, transport heme from inside the cell to outside for stereochemical attachment to cytochrome c, making them excellent models to study heme trafficking. System I is composed of eight integral membrane proteins (CcmA–H) and is proposed to transport heme via CcmC to an external “WWD” domain for presentation to the membrane-tethered heme chaperone, CcmE. Herein, we develop a new cysteine/heme crosslinking approach to trap and map endogenous heme in CcmC (WWD domain) and CcmE (defining “2-vinyl” and “4-vinyl” pockets for heme). Crosslinking occurs when either of the two vinyl groups of heme localize near a thiol of an engineered cysteine residue. Double crosslinking, whereby both vinyls crosslink to two engineered cysteines, facilitated a more detailed structural mapping of the heme binding sites, including stereospecificity. Using heme crosslinking results, heme ligand identification, and genomic coevolution data, we model the structure of the CcmCDE complex, including the WWD heme binding domain. We conclude that CcmC trafficks heme via its WWD domain and propose the structural basis for stereochemical attachment of heme.     

CCME, a nuclear-encoded heme-binding protein involved in cytochrome c maturation in plant mitochondria

The maturation of c-type cytochromes requires the covalent attachment of the heme cofactor to the apoprotein. For this process, plant mitochondria follow a pathway distinct from that of animal or yeast mitochondria, closer to that found in alpha- and gamma-proteobacteria. We report the first characterization of a nuclear-encoded component, namely AtCCME, the Arabidopsis thaliana orthologue of CcmE, a periplasmic heme chaperone in bacteria. AtCCME is targeted to mitochondria, and its N-terminal signal peptide is cleaved upon import. AtCCME is a peripheral protein of the mitochondrial inner membrane, and its major hydrophilic domain is oriented toward the intermembrane space. Although a AtCCME (Met(79)-Ser(256)) is not fully able to complement an Escherichia coli CcmE mutant strain for bacterial holocytochrome c production, it is able to bind heme covalently through a conserved histidine, a feature previously shown for E. coli CcmE. Our results suggest that AtCCME is important for cytochrome c maturation in A. thaliana mitochondria and that its heme-binding function has been conserved evolutionary between land plant mitochondria and alpha-proteobacteria.