The C-terminal flexible domain of the heme chaperone CcmE is important but not essential for its function

CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a beta-barrel and a flexible C-terminal domain with a short alpha-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence (131)DENYTPP(137) was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.     

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.     

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.     

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.     

Interspecies complementation of Escherichia coli ccm mutants: CcmE (CycJ) from Bradyrhizobium japonicum acts as a heme chaperone during cytochrome c maturation

Biogenesis of c-type cytochromes in alpha- and gamma-proteobacteria requires the function of a set of orthologous genes (ccm genes) that encode specific maturation factors. The Escherichia coli CcmE protein is a periplasmic heme chaperone. The membrane protein CcmC is required for loading CcmE with heme. By expressing CcmE (CycJ) from Bradyrhizobium japonicum in E. coli we demonstrated that heme is bound covalently to this protein at a strictly conserved histidine residue. The B. japonicum homologue can transfer heme to apocytochrome c in E. coli, suggesting that it functions as a heme chaperone. CcmC (CycZ) from B. japonicum expressed in E. coli was capable of inserting heme into CcmE.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Axial coordination of heme in ferric CcmE chaperone characterized by EPR spectroscopy

In Escherichia coli cytochrome c maturation requires a set of eight proteins including the heme chaperone CcmE, which binds heme transiently, yet covalently. Several variants of CcmE were purified and analyzed by continuous-wave electron paramagnetic resonance, electron nuclear double resonance, and hyperfine sublevel correlation spectroscopy to investigate the heme axial coordination. Results reveal the presence of a number of coordination environments, two high-spin heme centers with different rhombicities, and at least one low-spin heme center. The low-spin species was shown to be an artifact induced by the presence of available histidines in the vicinity of the iron. Both of the high-spin forms are five-coordinated, and comparison of the spectra of the wild-type CcmE with those of the mutant CcmE(Y134H) proves that the higher-rhombicity form is coordinated by Tyr134. The low-rhombicity (axial) form does not have a histidine residue or a water molecule as an axial ligand. However, we identified exchangeable protons coupled to the iron ion. We propose that the axial form can be coordinated by a carboxyl group of an acidic residue in the flexible domain of the protein. The two species would represent two different conformations of the flexible alpha-helix domain surrounding the heme. This conformational flexibility confers CcmE special dynamic properties that are certainly important for its function.     

Cytochrome c maturation: a complex pathway for a simple task?

Post-translational maturation of c-type cytochromes involves covalent attachment of haem to the apocytochrome polypeptide by two thioether bonds. In bacteria, haem attachment occurs in the periplasm, after the separate translocation of haem and the polypeptide across the cytoplasmic membrane. In Escherichia coli, delivery and attachment of the cofactor requires eight or nine specific proteins, which are believed to be organized in a membrane protein complex. After transport across the membrane, haem is attached covalently to the haem chaperone CcmE in an unusual way at a single histidine residue. However, haem binding to CcmE is transient and is succeeded by a further transfer to apocytochrome c. Both haem binding to and release from CcmE involve integral membrane proteins, CcmC and CcmF respectively, which carry a conserved tryptophan-rich motif in a periplasmic domain. Apocytochrome c polypeptides are synthesized as precursors and reach the periplasm by sec-dependent translocation. There they are prepared for haem binding by reduction of the cysteine residues in the motif Cys-Xaa-Xaa-Cys-His, which is characteristic of such proteins. This reduction is achieved in a thio-reduction pathway, whereby electrons are passed from cytoplasmic thioredoxin to the transmembrane protein DsbD, across the membrane, and on to the specific reductases CcmG/CcmH. The merging of the haem delivery and the thio-reduction pathways leads to the stereospecific insertion of haem into various type c cytochromes.     

The N-terminal -barrel domain of the Escherichia coli K88 periplasmic chaperone FaeE determines fimbrial subunit recognition and dimerization

The K88 periplasmic chaperone FaeE is a homodimer, whereas the K99 chaperone FanE is a monomer. The structural requirements for dimerization of the K88 fimbrial periplasmic chaperone and for fimbrial subunit-binding specificity were investigated by analysis of mutant chaperones. FaeE contains a C-terminal extension of 19 amino acid residues when compared to FanE and most other fimbrial chaperones. A C-terminal truncate of the K88 chaperone FaeE was constructed that lacked 19 C-terminal amino acid residues. Expression and complementation experiments revealed that this C-terminal shortened chaperone was still functional in binding the K88 major subunit FaeG and K88 biosynthesis. Two hybrid chaperones were constructed. Each hybrid protein contained one -barrel domain of FaeE and the other -barrel domain of FanE (Fae/FanE or Fan/FaeE, respectively). Expression and complementation experiments revealed that the Fae/FanE but not the Fan/FaeE hybrid chaperone was functional in the formation of K88 fimbriae. The Fan/FaeE hybrid chaperone was active in the biosynthesis of K99 fimbriae. The truncated FaeE mutant chaperone and the hybrid Fae/FanE chaperone were able to form stable periplasmic protein complexes with the K88 major fimbrial subunit FaeG. Cross-linking experiments suggested that the C-terminal shortened chaperone and the Fae/FanE hybrid chaperone were homodimers, as is the wild-type K88 chaperone. Altogether, the data suggested that the N-terminal -barrel domain of a fimbrial chaperone determines subunit specificity. In the case of the K88 periplasmic chaperone, this N-terminal domain also determines dimerization of the protein.