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.     

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.     

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.     

New insights into the role of CcmC, CcmD and CcmE in the haem delivery pathway during cytochrome c maturation by a complete mutational analysis of the conserved tryptophan-rich motif of CcmC

Maturation of c-type cytochromes in Escherichia coli is a complex process requiring eight membrane proteins encoded by the ccmABCDEFGH operon. CcmE is a mediator of haem delivery. It binds haem transiently at a conserved histidine residue and releases it for directed transfer to apocytochrome c. CcmC, an integral membrane protein with six transmembrane helices, is necessary and sufficient to incorporate haem covalently into CcmE. CcmC contains a highly conserved tryptophan-rich motif, WGXXWXWD, in its second periplasmic loop. Here, we present the results of a systematic mutational analysis of this motif. Changes of the non-conserved T121 and W122 to A resulted in wild-type CcmC activity. Changes of the single amino acids W119A, G120A, W123A, W125I and D126A or of the spacing within the motif by deleting V124 (DeltaV124) inhibited the covalent haem incorporation into CcmE. Enhanced expression of ccmD suppressed this mutant phenotype by increasing the amounts of CcmC and CcmE polypeptides in the membrane. The DeltaV124 mutant showed the strongest defect of all single mutants. Mutants in which six residues of the tryptophan-rich motif were changed showed no residual CcmC activity. This phenotype was independent of the level of ccmD expression. Our results demonstrate the functional importance of the tryptophan-rich motif for haem transfer to CcmE. We propose that the three membrane proteins CcmC, CcmD and CcmE interact directly with each other, establishing a cytoplasm to periplasm haem delivery pathway for cytochrome c maturation.     

Solution structure and characterization of the heme chaperone CcmE

The covalent attachment of the heme cofactor in c-type cytochromes is a surprisingly complex process, which in bacteria involves a number of different proteins. Among the latter, the ccmE gene product is known to perform a key role in the heme delivery pathway in Gram-negative bacteria. The solution structure of the soluble domain of apo-CcmE from Shewanella putrefaciens was determined through NMR spectroscopy on a 13C,15N-labeled sample. The structure is characterized by a compact core with large regions of beta structure, while the N-terminal and C-terminal regions are essentially unstructured. The overall folding is similar to that of the so-called oligo-binding proteins (OB fold). Solvent-exposed aromatic residues, conserved in all CcmE homologues, have been found in the proximity of His131, the putative heme-binding residue, that could have a role in the interaction with heme. No interaction between CcmE and heme, as well as between CcmE and holocytochrome c, could be detected in vitro by electronic spectroscopy or by NMR. The data available suggest that the heme transfer process is likely to involve a heterooligomeric protein complex and occur under a tight enzymatic control.     

CcmI subunit of CcmFHI heme ligation complex functions as an apocytochrome c chaperone during c-type cytochrome maturation

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

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.     

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.     

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.     

Comparing substrate specificity between cytochrome c maturation and cytochrome c heme lyase systems for cytochrome c biogenesis

Hemes c are characterized by their covalent attachment to a polypeptide via a widely conserved CXXCH motif. There are multiple biological systems that facilitate heme c biogenesis. System I, the cytochrome c maturation (CCM) system, is found in many bacteria and is commonly employed in the maturation of bacterial cytochromes c in Escherichia coli-based expression systems. System III, cytochrome c heme lyase (CCHL), is an enzyme found in the mitochondria of many eukaryotes and is used for heterologous expression of mitochondrial holocytochromes c. To test CCM specificity, a series of Hydrogenobacter thermophilus cytochrome c(552) variants was successfully expressed and matured by the CCM system with CX(n)CH motifs where n = 1-4, further extending the known substrate flexibility of the CCM system by successful maturation of a bacterial cytochrome c with a novel CXCH motif. Horse cytochrome c variants with both expanded and contracted attachment motifs (n = 1-3) were also tested for expression and maturation by both CCM and CCHL, allowing direct comparison of CCM and CCHL substrate specificities. Successful maturation of horse cytochrome c by CCHL with an extended CXXXCH motif was observed, demonstrating that CCHL shares the ability of CCM to mature hemes c with extended heme attachment motifs. In contrast, two single amino acid mutants were found in horse cytochrome c that severely limit maturation by CCHL, yet were efficiently matured with CCM. These results identify potentially important residues for the substrate recognition of CCHL.     

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.     

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.     

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.     

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.     

The interaction between heme and protein in cytochrome c1

The hormonal regulation of two regulatory enzymes of fatty acid synthesis acetyl-CoA carboxylase (EC 6.4.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49), has been investigated in human diploid fibroblasts. There was a 35% increase in acetyl-CoA carboxylase activity, 72 h following addition of 10 microU/ml insulin to the culture medium. Addition of 1 microgram/ml of 3,3'5-triiodothyronine for 72 h resulted in an increase in acetyl-CoA carboxylase activity to 166% of the controls. The simultaneous addition of 1 microgram/ml triiodothyronine and 10 mU/ml insulin caused the enzyme activity to rise to 240% of the controls. A dose-dependent reduction in acetyl-CoA carboxylase activity was brought about by 1 X 10(-4) to 1 X 10(-3) M dibutyryl cyclic AMP. The earliest effect of dibutyryl cyclic AMP was observed within 24 h. Glucose-6-phosphate dehydrogenase followed qualitatively the same pattern of response, whereas the constitutive enzyme, lactate dehydrogenase (EC 1.1.1.27), did not show significant changes in these experiments. The data demonstrate common features of hormonal regulation of lipogenesis in human fibroblasts with liver and adipose tissue and substantiate the growing evidence that thyroid hormones are of major importance for the regulation of this process.     

Electrostatic analysis of the interaction of cytochrome c with native and dimethyl ester heme substituted cytochrome b5

The stability of the complex formed between cytochrome c and dimethyl ester heme substituted cytochrome b5 (DME-cytochrome b5) has been determined under a variety of experimental conditions to evaluate the role of the cytochrome b5 heme propionate groups in the interaction of the two native proteins. Interaction between cytochrome c and the modified cytochrome b5 was found to produce a difference spectrum in the visible range that is very similar to that generated by the interaction of the native proteins and that can be used to monitor complex formation between the two proteins. At pH 8 [25 degrees C (HEPPS), I = 5 mM], DME-cytochrome b5 and cytochrome c form a 1:1 complex with an association constant KA of 3 (1) X 10(6) M-1. This pH is the optimal pH for complex formation between these two proteins and is significantly higher than that observed for the interaction between the two native proteins. The stability of the complex formed between DME-cytochrome b5 and cytochrome c is strongly dependent on ionic strength with KA ranging from 2.4 X 10(7) M-1 at I = 1 mM to 8.2 X 10(4) M-1 at I = 13 mM [pH 8.0 (HEPPS), 25 degrees C]. Calculations for the native, trypsin-solubilized form of cytochrome b5 and cytochrome c confirm that the intermolecular complex proposed by Salemme [Salemme, F. R. (1976) J. Mol. Biol. 102, 563] describes the protein-protein orientation that is electrostatically favored at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC

Here we show that during the meiotic maturation of Xenopus oocytes, histone H3 becomes phosphorylated on serine-10 at about the time of maturation promoting factor activation and meiosis I entry. However, overexpression of cAMP-dependent protein kinase that blocks entry into M phase, also leads to massive serine-10 phosphorylation of histone H3 in intact Xenopus oocytes but does not cause chromosome condensation. We also show that the phosphorylation of histone H3 during oocyte maturation requires the activation of the mitogen-activated protein kinase/p90Rsk pathway. Our results indicate that in G2-arrested oocytes, which are about to enter M phase, histone H3 phosphorylation is not sufficient for chromosome condensation.