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.     

A cytochrome b562 variant with a c-type cytochrome CXXCH heme-binding motif as a probe of the Escherichia coli cytochrome c maturation system

Cytochrome b562 is a periplasmic Escherichia coli protein; previous work has shown that heme can be attached covalently in vivo as a consequence of introduction of one or two cysteines into the heme-binding pocket. A heterogeneous mixture of products was obtained, and it was not established whether the covalent bond formation was catalyzed or spontaneous. Here, we show that coexpression from plasmids of a variant of cytochrome b562 containing a CXXCH heme-binding motif with the E. coli cytochrome c maturation (Ccm) proteins results in an essentially homogeneous product that is a correctly matured c-type cytochrome. Formation of the holocytochrome was accompanied by substantial production of its apo form, in which, for the protein as isolated, there is a disulfide bond between the two cysteines in the CXXCH motif. Following addition of heme to reduced CXXCH apoprotein, spontaneous covalent addition of heme to polypeptide occurred in vitro. Strikingly, the spectral properties were very similar to those of the material obtained from cells in which presumed uncatalyzed addition of heme (i.e. in the absence of Ccm) had been observed. The major product from uncatalyzed heme attachment was an incorrectly matured cytochrome with the heme rotated by 180 degrees relative to its normal orientation. The contrast between Ccm-dependent and Ccm-independent covalent attachment of heme indicates that the Ccm apparatus presents heme to the protein only in the orientation that results in formation of the correct product and also that heme does not become covalently attached to the apocytochrome b562 CXXCH variant without being handled by the Ccm system in the periplasm. The CXXCH variant of cytochrome b562 was also expressed in E. coli strains deficient in the periplasmic reductant DsbD or oxidant DsbA. In the DsbA- strain under aerobic conditions, c-type cytochromes were made abundantly and correctly when the Ccm proteins were expressed. This contrasts with previous reports indicating that DsbA is essential for cytochrome c biogenesis in E. coli.     

Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli

c-Type cytochromes are located partially or completely in the periplasm of gram-negative bacteria, and the heme prosthetic group is covalently bound to the protein. The cytochrome c maturation (Ccm) multiprotein system is required for transport of heme to the periplasm and its covalent linkage to the peptide. Other cytochromes and hemoglobins contain a noncovalently bound heme and do not require accessory proteins for assembly. Here we show that Bradyrhizobium japonicum cytochrome c550 polypeptide accumulation in Escherichia coli was heme dependent, with very low levels found in heme-deficient cells. However, apoproteins of the periplasmic E. coli cytochrome b562 or the cytosolic Vitreoscilla hemoglobin (Vhb) accumulated independently of the heme status. Mutation of the heme-binding cysteines of cytochrome c550 or the absence of Ccm also resulted in a low apoprotein level. These levels were restored in a degP mutant strain, showing that apocytochrome c550 is degraded by the periplasmic protease DegP. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b562 (c-b562) resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. This variant polypeptide was stable in heme-deficient cells but was degraded by DegP in the absence of Ccm. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm dependent nonetheless. The data show that the cytochrome c heme-binding motif is an instability element and that stabilization by Ccm does not require ligation of the heme moiety to the protein.     

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.     

Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function.

Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.     

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.     

The Escherichia coli cytochrome c maturation (Ccm) system does not detectably attach heme to single cysteine variants of an apocytochrome c

Cytochromes c are typically characterized by the covalent attachment of heme to polypeptide through two thioether bonds with the cysteine residues of a Cys-Xaa-Xaa-Cys-His peptide motif. In many Gram-negative bacteria, the heme is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (Ccm) proteins. Exceptionally, Hydrogenobacter thermophilus cytochrome c(552), which has a normal CXXCH heme-binding motif, and variants with AXXCH, CXXAH, and AXXAH motifs, can be expressed as stable holocytochromes in the cytoplasm of Escherichia coli. By targeting these proteins to the periplasm using a signal peptide, with or without co-expression of the Ccm proteins, we have assessed the ability of the Ccm system to attach heme to proteins with no, one, or two cysteine residues in the heme-binding motif. Only the wild-type protein, with two cysteines, was effectively processed and thus accumulated in the periplasm as a holocytochrome. This is strong evidence for disulfide bond formation involving the two cysteine residues of apocytochrome c as an intermediate in Ccm-type Gram-negative bacterial cytochrome c biogenesis and/or that only a pair of cysteines can be recognized by the heme attachment apparatus.     

Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis.

The genes coding for the photosynthetic reaction center cytochrome c subunit (pufC) and the soluble cytochrome c2 (cycA) from the purple non-sulfur bacterium Rhodopseudomonas viridis were expressed in Escherichia coli. Biosynthesis of the reaction center cytochrome without a signal peptide resulted in the formation of inclusion bodies in the cytoplasm amounting to 14% of the total cellular protein. A series of plasmids coding for the cytochrome subunit with varying N-terminal signal peptides was constructed in attempts to achieve translocation across the E. coli cytoplasmic membrane and heme attachment. However, the two major recombinant proteins with N-termini corresponding to the signal peptide and the cytochrome were synthesized in E. coli as non-specific aggregates without heme incorporation. An increased ratio of precursor as compared to 'processed' apo-cytochrome was obtained when expression was carried out in a proteinase-deficient strain. Cytochrome c2 from R. viridis was synthesized in E. coli as a precursor associated with the cytoplasmic membrane. An expression plasmid was designed encoding the N-terminal part of the 33 kDa precursor protein of the oxygen-evolving complex of Photosystem II from spinach followed by cytochrome c2. Two recombinant proteins without heme were found to aggregate as inclusion bodies with N-termini corresponding to the signal peptide and the mature 33 kDa protein.     

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.     

Mutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppress cytochrome c deficiency of CcdA-defective Bacillus subtilis cells

Cytochromes of the c type in the gram-positive bacterium Bacillus subtilis are all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane. They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds. The cysteinyls in the heme-binding site (CXXCH) in apocytochrome c must be reduced in order for the covalent attachment of the heme to occur. It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane. Strains deficient in the CcdA protein are defective in cytochrome c and spore synthesis. We have discovered that mutations in the bdbC and bdbD genes can suppress the defects caused by lack of CcdA. BdbC and BdbD are thiol-disulfide oxidoreductases. Our experimental findings indicate that these B. subtilis proteins functionally correspond to the well-characterized Escherichia coli DsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.     

Heme concentration dependence and metalloporphyrin inhibition of the system I and II cytochrome c assembly pathways

Studies have indicated that specific heme delivery to apocytochrome c is a critical feature of the cytochrome c biogenesis pathways called system I and II. To determine directly the heme requirements of each system, including whether other metal porphyrins can be incorporated into cytochromes c, we engineered Escherichia coli so that the natural system I (ccmABCDEFGH) was deleted and exogenous porphyrins were the sole source of porphyrins (Delta hemA). The engineered E. coli strains that produced recombinant system I (from E. coli) or system II (from Helicobacter) facilitated studies of the heme concentration dependence of each system. Using this exogenous porphyrin approach, it was shown that in system I the levels of heme used are at least fivefold lower than the levels used in system II, providing an important advantage for system I. Neither system could assemble holocytochromes c with other metal porphyrins, suggesting that the attachment mechanism is specific for Fe protoporphyrin. Surprisingly, Zn and Sn protoporphyrins are potent inhibitors of the pathways, and exogenous heme competes with this inhibition. We propose that the targets are the heme binding proteins in the pathways (CcmC, CcmE, and CcmF for system I and CcsA for system II).     

Cytochrome c biogenesis in mitochondria

As part of the respiratory chain, c-type cytochromes are essential electron transporters. They are characterized by the covalent attachment of a heme prosthetic group. The biogenesis of these proteins includes all the processes leading to this fixation. Yeast and animals have evolved a comparatively simple mechanism relying on cytochrome c heme lyases. In contrast, plant mitochondria have kept a maturation pathway inherited from their prokaryote ancestor. It involves Ccm proteins encoded in both the nuclear and the mitochondrial genomes of plants. These proteins compose a heme delivery pathway, include an ABC transporter, a redox protein and a putative heme lyase.     

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.     

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.     

Soluble cytochrome composition of the purple phototrophic bacterium, Rhodopseudomonas sphaeroides ATCC 17023

A detailed study of the soluble cytochrome composition of Rhodopseudomonas sphaeroides (ATCC 17023) indicates that there are five c-type cytochromes and one b-type cytochrome present. The molecular weights, heme contents, amino acid compositions, isoelectric points, and oxidation-reduction potentials were determined and the proteins were compared with those from other bacterial sources. Cytochromes c2 and c' have previously been well characterized. Cytochrome c-551.5 is a diheme protein which has a very low redox potential, similar to certain purple bacterial and algal cytochromes. Cytochrome c-554 is an oligomer, which is spectrally similar to the low-spin isozyme of cytochrome c' found in other purple bacteria (e.g., Rhodopseudomonas palustris cytochrome c-556). An unusual high-spin c-type heme protein has also been isolated. It is spectrally distinguishable from cytochrome c' and binds a variety of heme ligands including oxygen. A large molecular-weight cytochrome b-558 is also present which appears related to a similar protein from Rhodospirillum rubrum, and the bacterioferritin from Escherichia coli. None of the soluble proteins appear to be related to the abundant membrane-bound c-type cytochrome in Rps. sphaeroides which has a larger subunit molecular weight similar to mitochondrial cytochrome c1 and chloroplast cytochrome f.     

Immunological analysis of the heme proteins present in aerobically grown Escherichia coli.

Immunological methods were used to obtain information about Escherichia coli heme proteins. There is a membrane-bound catalase which consists of a single subunit (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis) which is also present in the soluble fraction. Antibodies raised against purified, soluble cytochrome b562 showed that this cytochrome is not related to any of the membrane-bound cytochromes, including the b562 component of the cytochrome o complex. Cytochrome b556 is immunologically unrelated to the cytochrome b556 NR associated with the nitrate reductase system. Cytochrome b556 and cytochrome o are not present in a constant ratio in the membrane.     

Bioelectrocatalysis by redox enzymes at modified electrodes

Self-assembled monolayers of thiolated compounds are used as promoters for protein-electrode reactions. They provide an anchor group based on thiol chemisorptions and also a functional group for effective interaction with the protein. These interactions are often governed by electrostatic attraction. For example, for positively charged proteins, such as cytochrome c and the selenoprotein glutathione peroxidase, mercaptoalkanoic acids have been used. Clay modification of the electrode surface has been found to facilitate the heterogeneous electron transfer process for heme proteins, e.g. cytochrome c, cytochrome P450 and myoglobin. Interestingly, nucleic acids at carbon electrodes and thiol-modified double stranded oligonucleotides act as promoters of the redox communication to proteins, whereas the mechanism is still subject to controversy interpretations. By interacting the protein immobilised at the electrode with species in solution, signal chains have been constructed. The interaction can result in a simple co-ordination or redox reaction, depending on the nature of the reaction partners. For analytical purposes, e.g. biosensors, the electrochemical redox conversion of the immobilised protein is evaluated.     

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.     

TPR domain of NrfG mediates complex formation between heme lyase and formate-dependent nitrite reductase in Escherichia coli O157:H7

Escherichia coli synthesize C-type cytochromes only during anaerobic growth in media supplemented with nitrate and nitrite. The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two separate periplasmic enzymes, nitrate reductase and nitrite reductase. The nitrite reductase involved, NrfA, contains cytochrome C and is synthesized coordinately with a membrane-associated cytochrome C, NrfB, during growth in the presence of nitrite or in limiting nitrate concentrations. The genes NrfE, NrfF, and NrfG are required for the formate-dependent nitrite reduction pathway, which involves at least two C-type cytochrome proteins, NrfA and NrfB. The NrfE, NrfF, and NrfG genes (heme lyase complex) are involved in the maturation of a special C-type cytochrome, apocytochrome C (apoNrfA), to cytochrome C (NrfA) by transferring a heme to the unusual heme binding motif of the Cys-Trp-Ser-Cys-Lys sequence in apoNrfA protein. Thus, in order to further investigate the roles of NrfG in the formation of heme lyase complex (NrfEFG) and in the interaction between heme lyase complex and formate-dependent nitrite reductase (NrfA), we determined the crystal structure of NrfG at 2.05 A. The structure of NrfG showed that the contact between heme lyase complex (NrfEFG) and NrfA is accomplished via a TPR domain in NrfG which serves as a binding site for the C-terminal motif of NrfA. The portion of NrfA that binds to TPR domain of NrfG has a unique secondary motif, a helix followed by about a six-residue C-terminal loop (the so called "hook conformation"). This study allows us to better understand the mechanism of special C-type cytochrome assembly during the maturation of formate-dependent nitrite reductase, and also adds a new TPR binding conformation to the list of TPR-mediated protein-protein interactions.     

Cytochrome c biogenesis in mitochondria--Systems III and V

In c-type cytochromes, heme becomes covalently attached to the polypeptide chain by a reaction between the vinyl groups of the heme and cysteine thiols from the protein. There are two such cytochromes in mitochondria: cytochrome c and cytochrome c(1). The heme attachment is a post-translational modification that is catalysed by different biogenesis proteins in different organisms. Three types of biogenesis system are found or predicted in mitochondria: System I (the cytochrome c maturation system); System III (termed holocytochrome c synthase (HCCS) or heme lyase); and System V. This review focuses primarily on cytochrome c maturation in mitochondria containing HCCS (System III). It describes what is known about the enzymology and substrate specificity of HCCS; the role of HCCS in human disease; import of HCCS into mitochondria; import of apocytochromes c and c(1) into mitochondria and the close relationships with HCCS-dependent heme attachment; and the role of the fungal cytochrome c biogenesis accessory protein Cyc2. System V is also discussed; this is the postulated mitochondrial cytochrome c biogenesis system of trypanosomes and related organisms. No cytochrome c biogenesis proteins have been identified in the genomes of these organisms whose c-type cytochromes also have a unique mode of heme attachment.