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

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

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.     

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.     

Cytochrome c maturation. The in vitro reactions of horse heart apocytochrome c and Paracoccus dentrificans apocytochrome c550 with heme

C-type cytochromes are characterized by having the heme moiety covalently attached via thioether bonds between the heme vinyl groups and the thiols of conserved cysteine residues of the polypeptide chain. Previously, we have shown the in vitro formation of Hydrogenobacter thermophilus cytochrome c(552) (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872-7876). In this work we report that thioether bonds can form spontaneously in vitro between heme and the apocytochromes c from horse heart and Paracoccus denitrificans via b-type cytochrome intermediates. Both apocytochromes, but not the holo forms, bind 8-anilino-1-naphthalenesulfonate, indicating that the apoproteins each have an affinity for a hydrophobic ligand. Furthermore, for both apocytochromes c an intramolecular disulfide can form between the cysteines of the CXXCH motif that is characteristic of c-type cytochromes. In vitro reaction of these apocytochromes c with heme to yield holocytochromes c, and the tendency to form a disulfide, have implications for the different systems responsible for cytochrome c maturation in vivo in various organisms.     

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.     

Characterization of recombinant horse cytochrome c synthesized with the assistance of Escherichia coli cytochrome c maturation factors

Cytochromes c are characterized by the presence of a protoporphyrin IX group covalently attached to the polypeptide via one or two thioether bonds to Cys side chains. The heme attachment process, known as cytochrome c maturation, occurs posttranslationally in the periplasm (for bacterial cytochromes c) or in the mitochondrial intermembrane space (for eukaryotic cytochromes c) through a pathway dependent on the organism. It is demonstrated in this work that a mitochondrial cytochrome c expressed in Escherichia coli that undergoes maturation under control of the E. coli cytochrome c maturation factors achieves a native-like structure and stability. The recombinant protein is characterized spectroscopically (by circular dichroism (CD), absorption, and nuclear magnetic resonance (NMR) spectroscopy) and it is verified that the heme and its environment are indistinguishable from authentic horse cytochrome c. Mass spectrometry reveals that the recombinant protein is not acetylated at the N terminus, however, no significant effect on protein structure or stability is detected as a result.     

A strategic protein in cytochrome c maturation: three-dimensional structure of CcmH and binding to apocytochrome c

CcmH (cytochromes c maturation protein H) is an essential component of the assembly line necessary for the maturation of c-type cytochromes in the periplasm of Gram-negative bacteria. The protein is a membrane-anchored thiol-oxidoreductase that has been hypothesized to be involved in the recognition and reduction of apocytochrome c, a prerequisite for covalent heme attachment. Here, we present the 1.7A crystal structure of the soluble periplasmic domain of CcmH from the opportunistic pathogen Pseudomonas aeruginosa (Pa-CcmH*). The protein contains a three-helix bundle, i.e. a fold that is different from that of all other thiol-oxidoreductases reported so far. The catalytic Cys residues of the conserved LRCXXC motif (Cys(25) and Cys(28)), located in a long loop connecting the first two helices, form a disulfide bond in the oxidized enzyme. We have determined the pK(a) values of these 2 Cys residues of Pa-CcmH* (both >8) and propose a possible mechanistic role for a conserved Ser(36) and a water molecule in the active site. The interaction between Pa-CcmH* and Pa-apocyt c(551) (where cyt c(551) represents cytochrome c(551)) was characterized in vitro following the binding kinetics by stopped-flow using a Trp-containing fluorescent variant of Pa-CcmH* and a dansylated peptide, mimicking the apocytochrome c(551) heme binding motif. The kinetic results show that the protein has a moderate affinity to its apocyt substrate, consistent with the role of Pa-CcmH as an intermediate component of the assembly line for c-type cytochrome biogenesis.     

Evidence for formation of two thioether bonds to link heme to apocytochrome c by partially purified cytochrome c synthetase

Cytochrome c synthetase has been solubilized from yeast mitochondria using Triton X-100 and fractionated with ammonium sulfate. Use of this partially purified enzyme has permitted us to isolate a quantity of iso-1-cytochrome c formed from 125I-labeled apocytochrome c and hemin in the presence of a NADPH-generating system. Visible absorption spectra (pH 8.0 or 5.0) including alpha, beta, and Soret bands and their molar absorption coefficients of this enzymatically synthesized cytochrome c in the oxidized and reduced states are the same, within experimental error, as those of native cytochrome c. Pyridine ferrohemochrome (pH 13) of the synthesized species also exhibits the same alpha and beta bands as those of iso-l-cytochrome c and similar to those reported for heme peptides of cytochrome c. If only one or no thioether bond were formed between the two vinyl side groups of heme and the cysteine residues of apocytochrome c, all these alpha and beta bands would have shifted to red (Pettigrew, G. W., Leaver, J. L., Meyer, T. E., and Ryle, T. E. (1975) Biochem J. 147, 291-302). Thus, two thioether bonds appear to be formed to link heme to apocytochrome c by cytochrome c synthetase, completing information of the three-dimensional structure of cytochrome c.     

High yield of Methylophilus methylotrophus cytochrome c by coexpression with cytochrome c maturation gene cluster from Escherichia coli

Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c" isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS-PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and (1)H-NMR spectroscopy. The success in expressing the mature cytochrome c in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship.     

The interplay between the disulfide bond formation pathway and cytochrome c maturation in Escherichia coli

Heme attachment to c-type cytochromes in bacteria requires cysteine thiols in the CXXCH motif of the protein. The involvement of the periplasmic disulfide generation system in this process remains unclear. We undertake a systematic evaluation of the role of DsbA and DsbD in cytochrome c biogenesis in Escherichia coli and show unequivocally that DsbA is not essential for holocytochrome production under aerobic or anaerobic conditions. We also prove that DsbD is important but not essential for maturation of c-type cytochromes. We discuss the findings in the context of a model in which heme attachment to, and oxidation of, the apocytochrome are competing processes.     

A 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.     

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.     

The acidic nature of the CcmG redox-active center is important for cytochrome c maturation in Escherichia coli

Cytochrome c biogenesis in Escherichia coli is a complex process requiring at least eight genes (ccmABCDEFGH). One of these genes, ccmG, encodes a thioredoxin-like protein with unusually specific redox activity. Here, we investigate the basis for CcmG function and demonstrate the importance of acidic residues surrounding the redox-active center.     

The low-spin heme site of cytochrome o from Escherichia coli is promiscuous with respect to heme type.

Cytochrome o of Escherichia coli is able to incorporate two different structures of heme, either heme B (protoheme) or heme O, in its low-spin heme site. In contrast, the heme of the binuclear O2 reduction site is invariably heme O. Heme O is a newly discovered heme that is related to heme A, but with the formyl group of the latter replaced by methyl. Enzyme isolated from wild type E. coli has predominantly heme B in the low-spin site, whereas enzyme isolated from various overexpressing strains contains both types of enzyme in different proportions. In some strains, 70% of the enzyme has heme O in the low-spin site. Despite this variation in the structure of one of the prosthetic groups, the enzymatic activity and polypeptide composition of the enzyme remain virtually constant. EPR and activity data both indicate that heme B and heme O occupy the same low-spin heme site in the enzyme. With heme O in this site, the alpha-absorption band is narrower and further to the blue, and the Em,7 is lower, than when there is heme B in the site. In contrast to previous proposals, we show here that the enzyme does not exhibit significant spectral interactions between the hemes. The structural heterogeneity of the low-spin heme accounts for the variation in the optical spectra and redox properties of the enzyme as isolated from different strains of E. coli.     

Recognition and binding of apocytochrome c to P. aeruginosa CcmI,a component of cytochrome c maturation machinery

The biogenesis of c-type cytochromes (Cytc) is a process that in Gram-negative bacteria demands the coordinated action of different periplasmic proteins (CcmA-I), whose specific roles are still being investigated. Activities of Ccm proteins span from the chaperoning of heme b in the periplasm to the specific reduction of oxidized apocytochrome (apoCyt) cysteine residues and to chaperoning and recognition of the unfolded apoCyt before covalent attachment of the heme to the cysteine thiols can occur. We present here the functional characterization of the periplasmic domain of CcmI from the pathogen Pseudomonas aeruginosa (Pa-CcmI*). Pa-CcmI* is composed of a TPR domain and a peculiar C-terminal domain. Pa-CcmI* fulfills both the ability to recognize and bind to P. aeruginosa apo-cytochrome c551 (Pa-apoCyt) and a chaperoning activity towards unfolded proteins, as it prevents citrate synthase aggregation in a concentration-dependent manner. Equilibrium and kinetic experiments with Pa-CcmI*, or its isolated domains, with peptides mimicking portions of Pa-apoCyt sequence allow us to quantify the molecular details of the interaction between Pa-apoCyt and Pa-CcmI*. Binding experiments show that the interaction occurs at the level of the TPR domain and that the recognition is mediated mainly by the C-terminal sequence of Pa-apoCyt. The affinity of Pa-CcmI* to full-length Pa-apoCyt or to its C-terminal sequence is in the range expected for a component of a multi-protein complex, whose task is to receive the apoCyt and to deliver it to other components of the apoCyt:heme b ligation protein machinery.     

Change in charge of an unvaried heme contact residue does not cause a major change of conformation in cytochrome c.

The structure of the Ala38 variant of yeast iso-1-cytochrome c, in which the previously unchanged Arg38 has been replaced, has been characterised by NMR. The NMR data indicate that the structure of the Ala38 variant is very similar to that of the wild type protein. In particular, the heme environment and interactions of the heme macrocycle are shown to be preserved. Analysis of the chemical shift perturbations to the resonances of Ile35 is shown to be consistent with the change in charge at position 38. The only significant area of conformational change detected was at residues 39 and 58, close to the site of modification. Therefore the redox potential change accompanying the modification [1988, Biochemistry 28, 3188-3197] appears to be a direct consequence of the altered side-chain of residue 38 and not a result of secondary conformational changes induced by the modification.     

Cytochrome c biogenesis in Campylobacter jejuni requires cytochrome c6 (CccA; Cj1153) to maintain apocytochrome cysteine thiols in a reduced state for haem attachment

The microaerophilic food‐borne pathogen Campylobacter jejuni uses complex cytochrome‐rich respiratory chains for growth and host colonisation. Cytochrome c biogenesis requires haem ligation to reduced apocytochrome cysteines, catalysed by the cytochrome c synthase, CcsBA. While ccsBA could not be deleted, we showed that the thiol reductase DsbD and the CcsX homologue Cj1207 are involved in, but not essential for, cytochromes c biogenesis. Mutant phenotypic analyses and biochemical studies with purified proteins revealed that the mono‐haem c‐type cytochromes Cj1153 (CccA) and Cj1020 (CccB) and the di‐haem Cj0037 (CccC) are electron donors to the cb‐oxidase (CcoNOQP), with CccC being more efficient than CccA. Remarkably, cccA deletion or site‐directed mutagenesis resulted in an almost complete loss of all other c‐type cytochromes. Cytochrome c structural and biogenesis genes were still transcribed in the cccA deletion mutant and the quinol oxidase genes (cioAB) were up‐regulated. Cytochrome c production could be rescued in this mutant by growth with exogenous dithiothreitol or L‐cysteine, suggesting that in the absence of CccA, apocytochrome c haem binding motifs become oxidised, preventing haem attachment. Our results identify CccA, the most abundant periplasmic c‐type cytochrome in C. jejuni, as a novel and unexpected protein required for cytochrome c biogenesis in this pathogen.     

The Escherichia coli biofilm-promoting protein Antigen 43 does not contribute to intestinal colonization

Abstract Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells.