Cytochrome c Biogenesis

JULIE M. STEVENS* AND STUART J. FERGUSON

[SECTION EDITOR: T. BEGLEY]

Posted November 7, 2008

Department of Biochemistry, University of Oxford, Oxford, United Kingdom

*Corresponding author. Mailing address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom. Phone: 44 (0) 1865 275 242, Fax: 44 (0) 1865 275 259, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

CYTOCHROMES c AND THEIR BIOGENESIS

Cytochromes c are defined as heme-containing proteins that are covalently linked to the heme cofactor (Fe-protoporphyrin IX) by, in general, two thioether bonds. Heme is used as a cofactor in a wide variety of proteins, and in many cases, such as in hemoglobin and b-type cytochromes, the heme is noncovalently bound. However, many cytochromes in various respiratory chains are of the c type, in which heme is found covalently attached to the cytochrome polypeptide. Several explanations for the covalent attachment have been proposed previously (2, 8), including heme retention, protein stabilization, and the modulation of the heme reduction potential, as well as the enabling of high-density heme packing within multiheme cytochromes, presumably to optimize electron transfer rates, although this is not yet fully substantiated. The latter possibility appears to be the most likely explanation, as demonstrated by the structures of multiheme-containing cytochromes c (34). In cytochromes c, the heme is attached via bonds between the heme vinyl groups and the cysteine thiols in characteristic CXXCH protein motifs, as shown in Fig. 1. The histidine side chain almost always acts as a ligand for the heme iron.

Fig. 1Covalent heme attachment to the cysteine side chains of a c-type cytochrome.

Escherichia coli employs several c-type cytochromes, which are found in the periplasm or on the periplasmic side of the cytoplasmic membrane; they are used for respiration under different growth conditions. These cytochromes include components of nitrate reductase systems (NapB and NapC), trimethylamine N-oxide reductase systems (TorC and TorY), and formate-dependent nitrite reductase systems (NrfA and NrfB) which function under anaerobic conditions when the relevant electron acceptor is present (26). In addition, a cytochrome c peroxidase has been identified (35). All E. coli c-type cytochromes are multiheme cytochromes; E. coli does not have a monoheme cytochrome c of the kind found in mitochondria. Unlike many other bacterial species, E. coli does not use cytochromes c for aerobic growth; it lacks a bc₁ complex and utilizes cytochrome bo₃ and cytochrome bd as oxidases. In common with many bacterial species, E. coli makes a greater variety of c-type cytochromes than mammals, which have a relatively inflexible respiratory chain using only oxygen as the terminal electron acceptor. Another characteristic of bacterial cytochromes c in general is that they can contain more than one heme per polypeptide; in some cases, the number of hemes can be remarkably high (27 in a Geobacter cytochrome, for example [34]). The need to produce multiheme cytochromes c probably accounts in part for the intricacies of the process of covalent heme attachment to these proteins, which is remarkably complex in E. coli. The eight proteins required, CcmABCDEFGH, are expressed from the ccm (cytochrome c maturation) operon; they perform the posttranslational heme attachment process (46). A very similar operon exists in Salmonella species, but the E. coli system is by far the best-studied. Below, we summarize what is known about the components of this heme attachment system, which is known as cytochrome c biogenesis system I.

THE E.COLI Ccm SYSTEM

The eight proteins encoded by the ccm operon either are integral membrane proteins or are anchored to the cytoplasmic membrane with soluble periplasmic domains (as shown in Fig. 2). Structures of some of the domains of these proteins have been solved (as indicated in the figure), which has given some clues into their functions. Fifteen years of research into these proteins have provided some insights into how the heme attachment reaction is performed, albeit several elements of the pathway remain poorly understood, partly because of the challenges of studying integral membrane proteins.

Fig. 2Cytochrome c biogenesis system of E. coli showing the eight Ccm proteins and the disulfide bond (Dsb) proteins DsbA, DsbB, and DsbD. The cysteine residues in the thiol oxidoreductases are shown in yellow; there is a conserved pair of cysteines in the transmembrane domain of DsbD that are involved in the disulfide cascade. The identity of the transported substrate of CcmAB (and whether there is one) is unknown. The heme-binding histidine of CcmE is shown in blue. CcmC facilitates heme provision to CcmE, and CcmF is thought to facilitate heme release and transfer to the apocytochrome. DsbA oxidizes the apocytochrome, which is subsequently reversed by an electron flow from DsbD via CcmG. DsbB transfers electrons from DsbA to ubiquinone in the membrane. It is not known how heme is exported from its cytoplasmic site of synthesis. The structures shown, which are from E. coli unless otherwise indicated, are from the Protein Data Bank entries for the following proteins and domains: CcmE (1SR3) (19), CcmH (from Pseudomonas aeruginosa; 2E2E) (16), CcmG (1KNG) (18), the N-terminal domain of DsbD (1I6P) (24), the C-terminal domain of DsbD (1UC7) (28), TrxA (thioredoxin; 2TRX) (27), and DsbAB (2H17) (25). The cytochrome c shown is cytochrome c₅₅₀ from Paracoccus denitrificans (155C) (47). The structures were rendered in PyMOL (W. L. DeLano, The PyMOL Molecular Graphics System, 2002; http://www.pymol.org).

Transport of the Substrates to the Periplasm

The attachment of heme to cytochromes c occurs in the periplasm, and so the apoprotein must be transported across the cytoplasmic membrane; this step is mediated by the Sec system (45), which transports unfolded proteins across the membrane. The question of how heme reaches the periplasm following its synthesis in the cytoplasm remains unanswered. The proteins CcmAB, which constitute an ABC protein, were the prime candidates for the heme transport function; several lines of evidence now indicate that they do not transport heme (12, 13, 21). The precise function of CcmAB has not been resolved, but it may not be a transporter. ATPase activity has been demonstrated previously and appears to relate to the properties of the heme chaperone CcmE (12, 21). The annotation of CcmA and CcmB in DNA and protein databases as heme transport proteins persists, despite the lack of evidence. No protein is known to export heme across the cytoplasmic membrane to the periplasm in E. coli, although it appears that a dipeptide permease is able to transport heme in the opposite direction (33).

Heme Processing

The protein CcmE has been found to bind heme covalently via a single bond and then transfer the heme to apocytochromes (39). This protein has become known as the heme chaperone and is one of the most studied proteins in the system (41). That CcmE binds heme covalently, but transiently, is perplexing. Possible explanations for this observation may relate to the stereochemical control of the heme attachment to the cysteines of the apocytochrome (which is specific), to mechanistic necessity, or to heme storage. Another unusual feature of CcmE is that it binds heme covalently via a histidine residue (shown in Fig. 2) to a heme vinyl group (15, 39) by a unique bond (32). The Fe of heme covalently attached to CcmE is ligated by a tyrosine residue (22, 48), and the ligation is redox dependent, which is likely to be physiologically significant as it is expected that the heme transferred to apocytochromes would be in the reduced form. It appears that heme is delivered to CcmE by the membrane protein CcmC (37). The ATPase activity of CcmAB is not required for the formation of the covalent CcmE-heme complex but is implicated in the subsequent transfer of heme to the apocytochrome. The study of ccmC in other organisms (e.g., Pseudomonas species) has been insightful, suggesting the involvement of this gene in additional phenotypes (7). CcmD is a small but essential membrane protein that appears to assist in the formation of complexes between other Ccm proteins (1). CcmF, a large integral membrane protein, is thought to be involved in the reaction resulting in heme transfer to the apocytochrome (36), but its exact role is unknown. The NrfA nitrite reductase is an exceptional c-type cytochrome that possesses a CXXCK motif. Three specialized biogenesis proteins, NrfEFG, are required for heme attachment to this motif (17); NrfE is a paralog of CcmF, and NrfF and NrfG are paralogs of the N- and C-terminal domains of CcmH. Note that in many organisms, CcmH comprises only the equivalent of the N-terminal region of E. coli CcmH, with the equivalent of the C-terminal domain being a separate protein called CcmI. The Ccm proteins in Salmonella species are very similar to those in E. coli.

Redox Control of the CXXCH Cytochrome Motif

The cysteine thiols in the apocytochrome CXXCH motif have a tendency to form disulfides (14), which presumably occurs in the oxidizing environment of the periplasm, due at least partly to the presence of the strong oxidant DsbA. The DsbA protein is maintained in its oxidized form by the membrane protein DsbB (25) by electron transfer to ubiquinone; the structures are shown in Fig. 2. DsbA is required in the periplasm for the production of extracytoplasmic disulfide bond-containing proteins (pili and secretion machinery proteins), but this necessity has to be counterbalanced by reducing proteins because an apocytochrome containing a disulfide cannot bind heme covalently. For this purpose, the three-domain membrane protein DsbD provides reductant to periplasmic thiol groups (20) with electrons it acquires from thioredoxin in the cytoplasm (38). The protein CcmG acts as a junction between the Ccm system and the reductant provision pathway by interacting with the N-terminal domain of DsbD (42). CcmG has a thioredoxin-like fold (18) and is thought, along with the protein CcmH, to reduce a disulfide formed within the CXXCH motif of apocytochromes. CcmH has two domains, one containing a pair of cysteines that are thought to be involved in the thiol reduction pathway. The structure of the protein NrfG (16), which is a paralog of the C-terminal domain of CcmH that functions in heme attachment to an alternate heme-binding motif (CXXCK) found in a nitrite reductase, suggests that CcmH contains a tetratricopeptide domain that interacts with apocytochromes, possibly giving it a chaperone-like function. In Fig. 2, the structure of NrfG is shown for the C-terminal domain of CcmH, which is likely to be similar.

Substrate Specificity of Cytochrome c Biogenesis

E. coli has the ability to produce heterologously both mono- and multiheme cytochromes c from many organisms, ranging from other bacteria to mammals. Note that under aerobic conditions, the ccm operon must be overexpressed from a plasmid for this purpose (6) because the endogenous operon is significantly expressed only in anaerobiosis (11, 44). E. coli is unable to attach heme to cytochromes normally containing a single heme-protein thioether bond (40), and there is a dependence of heme attachment upon the presence of the histidine axial heme ligand in the CXXCH motif (5). The Ccm system is surprisingly able to attach heme to short peptides (of just 12 amino acids [10]), corroborating the general principle that the biogenesis system recognizes the short linear cysteine-containing protein motif for heme attachment, which involves the highly complex posttranslational modification described here. The acquisition of the final three-dimensional structure will follow the attachment of all the heme groups.

CYTOCHROME c BIOGENESIS SYSTEMS IN OTHER ORGANISMS

It should be mentioned that far less complex systems for cytochrome c biogenesis exist in other organisms and that enterobacteria do not function as a representative model system for the process in general, although plant mitochondria use the Ccm system found in E. coli. Several reviews address the distribution in nature of cytochrome c biogenesis pathways (3, 4, 23, 29, 40, 43). In many eukaryotes, a single enzyme, called heme lyase (known as system III), catalyzes the attachment reaction; in some cases, for example, in yeast, separate lyases are produced to synthesize cytochromes c or c₁. In gram-positive (31) and some gram-negative bacteria, as well as chloroplasts (23), a system involving at least four proteins exists for cytochrome c biogenesis; this is known as system II. A fourth distinct system has been identified that attaches one of the three hemes to the cytochrome b subunit of the unusual cytochrome b₆f complex (30). A further system must occur in the Euglenozoa, in which the cytochrome c has a single thioether bond to the heme moiety and none of the known biogenesis systems are present (4, 40). The variety and distribution of cytochromes and their biogenesis systems (9) reflect their significance and centrality in cellular bioenergetics, though the necessity for and origin of the diverse biogenesis systems are enigmatic.