Redox Processes Controlling the Biogenesis of c -Type Cytochromes

Characteristics of c-type cytochromes

Cytochromes are redox active hemoproteins. Their activity is based on the change of their heme-iron valence. Mitochondria and plastids possess different types of cytochromes characterized by their heme prosthetic groups. While hemes A, B, and C are found in mitochondria, plastids only contain hemes B and C.

Mitochondrial cytochromes

The mitochondrial complex IV (cytochrome c oxidase) contains two A-type hemes (in cytochrome a and cytochrome a ₃). In complex III (ubiquinol: cytochrome c oxidoreductase or bc ₁ complex), electrons flow from ubiquinol following two paths, via the Rieske iron–sulfur protein, cytochrome c ₁ and to cytochrome c, and via two b-type cytochromes to ubiquinone. Cytochromes b and cytochromes c possess an iron protoporphyrin IX or protoheme as a prosthetic group. Iron protoporphyrin IX is the direct product of the ferrochelatase (Fig. 1). In contrast, heme A is a protoheme modified by addition of a hydroxyethylfarnesyl chain and a formyl group. Aside from the heme structure, another criterion for cytochrome classification is the type of ligand involved in the iron-atom coordination. The central iron atom is coordinated by the four nitrogen atoms of the tetrapyrrole ring and by one or two so-called axial ligands that participate in a noncovalent bond between the heme and the protein. In the case of c-type cytochromes, in addition to the axial ligands, heme is covalently bound to the apoprotein (Fig. 1). In most organisms, two thioether bonds stereospecifically connect the cysteines of a CXXCH motif in the apocytochrome to the vinyl lateral chains of the porphyrin rings A and B. In euglenozoans, the XXXCH heme binding motif of cytochromes c provides a unique covalent bond for the heme (4, 39). For mitochondrial c-type cytochromes, the histidine of the heme binding motif provides the proximal axial ligand. The sixth axial ligand is the sulfur of a methionine residue. Whereas hemes A and hemes B are embedded within the respiratory chain complexes, cytochrome c and cytochrome c ₁ expose their heme to the mitochondrial IMS (Fig. 2). Cytochrome c is a soluble protein of the IMS. Cytochrome c ₁ is anchored in the inner membrane via a C-terminal alpha helix. The N-terminal domain carrying the prosthetic group is located in the IMS.

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FIG. 1.

The actions of ferrochelatase and heme lyase result in the formation of holocytochrome c . The last step of heme synthesis involves the insertion of ferrous iron into protoporphyrin IX by the ferrochelatase. The heme B is then covalently bound to the cysteines of the CXXCH motif of the apocytochrome by the action of a heme lyase to yield heme C. The nitrogen atom of the histidine imidazole group and the sulfur atom of the methionine, which provide axial ligation of heme iron, are indicated.

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FIG. 2.

Position of cytochromes c and c ₁ in mitochondrial complex III dimer. The structure of Saccharomyces cerevisiae complex III dimer (light gray) is deduced from X-ray crystallography (PDB accession code 3CX5). For simplicity, cytochromes c and c ₁ are presented in ribbon display (dark gray) in a complex III monomer. Cytochrome c is shown bound to complex III on the intermembrane space (IMS). Cytochrome c ₁ is anchored in the mitochondrial inner membrane by a C-terminal helix with its heme-containing domain facing the IMS. The heme molecule is indicated in black in both cytochromes.

Three different pathways for c-type cytochrome biogenesis in mitochondria.

The covalent linkage of heme to apocytochrome requires dedicated machineries. Up to now, four different biogenesis pathways (I, II, III, IV) have been identified and the existence of another one (V) has been inferred from analysis of predicted proteomes (45, 57). These systems operate in prokaryotes, and eukaryotic organelles (Table 1). Their distribution do not follow the evolutionary tree of life and lateral transfer of complete biogenesis pathways was proposed (6, 15, 43).

Features of c-type cytochrome biogenesis proteins

Based on our understanding of the biochemical requirements for apo to holocytochrome c conversion, it is expected that the cytochrome c biogenesis proteins fulfill at least one of the following tasks: transport of heme across a membrane, delivery of reduced heme to the site of assembly, maintenance of apocytochrome cysteines in a reduced form, and catalysis of the heme ligation. Therefore, conserved motifs or amino acids speaking to one of this above mentioned functions were identified and their functional importance analyzed via site-directed mutagenesis. A common characteristic of the biogenesis proteins is their attachment to the energy-transducing membrane. Indeed, the majority of these proteins possess transmembrane (TM) domains (from 1 to 13). For several of them, the topological arrangement has been experimentally deduced and conserved domains/motifs could be positioned with respect to the membrane (Fig. 3).

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FIG. 3.

Topology of proteins involved in c -type cytochrome assembly. (A) System I proteins RcCcmC (42), EcCcmF (79), and System II proteins CrCcsA (46), CrCcs1 (26), BsResB (1) which exhibit common motifs or residues indicative of heme handling activity; (B) BsResA (32), RcCcmG and RcCcmH (65), EcDsbD (53) and RcCcdA (24) which display motifs indicative of thioredox activity in System I or System II; (C) EcCcmE (87), ScCCHL (27) and ScCyc2p (14) are unique factors present in only one system. Proteins from the following model organisms Escherichia coli (Ec), Rhodobacter capsulatus (Rc), Chlamydomonas reinhardtii (Cr), Bacillus subtilis (Bs), and Saccharomyces cerevisiae (Sc) are shown. Residues, motifs, and cofactor binding sites which are important for function, are indicated: C, cysteine; H, histidine; Y, tyrosine; CPV, heme responsive element (HRM); WWD, tryptophan-rich motif; FAD, flavin adenine dinucleotide binding site. The thioredoxin-like domain is highlighted in white. Heme is symbolized by a cross surrounding an iron atom (Fe), and Q indicates a putative quinone binding site in CcmF. N-ter and C-ter point to the N- and C-terminal ends of the protein.

Important motifs and residues indicative of heme handling/delivery including hydrophobic residues and potential heme axial ligands have been identified and localized on both sides of the membrane. Some of these features appear to be common to System I and System II components (Fig. 3A) (59, 98). The tryptophan-rich or so-called “WWD” motif is found in two System I proteins (CcmC and CcmF) and one System II protein (CcsA). This motif is located between two transmembrane domains in a loop oriented toward the p-side of the membrane (11, 26, 42, 45, 78). The CcsBA protein from Helicobacter hepaticus, a natural fusion between CcsB and CcsA, contains histidines located within transmembrane domains close to the n-side of the membrane (38). These histidines are conserved in the corresponding ResB and ResC proteins from Bacillus subtilis. In addition, a cysteine was identified as a heme binding residue in ResB (1). In CcmF, a conserved histidine was shown to be the axial ligand of a B-type heme on the n-side of the membrane (78). The role of the WWD domain and the two pairs of conserved histidines in heme transport and delivery will be presented below.

As for heme delivery, common motifs indicative of a thioredox activity are found in both System I and II components. Putative thiol–disulfide oxidoreductase motifs and a thioredoxin-like domain have been recognized in protein domains facing the p-side of the membrane (Fig. 3B). Thioredoxin domains are present in CcmG and ResA from System I and II, respectively (20, 34, 61). These proteins are membrane anchored and their active domain is extracytoplasmic. Structures of E. coli, B. japonicum, and M. tuberculosis CcmG (29, 44, 71) and of B. subtilis ResA (19) have revealed a typical thioredoxin-like fold for these components. CcmH exhibits a RCXXC motif, but the protein does not fold like a thioredoxin (2, 25). CcmH is the unique component of System I thioredox pathway that is conserved in plant mitochondria. DsbD and CcdA are polytopic membrane proteins that contain conserved cysteines in their central hydrophobic domain (53, 66).

Some biogenesis proteins are specific of a single pathway. This is the case for the ABC transporter subunits CcmA and CcmB, CcmD and the heme chaperone CcmE from System I (Fig. 3C). CcmE is a unique heme chaperone that binds heme transiently in a covalent fashion on a conserved histidine with axial ligation via a conserved tyrosine (31, 48, 87, 102). CcmC is required to load heme on CcmE, while the activity of the CcmABCD ABC transporter complex is needed to release holoCcmE from CcmC, allowing the transfer of heme for ligation onto the apocytochrome (17, 37). CcmD is involved in the stability of the heme delivery protein complex.

The System III pathway involves unique proteins (CCHLs) that do not share any features with System I or II components. CCHLs are not integral membrane proteins, although they are bound to the mitochondrial inner membrane (13, 28, 69). A heme responsive motif (HRM) defined by CPX (where X is I, V, M, L) is present in CCHLs (Fig. 3C). In other proteins, the cysteine in HRM was shown to be necessary for heme binding (106) but it does not appear to be strictly essential for CCHL function in cytochromes c biogenesis (unpublished studies). In yeast, an additional protein Cyc2p is associated to CCHL for holocytochrome c formation. Cyc2p is anchored to the inner membrane and contains a FAD molecule in its C-terminal domain located in the IMS (14). Cyc2p is proposed to play a role in heme or apocytochrome reduction. Cyc2p seems to be restricted to mitochondria of fungi where two heme lyases CCHL and CC₁HL operate. There is no structural homolog of Cyc2p in multicellular eukaryotes where HCCS that diplays specificity towards both apocytochrome c and c ₁ is the only heme lyase (13, 14).

Requirements for the covalent linkage of heme to apocytochrome c

The covalent attachment of heme to the apocytochrome occurs through two thioether bonds formed between the heme vinyl groups and the thiols of the CXXCH binding motif. This reaction is stereospecific (i.e., the α2 and α4 carbon are linked with the sulfur of the first and second cysteines of the motif, respectively (Fig. 1). In vivo this post-translational modification requires dedicated machineries, however uncatalyzed holocytochrome c synthesis has been obtained and the analysis of the conditions required for correct stereochemical attachment has provided important information.

Holocytochrome c ₅₅₂ of the thermophilic bacteria Hydrogenobacter thermophilus can be produced in the cytoplasm of E. coli (52, 85). Due to this ectopic expression, apocytochrome c ₅₅₂ is not accessible to the action of the periplasmic System I and the correct heme attachment occurs independently of this pathway in the cytoplasm. It was proposed that holocytochrome c ₅₅₂ is assembled spontaneously, due to the partial folding of the apoform of the molecule, in contrast to the unfolded state of mitochondrial apocytochrome c (103). Indeed, when other apocytochromes c are expressed in E. coli cytoplasm, the heme attachment does not take place or is nonstereospecific (54, 85). Nevertheless, in vitro formation of a c-type cytochrome has been achieved with several cytochromes c, including cytochrome c ₅₅₂ of H. thermophilus (21), but also with Paracoccus denitrificans cytochrome c ₅₅₀ and the horse mitochondrial cytochrome c (22). The in vitro reaction occurs in two steps. First, a b-type cytochrome is formed involving coordination of heme iron with amino acids in the protein. Next, the thioether bonds are spontaneously formed, provided that the heme in the b-type cytochrome intermediate is in the correct orientation. The divalent iron heme and the free thiol groups are required to avoid the formation of incorrect products and acidic pH appears to be needed to promote the reaction (21). If exogenous reductant is omitted from the in vitro reaction, intramolecular disulfide between the cysteines of the CXXCH motif is detected and holocytochrome c formation does not take place.

Evidence for the implication of thiol–disulfide chemistry in bacterial System I and II maturation pathways

Rational for thioredox chemistry in the context of cytochrome c assembly

In the bacterial periplasm, thiol–disulfide homeostasis is under the control of several redox enzymes that promote disulfide bond formation and the isomerization of proteins with incorrect disulfide bonds (50, 51, 56, 66, 67). The thiol-oxidizing pathway is defined by the Dsb system that consists in thiol oxidase DsbA and recycling enzyme DsbB.* DsbA catalyzes disulfide bridge formation on cysteine-containing substrates that are translocated across the plasma membrane into the periplasm. DsbB operates by recycling reduced DsbA to its oxidized form. The electrons derived from DsbA reoxidation are shuttled to the quinone/menaquinone pool via the electron transport chain (Fig. 4). Several hundred proteins are substrates of the Dsb system, but genetic inactivation of this system is surprisingly not lethal but yields redox disturbance in the periplasmic space (50, 51, 56, 66, 67). Because apocytochromes c contain cysteines at their heme binding sites, they are believed to be substrates of the Dsb machinery and need to be consequently reduced to provide free sulfhydryls for the heme ligation.

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FIG. 4.

Bacterial apocytochrome oxidation and reduction pathways. The bacterial thio-oxidation pathway (DsbA and DsbB) is shown in gray and the reduction cascade (CcdA, CcmG and CcmH) in black. (A) For System I, the model where three partners (CcdA, CcmG, and CcmH) are required sequentially to reduce the apocytochrome c (Cyt.) is represented. It has also been proposed that CcmH functions independently from CcdA and CcmG. The source of reducing power in the cytoplasm is NADPH. (B) In System II, only two factors (CcdA and ResA/CcsX) are involved in the electron transfer from the NADPH to the apocytochrome c. In both System I and System II, the apocytochrome is believed to be oxidized by the DsbAB with a quinone or menaquinone (Q) as a final electron acceptor.

The formation of a disulfide bond at the CXXCH motif was believed initially to be a necessary step in the formation of holocytochrome c (reviewed in (45)). The proposed model was that apocytochrome c needs to be oxidized in order to be consequently reduced and reacted upon with heme. However, there is now multiple experimental evidence indicating that the disulfide bond forming catalysts are not essential for cytochrome c maturation (7, 24, 32, 36, 58). The fact that dsbAB mutants accumulate reduced levels of holocytochrome c suggests that the disulfide bond forming machinery is nevertheless required for optimal production of cytochromes c (101).

The reduced state of apocytochrome c is an absolute requirement for the heme ligation reaction. Consistent with this view is the finding that cytochrome c maturation requires the operation of several thiol–disulfide oxidoreductases that define a transmembrane subpathway for the delivery of reducing equivalents from the cytosol to the CXXCH targets in the periplasm (7, 34, 51, 66, 81). The current view is that the oxidized apocytochrome c CXXCH motif is reduced by the activity of thiol–disulfide oxidoreductase, CcmH in System I or thioredoxin-like ResA/CcsX in System II (Fig. 4). The observation that cytochrome c deficiency due to an inactivation of the thio-redox pathway can be compensated by exogenous thiols is the most convincing evidence that the disulfide in the CXXCH motif is the relevant target of action of this pathway (8, 10, 24, 32, 33, 36). The demonstration that recombinant forms of CcmH (65) and ResA (32) are active in vitro in thiol–disulfide exchange reactions solidifies this proposal. However, there is still no direct in vivo evidence that apocytochromes c are reduced by the proposed redox enzymes CcmH or ResA/CcsX. When the thio-reduction pathway does not operate, apocytochromes c are believed to become disulfide bonded by the Dsb enzymes and are unable to be acted upon by heme. In accord with this scenario is the finding that cytochrome c deficiency due to loss of the thio-reducing pathway can be compensated by mutations inactivating the thio-oxidation pathway (24, 33, 101). So it appears that the thioreduction branch in the context of cytochrome c assembly is only required to counteract the activity of the thio-oxidation pathway.

The involvement of redox chemistry in mitochondrial System I and System III assembly pathways

The question of redox chemistry in the context of mitochondrial cytochrome c assembly received little attention because of the accepted dogma that the IMS was a “naturally” reducing compartment due to the permeability of the outer membrane to cytosolic reducing components such as glutathione and pyridine nucleotide (12). However, the use of redox sensor showed that redox homeostasis in the IMS is independent from that of the cytosol (49). Moreover, the fact that thiol–disulfide chemistry is a catalyzed process has gained appreciation with the discovery that Mia40p and Erv1p drive the oxidative folding of a subset of IMS resident proteins (90). The Mia40 import pathway specifically interacts with small IMS proteins that possess a twin CX₃C and CX₉C motif but also other cysteine arrangement such as Erv1p (40, 63, 97). Apocytochrome c is not dependent upon Mia40p for its import (at least in yeast mitochondria (16)). However, it is possible that mitochondrial apocytochrome c/c ₁ become a target of Mia40 or Erv1 in conditions where they can no longer be assembled (Fig. 5), similarly to bacterial apocytochromes c that are believed to become disulfide bonded when they are not maintained reduced by the redox assembly components (24, 33, 101).

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FIG. 5.

Mitochondrial apocytochrome oxidation and reduction pathways. The mitochondrial apocytochrome (Cyt.) oxidation and reduction pathways are shown in gray and in black, respectively. Oxidizing proteins (MIA40, ERV1) are present in the IMS but their apocytochrome oxidation activity is speculative. (A) In A. thaliana System I, the only member of the reduction cascade is CCMH. An apocytochrome reduction activity has been evidenced in vitro but no data exist in vivo. (B) In S. cerevisiae System III, a NADPH-dependent apocytochrome disulfide reductase function has been suggested for Cyc2p. Note that, unlike apocytochrome c ₁ that does not depend upon heme attachment for import, in S. cerevisiae, apocytochrome c is not imported to the IMS in conditions where it cannot be assembled (reviewed in (45)). Hence in S. cerevisiae, apocytochrome c cannot be a substrate of the Mia40/Erv1 pathway. It is not known if this is also the case for A. thaliana cytochrome c and c ₁.

Heme delivery to cytochrome c assembly pathways requires transport through a membrane

The last step of heme synthesis is catalyzed by the ferrochelatase that inserts Fe²⁺ into protoporphyrin IX to form protoheme (Fig. 1). In prokaryotes, ferrochelatase is located on the cytoplasmic side of the membrane and heme must be transported across the cytoplasmic membrane to be attached to apocytochrome (70, 100). In eukaryotes, ferrochelatase is found in plastid or in mitochondria. Plastid ferrochelatase is reported to be associated with both envelope and thylakoid membranes, but the majority of enzyme activity is found in thylakoids (95). The ferrochelatase is believed to be located on the stromal side. Hence, there is a need for heme to cross the thylakoid membrane to reach the lumen side of the membrane where assembly through system II takes place. In mitochondria, two different situations are encountered depending whether the organelle compartment is the site of heme synthesis. In most animals and fungi, mitochondria are the only source of heme for the entire cell. The mitochondrial ferrochelatase is located on the matrix side of the inner mitochondrial membrane and heme export to the IMS for c-type cytochrome maturation and to the cytosol for cellular hemoproteins assembly is obligatory (3, 47). In photosynthetic eukaryotes, it is believed that mitochondrial heme originates from plastid (45, 96). Such an extramitochondrial source of heme is also required for cytochrome biogenesis in some organisms that are natural heme auxotrophs such as the nematode Caenorhabditis elegans (72). The delivery of heme originating from outside the mitochondria to the mitochondrial IMS could follow different routes involving at least transport through the outer mitochondrial membrane (45). It should be noted that there is no correlation between the presumed origin of heme and the cytochrome c maturation system used in mitochondria (I or III).

In the context of system III, heme transport across mitochondrial membranes is not understood, while for system I and II, proteins involved in transport, binding and presentation of heme have been identified (30, 37, 42, 98). However, Systems I and II components involved in heme delivery/handling appear to be specific to the cytochrome c maturation pathway. It is possible that in System III, the provision of heme to the IMS is depending upon a general heme exporter that has escaped genetic identification in saturating screens for mutants deficient in mitochondrial cytochromes c (89). Recently, the functions of the Ccm and Ccs proteins in heme provision to the p-side of the membrane have been deciphered and new roles have emerged (59). In System I, a heme iron redox control has been defined and a new function for CcmF protein as a quinol:heme oxido reductase has been unveiled.

System I: the determinant role of the heme chaperone CcmE and the operation of a heme iron redox system

In System I, CcmABCDE proteins are implicated in heme delivery to the heme lyase unit composed of CcmFH. CcmE appears to occupy a key and central position in this heme delivery branch. CcmE is a periplasmic heme chaperone that binds heme covalently on a single histidine residue (87). A covalent adduct is formed between one vinyl of heme and a conserved histidine on CcmE. CcmE first interacts with CcmC to capture heme and with CcmF to transfer it to apocytochrome for covalent ligation (76, 77). In E. coli, overexpression of CcmC is necessary and sufficient to promote covalent heme incorporation into CcmE, while CcmD favors the formation of holo-CcmE (86). CcmC is an integral membrane protein that contains the hydrophobic WWD domain in its second periplasmic loop (Fig. 3). The ability of CcmC to bind heme has been shown for the first time by affinity chromatography on hemin agarose (77). Recent advance from the Kranz lab has uncovered the role of axial ligation in maintaining the oxidation state of heme iron in System I (78). The expression of a subset of Ccm proteins allowed the authors to trap different intermediate complexes with bound heme. Biochemical and spectral analyses of these complexes have led to the following sequence of events (Fig. 6). Heme is transferred through CcmC and can be detected in a CcmCDE complex before the covalent heme attachment onto CcmE. The external heme binding domain of CcmC is composed of the periplasmic WWD domain proposed to provide a hydrophobic platform and two conserved histidines located in the adjacent periplasmic loops 1 and 3, which provide the fifth and sixth axial ligand to heme iron (Fig. 6). When holoCcmE is formed, heme is still coordinated by CcmC histidines. The change in axial ligand only occurs when holoCcmE is released from CcmC by the action of the ATPase activity of CcmAB (17, 37). In free holoCcmE, heme is ligated by CcmE Tyr as already proposed from structural and spectroscopic data (31, 102). The work by Richard–Fogal et al. revealed that, although heme is transferred to CcmC periplasmic heme binding site in a reduced form, all heme detected on holoCcmE or holoCcmE containing complexes is oxidized (78). This observation implies that the covalent ligation of heme to the His130 of CcmE is somehow accompanied by heme oxidation, yet the oxidant for ferric heme formation is not identified. In accord with this finding is the fact that apo-CcmE has a higher affinity to ferric heme when covalent attachment of heme to CcmE is achieved in vitro (23). The importance of the iron state for the chemistry of heme adduct on CcmE has been reviewed in (59).

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FIG. 6.

Delivery of reduced heme in System I (adapted from (78)). CcmC, CcmE, and CcmF proteins are represented at three stages of the process. In the top panel, ferroheme is bound to CcmC in a CcmCDE complex (CcmD is not represented). CcmC histidines provide the axial ligands to heme. In the following step, heme is covalently bound to CcmE and still coordinated by CcmC histidines. Heme is oxidized in holoCcmE. After the action of CcmAB (not shown), CcmE is released from the complex and its conserved tyrosine is proposed to act as axial ligand in this transient step (not represented). CcmE subsequently can transfer heme on CcmF and heme axial ligands switch to CcmF histidines. By transferring electron from ubiquinol (Q) through a B-type structural heme, CcmF is proposed to reduce the ferriheme before its attachment to apocytochrome (panel 3). See Figure 3 for legend.

As described in the first section, reduced heme is a prerequisite for the heme ligation reaction. Therefore, a holoCcmE reductase function is needed to complete holocytochrome c formation. Similarly to CcmC, CcmF possesses a periplasmic heme-binding site that provides axial ligands to holoCcmE heme. CcmF also contains a B heme ligated by at least one histidine located in TM5 and possibly a second one in TM12 (78) (Fig. 6). The structural heme of CcmF is reduced in vitro by quinol. This led to the proposal that CcmF acts as a quinol:heme oxidoreductase that relays electrons from quinol to holoCcmE heme through the B heme. However, there is still no direct proof that the B heme in CcmF reduces ferriheme prior to its ligation onto apocytochrome c.

Most of the components of the heme delivery pathway (i.e., CcmABCEF) have counterparts in plant mitochondria (41). The most hydrophobic proteins (CcmB, -C, -F_N1, -F_N2, -F_C) are still encoded by the mitochondrial genome while the peripheral CCMA and the monotopic CCME are nuclear encoded (41). The topology and motifs depicted in Figure 3 are conserved in bacterial and mitochondrial proteins (64, 73, 74, 91). Since CcmF is split into three polypeptides, the potential histidine ligands of heme in holoCCME and those of the structural heme are located on two different polypeptides. The three CcmF polypeptides are present in a high molecular weight complex including the CCMH protein (64, 73). Yeast two-hybrid experiments suggest that CcmF_N2 interacts with CcmF_N1 and CcmF_C, while CCMH interacts with CcmF_N1 and CcmF_N2. Both apocytochromes c and c ₁ could also interact with CCMH, CcmF_N1 and CcmF_N2. The association of the four CCM proteins with CcmE, as a possible heme lyase or scaffold for heme and apocytochrome assembly seems very likely in mitochondria, similarly to the bacterial situation.

System II: Heme channeling via histidine ligands protects iron from oxidation

In bacteria, two out of the four proteins involved in System II, CcsA and CcsB, have been identified as the minimal set required for heme transport and ligation (33, 37) (Table 1). In Helicobacter hepaticus, the two proteins are fused into a single CcsBA protein whose topology has been determined (38). CcsBA has 10 transmembrane domains, the WWD motif flanked by two conserved histidines present in adjacent loops in the periplasm, and two other histidines located in transmembrane segments closer to the cytosolic face. If the overall topologies of B. subtilis ResB (1), M. leprae and C. reinhardtii CcsA (43, 46) differ from H. hepaticus CcsB and CcsA corresponding domains, the location of WWD and histidines that are essential for c-type cytochrome maturation appears to be conserved in all the orthologs (Fig. 3). Frawley and Kranz have recently described a heme transfer pathway in System II using the naturally fused CcsBA protein (38). When expressed in E. coli, CcsBA binds one reduced heme. Mutation of the conserved histidines in CcsBA revealed different roles for the two pairs of residues (Fig. 7). When either of the periplasmic histidines is mutated, CcsBA still binds heme but displays a change in its absorption spectra. This experiment underscores the role of the two periplasmic histidines as axial ligands of heme. Interestingly, heme associated to the CcsBA histidine mutants is found under the oxidized form. This led to the proposal that the axial ligands protect iron against oxidation. An alternative interpretation is that mutation of the periplasmic histidines induces a change in a heme binding interface resulting in heme exposure to air oxidation.

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FIG. 7.

Heme delivery in System II (adapted from (38)). C. reinhardtii proteins have been used as models for System II-dependent heme delivery. The top panel represents the initial binding of reduced heme in the CcsA/Ccs1 complex that operates as a heme channel. Heme is coordinated by TM histidines that reside on the n-side of the membrane in CcsA and Ccs1. Heme is then proposed to be transferred across the membrane from the n-side to an external binding site on the p-side consisting of the WWD domain and a pair of histidines in CcsA. The histidines on the p-side are postulated to coordinate the heme. Heme on the external binding site is acted upon by apocytochrome c to yield holocytochrome c. Hence CcsA/Ccs1 is also acting as a heme lyase. Histidine-based coordination of the heme co-factor is assumed to prevent oxidation of heme as it is transferred from the site of synthesis to its site of assembly to apocytochrome c.

Conversely, when either transmembrane histidine on the cytosolic side is mutated, a reduction of heme binding to CcsBA is observed. In one histidine mutant, the spectral characteristics of CcsBA can be measured and are similar to those of wild type, although some CcsBA bound heme appears to be oxidized. The transmembrane histidines have been proposed to participate in channeling heme to the external heme-binding site and to also play a protective role against heme oxidation (Fig. 7). Interestingly, provision of imidazole restores the function of the TM histidines mutants for cytochrome c production, but this complementation is less efficient under aerobic growth conditions, an observation that was attributed to a possible heme oxidation. A general protective role of histidine axial ligands of heme iron against oxidation has been proposed (38, 62). H. hepaticus CcsBA assures both heme transport and cytochrome c assembly without the help of a heme chaperone like CcmE. The heme attachment reaction in System II appears to be mechanistically simpler than in System I.

This view of a CcsB/CcsA-mediated transport/ligation via the histidine ligands might be oversimplified based on a recent study using B. subtilis ResB and ResC that are orthologous to CcsB and CcsA, respectively. Expression of ResB and ResC in E.coli does not complement cytochrome c maturation, unlike CcsBA (1). Surprisingly, ResB accumulates with a heme covalently attached to a conserved cysteine residue located in the second transmembrane close to the cytosolic side of the membrane (1). This cysteine is not essential for cytochrome c assembly function, as previously shown for Ccs1, the C. reinhardtii ResB ortholog (26). The biological significance of this finding is unclear but it is possible that the cysteine participates in capturing heme on the cytosolic side and becomes reactive to this molecule in conditions where the ResB cannot function as a heme channel.

System III: Role of Cyc2p

The redox role of Cyc2p in cytochrome c maturation is supported by the fact that a recombinant form of the protein is redox active in an in vitro assay (14). However, there is still no direct evidence that heme and/or apocytochrome c sulfhydryls are direct relevant targets of action in vivo and there are arguments in support of both hypotheses (14). The sequence similarity of Cyc2p to NADH-cytochrome b ₅ reductase led to the hypothesis that heme, the substrate of the heme ligation reaction could be reduced by the activity of Cyc2p (13, 14). Cytochrome b ₅ reductase (EC 1.6.2.2), is a family of ER enzymes that catalyzes the reduction of cytochrome b ₅, a b-type cytochrome using reduced pyridine nucleotide as the physiological electron donor. The sequence relationship of Cyc2p to this family of enzymes suggests that a b-type cytochrome could be a target of Cyc2p reducing activity. It is conceivable that a b-type cytochrome is formed as an intermediate of the heme ligation reaction similar to what has been observed in bacterial System I. Another possibility is that CCHL, the mitochondrial heme lyase contains, similarly to CcmF (78), a B-type heme that is reduced by Cyc2p. The presence of conserved histidines in CCHL is compatible with such a scenario but this awaits spectroscopic analysis on recombinant CCHL. That heme is reduced by a flavin-linked NADPH dependent electron transfer is compatible with the observation that both redox molecules are required for the in organello reconstitution of holocytochrome c assembly (69).

Reduction of apocytochrome sulfhydryls

Thioredox chemistry was historically defined, perhaps naively as a sole need to maintain apocytochrome c under the reduced dithiol form prior to the heme ligation reaction. A comparison of thioredox components involved in System I and System II now indicates that this perception needs to be expanded. In System I, a role for CcmH as a redox factor controlling a yet-to-be-defined step in the heme lyase reaction is now beginning to emerge. The exact activity of CcmH is still enigmatic but a “simple” function in converting apocytochrome c CXXCH disulfide to dithiol seems unlikely. Several arguments indicate that the activity of CcmH is dependent upon the heme lyase CcmF and heme chaperone CcmE. It is possible to envision that the CcmE-dependence in relaying heme to the CcmF lyase dictates a specific redox requirement to ensure stereospecificity of the heme attachment reaction. CcmH is likely to have an essential role as it is the only redox component that appears to be conserved in System I mitochondria. There is no transmembrane thio-reducing pathway in mitochondria, and one has to envision that “simple” reduction of the CXXCH cysteines is under the control of unrecognized components, possibly involving Cyc2p in System III. Alternatively, the reduction of CXXCH cysteines might no longer be needed because apocytochrome is rapidly trapped by the assembly machinery and thereby protected from oxidation. Conceivably, CCHL in system III or CcmF/H for System I would exert this protection on the CXXCH cysteines and maintain the apocytochrome substrate in the competent conformation for heme ligation to occur.

Reduction of the heme cofactor

Functional dissection of the heme relay subpathway in bacterial System I and System II has considerably advanced our view on this question. In System I, the involvement of a direct heme reduction mechanism was deduced from the discovery that heme is oxidized when bound to the CcmE chaperone. Because CcmE defines the last step of heme transfer before ligation onto apocytochrome c, a mechanism for heme reduction has to be postulated. There is support but yet no direct proof that heme reduction is achieved by an electron transfer reaction from quinone via the CcmF heme lyase. Since System I mitochondria have kept a number of the heme handling CCM proteins from their bacterial ancestor, it is very likely that the mechanism of heme reduction in System I mitochondria and bacteria is similar. Investigation in the plant experimental system is required to corroborate this view and it is conceivable that some differences might be revealed between System I mitochondria and bacteria. In bacterial System II, it appears that heme is not enzymatically reduced but protected from oxidation, presumably from its site of synthesis (on ferrochelatase) to its site of assembly onto apocytochrome c. It is not known if System III mitochondria require an active mechanism for heme reduction similarly to bacterial System I. The need for a pyridine nucleotide and flavin molecules to reduce heme for holocytochrome c reconstitution in the in organello reaction is indicative of such an active mechanism (68, 69). The discovery that Cyc2p, a NADPH-dependent flavoprotein is required for cytochrome c assembly suggests that this component could function as a heme reductase. However, this proposal still awaits experimental testing as there are also indications that the protein could display disulfide reductase activity. The fact that Cyc2p appears to be restricted to fungal mitochondria raises the question as to the universality of such a mechanism. Other factors must operate in System III mitochondria that have not retained Cyc2p but their molecular identity is currently unknown.

A cytochrome c centric view?

The reason why there is such a diversity in cytochrome c maturation pathways has no obvious and straightforward answer. One attractive hypothesis is that the cytochrome c assembly machineries have evolved with additional functions that remain to be deciphered. In some System I bacteria, there are several reports that the heme handling Ccm proteins participate in cellular processes that are distinct from the cytochrome c maturation (18). These additional functions were postulated on the basis that mutations in the corresponding Ccm genes result in new phenotypes such as disturbance of siderophore metabolism and metal homeostasis that cannot be attributed to loss of cytochrome c production. Considering that the functional repertoire of c-type cytochromes keeps expanding, it is not surprising to envision that their dedicated assembly components could also be recruited for functions beside the well-documented activity in co-factor attachment.