Cytochrome c biogenesis: mechanisms for covalent modifications and trafficking of heme and for heme-iron redox control

Abstract

Heme is the prosthetic group for cytochromes, which are directly involved in oxidation/reduction reactions inside and outside the cell. Many cytochromes contain heme with covalent additions at one or both vinyl groups. These include farnesylation at one vinyl in hemes o and a and thioether linkages to each vinyl in cytochrome c (at CXXCH of the protein). Here we review the mechanisms for these covalent attachments, with emphasis on the three unique cytochrome c assembly pathways called systems I, II, and III. All proteins in system I (called Ccm proteins) and system II (Ccs proteins) are integral membrane proteins. Recent biochemical analyses suggest mechanisms for heme channeling to the outside, heme-iron redox control, and attachment to the CXXCH. For system II, the CcsB and CcsA proteins form a cytochrome c synthetase complex which specifically channels heme to an external heme binding domain; in this conserved tryptophan-rich "WWD domain" (in CcsA), the heme is maintained in the reduced state by two external histidines and then ligated to the CXXCH motif. In system I, a two-step process is described. Step 1 is the CcmABCD-mediated synthesis and release of oxidized holoCcmE (heme in the Fe(+3) state). We describe how external histidines in CcmC are involved in heme attachment to CcmE, and the chemical mechanism to form oxidized holoCcmE is discussed. Step 2 includes the CcmFH-mediated reduction (to Fe(+2)) of holoCcmE and ligation of the heme to CXXCH. The evolutionary and ecological advantages for each system are discussed with respect to iron limitation and oxidizing environments.

FIG. 1.

Types of heme (b, o, a, and c), modifications and genes/enzymes required for modification, as discussed in the text. The Fischer numbering system was used.

FIG. 2.

Working models for cytochrome c biogenesis by systems I, II, and III. Models include trafficking and oxidation states of heme, as well as the subpathways for apocytochrome reduction (in red for system I and system II). Representative genera possessing each system are listed under the models.

FIG. 3.

Topology of the system II CcsBA fusion protein from Helicobacter hepaticus and completely conserved residues (red). This topology is based on the experimentally determined topologies for the CcsBA fusion (59), the CcsB protein of B. pertussis, and the CcsA proteins of Mycobacterium leprae and Chlamydomonas reinhardtii as described in the text. The delineation between the CcsB and CcsA portions of the fusion protein is noted. Absolutely conserved (identical) residues in bacterial system II are colored red. Four absolutely conserved histidines that are potential axial ligands to the heme iron are starred. The highly conserved WWD domain in CcsA is shaded, as are the hydrophobic patches. Reduced heme is proposed to move through CcsBA (using H77 and H858 as initial axial ligands) to the WWD domain where H761 and H897 ligand the heme iron. Conservation was determined by alignments of the CcsB and CcsA proteins from the following organisms, which were chosen as representatives across the kingdoms and genera shown in Fig. 2: Arthrobacter aurescens, Arthrobacter butzleri, Bdellovibrio bacteriovorus HD100, Bordetella pertussis Tohama I, Campylobacter jejuni NCTC 11168, Chlamydomonas reinhardtii, Chlorobium tepidum TLS, Desulfitobacterium hafniense Y51, Geobacter sulfurreducens PCA, Helicobacter acinonychis Sheeba, Helicobacter hepaticus ATCC 51449, Helicobacter pylori 26695, Leifsonia xyli CTCB07, Mycobacterium leprae TN, Neisseria gonorrhoeae FA1090, Ralstonia metallidurans CH34, Synechocystis sp. strain PCC6803, Thiobacillus denitrificans ATCC 25259, Thiomicrospira denitrificans ATCC 33889, and Wolinella succinogenes DSMZ 1740.

FIG. 4.

Topologies of the CcmABCDE integral membrane proteins required for system I cytochrome c biogenesis step 1, i.e., heme delivery to apoCcmE and release of holoCcmE by CcmABCD. Completely conserved residues are in red. The indicated sequences are from Escherichia coli. The topologies of CcmB, CcmC, CcmD, and CcmE are based on experimentally determined topologies as described in the text (see, e.g., reference 125). Completely conserved (i.e., identical) amino acid residues (red) were identified by individual protein alignments with bacterial sequences from genera shown in Fig. 2. Histidine residues that potentially act as axial heme iron ligands are starred, and some are shown with dashed lines to specific heme molecules. CcmE has a completely conserved His or Cys (deinococci) residue (green) for covalent heme attachment and a Tyr (Y134) residue as an axial heme iron ligand (dashed line). The oxidation states of heme are shown, and other important residues, domains, or motifs are labeled for descriptions used in the text. Genes from the following organisms were used for determining conserved residues: the alphaproteobacteria, Agrobacterium tumefaciens C58, R. capsulatus, Caulobacter crescentus CB15, and Bradyrhizobium japonicum (blr 0467); the betaproteobacteria, Nitrospira multiformis ATCC 25196 and Nitrosomonas europaea ATCC 19718; the gammaproteobacteria, E. coli K-12 MG1655, Pseudomonas fluorescens Pf01, Shewanella oneidensis MR-1, and Vibrio parahaemolyticus RIMD 2210633; the deltaproteobacteria, Myxococcus xanthus and Desulfovibrio desulfuricans; and the deinococci, Deinococcus geothermalis and Thermus thermophilus.

FIG. 5.

Proposed radical and nucleophilic reaction mechanisms for holoCcmE (His130) adduct (A) and cytochrome c (B) linkage to heme vinyl groups. Noted are the oxidation states of iron (Fe³⁺ or Fe²⁺); red half arrows are one-electron transfers, and full red arrows are two electron transfers. Transfer of the proton from the imidazolium to the alpha carbon is probably solvent or protein mediated (i.e., the proton may be abstracted at an early step, with a solvent- or protein-mediated protonation at the alpha carbon at a later step). The final Fe³⁺ state could be either reduced back to the Fe²⁺ state by Q⁻ or kept in the Fe³⁺ state to stabilize the imidazole adduct until it is ejected at the next step (reverse blue arrow). Only a single vinyl of heme and, for the radical step, only one set of one-electron arrows are shown for simplicity.

FIG. 6.

Comparative models of the system I CcmABCD holoCcmE release pathway and the Lol lipoprotein release system (LolABCDE).

FIG. 7.

Topologies of the CcmF and CcmH integral membrane proteins required for system I cytochrome c biogenesis for step 2, i.e., heme attachment to apocytochrome c by CcmFH. The indicated sequences are from Escherichia coli. The topologies of CcmH and CcmF are experimentally established, as described in the text (see, e.g., reference 125). Completely conserved (i.e., identical) amino acid residues (red) were identified by individual protein alignments with bacterial sequences. The potential histidine axial ligands to the b heme and heme in holoCcmE are starred. The highly conserved WWD domain in CcmF is shaded, as are the hydrophobic patches. The following organisms were used for determining conserved residues: the alphaproteobacteria, Agrobacterium tumefaciens C58, R. capsulatus, Caulobacter crescentus CB15, and Bradyrhizobium japonicum (blr 0467); the betaproteobacteria, Nitrospira multiformis ATCC 25196 and Nitrosomonas europaea ATCC 19718; the gammaproteobacteria, E. coli K-12 MG1655, Pseudomonas fluorescens Pf01, Shewanella oneidensis MR-1, and Vibrio parahaemolyticus RIMD 2210633; the deltaproteobacteria, Myxococcus xanthus and Desulfovibrio desulfuricans; and the deinococci, Deinococcus geothermalis and Thermus thermophilus.