Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms

Abstract

Cytochromes c (Cyt c) are ubiquitous heme-containing proteins, mainly involved in electron transfer processes, whose structure and functions have been and still are intensely studied. Surprisingly, our understanding of the molecular mechanism whereby the heme group is covalently attached to the apoprotein (apoCyt) in the cell is still largely unknown. This posttranslational process, known as Cyt c biogenesis or Cyt c maturation, ensures the stereospecific formation of the thioether bonds between the heme vinyl groups and the cysteine thiols of the apoCyt heme binding motif. To accomplish this task, prokaryotic and eukaryotic cells have evolved distinctive protein machineries composed of different proteins. In this review, the structural and functional properties of the main maturation apparatuses found in gram-negative and gram-positive bacteria and in the mitochondria of eukaryotic cells will be presented, dissecting the Cyt c maturation process into three functional steps: (i) heme translocation and delivery, (ii) apoCyt thioreductive pathway, and (iii) apoCyt chaperoning and heme ligation. Moreover, current hypotheses and open questions about the molecular mechanisms of each of the three steps will be discussed, with special attention to System I, the maturation apparatus found in gram-negative bacteria.

Figure 1

The heme-binding site typically observed in c-type cytochromes, as exemplified by a close-up view of the structure of P. aeruginosa Cyt c551 (Pa-Cytc; PDB 351c). The heme is shown in red, while the atoms of the residues from the heme-binding motif of Pa-Cytc (C₁₂VAC₁₅H) and the distal Met61 are color-coded (C: green; O: red; N: blue; S: yellow). The figure highlights the thioether bonds between the Cys12 (on the right) and the vinyl-2, and between Cys15 (on the left) and the vinyl-4. The iron atom of the heme (in gray) is axially coordinated by the distal methionine residue (Met61; shown above the heme plane) and by the proximal histidine residue (His16; shown below the heme plane).

Figure 2

Schematic representation of the protein components of System I. Proteins involved in the heme translocation and delivery pathway are shown in light brown; proteins involved in the apoCyt thioreduction pathway are shown in green; proteins involved in apoCyt chaperoning and heme attachment processes are shown in light purple. Cyt c (the 3D structure is that of the Cyt c551 from P. aeruginosa), Protein Data Bank accession number 2EXV [20] and apoCyt (represented as a cartoon) are shown in blue. The translocation process of heme (shown in red) is unknown. The 3D structures of the soluble periplasmic domains of Ec-CcmE, Pa-CcmG and Pa-CcmH are shown (Protein Data Bank accession numbers are 1LIZ [21], 3KH7 [22], and 2HL7 [23], resp.). Organisms employing System I: α- and γ-proteobacteria, some β-proteobacteria (e.g., Nitrosomonas) and δ-proteobacteria (e.g., Desulfovibrio), and Deinococci and Archaea. Additionally, System I is observed in plant mitochondria and in the mitochondria of some protozoa (e.g., Tetrahymena).

Figure 3

Schematic representation of the protein components of System II. Proteins involved in the heme translocation and delivery and in the apoCyt chaperoning and heme attachment processes are shown in light brown; proteins involved in the apoCyt thioreduction pathway are shown in green. Cyt c and apoCyt (represented as a cartoon) are shown in blue. The 3D structure of the soluble periplasmic domain of Bs-ResA is shown in green (Protein Data Bank accession number is 1ST9 [24]. System II is found in plant chloroplasts, in gram-positive bacteria, cyanobacteria, ε-proteobacteria, most β-proteobacteria (e.g., Bordetella, Burkholderia), and some δ-proteobacteria (e.g., Geobacter).

Figure 4

Schematic representation of System III. A single protein (HCCS) associated to the mitochondrial inner membrane is required for Cyt c maturation. The translocation process of heme (shown in red) is unknown. System III is found in the mitochondria of fungi (e.g., S. cerevisiae), vertebrates (e.g., human), and invertebrates (e.g., C. elegans, Drosophila).

Figure 5

Three-dimensional structure of Pa-CcmH shown in ribbon representation. The figure shows the three-helix bundle forming the characteristic fold of Pa-CcmH. The active site disulfide bond between residues Cys25 and Cys28 in the long loop connecting helices α-helix1 and α-helix 2 is highlighted in yellow.

Figure 6

: Alternative thioreduction pathways which may be operative in System I and hypothesized on the basis of structural and functional characterization of the redox-active Ccm proteins from P. aeruginosa [22, 23, 25]. Scheme 1 is a linear redox cascade whereby CcmG is the direct reductant of CcmH, which reduces oxidized apoCyt. Scheme 2 envisages a more complex scenario involving the formation of a mixed-disulfide complex between CcmH and apoCyt (Step 1). This complex is the substrate for the attack by reduced CcmG (Step 2) that liberates reduced apoCyt. The resulting disulfide bond between CcmH and CcmG is then resolved by the free Cys thiol of CcmG (probably Cys77 in Pa-CcmG). Adapted from [25].