The thioreduction component CcmG confers efficiency and the heme ligation component CcmH ensures stereo-specificity during cytochrome c maturation

• Published in: The Journal of biological chemistry 2017-08, Vol.292 (32), p.13154-13167
• Main Authors: Verissimo, Andreia F., Khalfaoui-Hassani, Bahia, Hwang, Josephine, Steimle, Stefan, Selamoglu, Nur, Sanders, Carsten, Khatchikian, Camilo E., Daldal, Fevzi
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; apocytochrome; Apoenzymes - chemistry; Apoenzymes - metabolism; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; BASIC BIOLOGICAL SCIENCES; Binding Sites; Biochemistry & Molecular Biology; Bioenergetics; Cysteine - chemistry; Cysteine - metabolism; Cystine - chemistry; Cystine - metabolism; cytochrome c; cytochrome c maturation; Cytochromes c - chemistry; Cytochromes c - metabolism; disulfide; heme; Heme - metabolism; INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Life Sciences; Models, Biological; Mutation; Oxidation-Reduction; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Disulfide Reductase (Glutathione) - chemistry; Protein Disulfide Reductase (Glutathione) - genetics; Protein Disulfide Reductase (Glutathione) - metabolism; Protein Interaction Domains and Motifs; Protein Multimerization; protein–protein interaction; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Rhodobacter capsulatus; Rhodobacter capsulatus - enzymology; Stereoisomerism; thiol; thiol–disulfide exchange; thioreduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M117.794586

In many Gram-negative bacteria, including Rhodobacter capsulatus, cytochrome c maturation (Ccm) is carried out by a membrane-integral machinery composed of nine proteins (CcmA to I). During this process, the periplasmic thiol-disulfide oxidoreductase DsbA is thought to catalyze the formation of a disulfide bond between the Cys residues at the apocytochrome c heme-binding site (CXXCH). Subsequently, a Ccm-specific thioreductive pathway involving CcmG and CcmH reduces this disulfide bond to allow covalent heme ligation. Currently, the sequence of thioredox reactions occurring between these components and apocytochrome c and the identity of their active Cys residues are unknown. In this work, we first investigated protein–protein interactions among the apocytochrome c, CcmG, and the heme-ligation components CcmF, CcmH, and CcmI. We found that they all interact with each other, forming a CcmFGHI–apocytochrome c complex. Using purified wild-type CcmG, CcmH, and apocytochrome c, as well as their respective Cys mutant variants, we determined the rates of thiol-disulfide exchange reactions between selected pairs of Cys residues from these proteins. We established that CcmG can efficiently reduce the disulfide bond of apocytochrome c and also resolve a mixed disulfide bond formed between apocytochrome c and CcmH. We further show that Cys-45 of CcmH and Cys-34 of apocytochrome c are most likely to form this mixed disulfide bond, which is consistent with the stereo-specificity of the heme–apocytochrome c ligation reaction. We conclude that CcmG confers efficiency, and CcmH ensures stereo-specificity during Ccm and present a comprehensive model for thioreduction reactions that lead to heme–apocytochrome c ligation.