CcmI Subunit of CcmFHI Heme Ligation Complex Functions as an Apocytochrome c Chaperone during c-Type Cytochrome Maturation

Cytochrome c maturation (Ccm) is a sophisticated post-translational process. It occurs after translocation of apocytochromes c to the p side of energy transducing membranes and forms stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of cysteines at their conserved heme binding sites. In many organisms this process involves up to 10 (CcmABCDEFGHI and CcdA) membrane proteins. One of these proteins is CcmI, which has an N-terminal membrane-embedded domain with two transmembrane helices and a large C-terminal periplasmic domain with protein-protein interaction motifs. Together with CcmF and CcmH, CcmI forms a multisubunit heme ligation complex. How the CcmFHI complex recognizes its apocytochrome c substrates remained unknown. In this study, using Rhodobacter capsulatus apocytochrome c2 as a Ccm substrate, we demonstrate for the first time that CcmI binds apocytochrome c2 but not holocytochrome c2. Mainly the C-terminal portions of both CcmI and apocytochrome c2 mediate this binding. Other physical interactions via the conserved structural elements in apocytochrome c2, like the heme ligating cysteines or heme iron axial ligands, are less crucial. Furthermore, we show that the N-terminal domain of CcmI can also weakly bind apocytochrome c2, but this interaction requires a free thiol group at apocytochrome c2 heme binding site. We conclude that the CcmI subunit of the CcmFHI complex functions as an apocytochrome c chaperone during the Ccm process used by proteobacteria, archaea, mitochondria of plants and red algae. Background: Cytochrome c maturation (Ccm) is the covalent ligation of heme b to an apocytochrome c by the Ccm apparatus. Results: CcmI subunit of CcmFHI heme ligation complex recognizes and binds specifically apo- and not holocytochrome c2. Conclusion: CcmI and its homologues are apocytochrome c chaperones. Significance: A first glimpse to how the heme ligation complex recognizes an apocytochrome c before, and releases a holocytochrome c after, cofactor addition.