Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site, CysXXCysHis [Bioenergetics]

August 28th, 2014 by Babbitt, S. E., San Francisco, B., Mendez, D. L., Lukat-Rodgers, G. S., Rodgers, K. R., Bretsnyder, E. C., Kranz, R. G.

Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-vis spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand previously shown to be from HCCS residue His154. Substitutions of His19 in cytochrome c to seven other residues (G,A,M,R,K,C,Y) were used with various approaches to establish other roles played by His19. Three roles for His19 in HCCS-mediated assembly are suggested: i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His19.
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