title: Human mitochondrial holocytochrome c synthase's heme binding, maturation determinants, and complex formation with cytochrome c.
abstract: Proper functioning of the mitochondrion requires the orchestrated assembly of respiratory complexes with their cofactors. Cytochrome c, an essential electron carrier in mitochondria and a critical component of the apoptotic pathway, contains a heme cofactor covalently attached to the protein at a conserved CXXCH motif. Although it has been known for more than two decades that heme attachment requires the mitochondrial protein holocytochrome c synthase (HCCS), the mechanism remained unknown. We purified membrane-bound human HCCS with endogenous heme and in complex with its cognate human apocytochrome c. Spectroscopic analyses of HCCS alone and complexes of HCCS with site-directed variants of cytochrome c revealed the fundamental steps of heme attachment and maturation. A conserved histidine in HCCS (His154) provided the key ligand to the heme iron. Formation of the HCCS:heme complex served as the platform for interaction with apocytochrome c. Heme was the central molecule mediating contact between HCCS and apocytochrome c. A conserved histidine in apocytochrome c (His19 of CXXCH) supplied the second axial ligand to heme in the trapped HCCS:heme:cytochrome c complex. We also examined the substrate specificity of human HCCS and converted a bacterial cytochrome c into a robust substrate for the HCCS. The results allow us to describe the molecular mechanisms underlying the HCCS reaction.
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: San Francisco
authors.forename: Eric C
authors.initials: EC
authors.lastname: Bretsnyder
authors.forename: Robert G
authors.initials: RG
authors.lastname: Kranz
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D045304:Cytochromes c / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008190:Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D016354:Rhodobacter capsulatus / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D006418:Heme; D006639:Histidine; D045304:Cytochromes c; D008190:Lyases; C031581:cytochrome C synthetase