title: Conserved residues of the human mitochondrial holocytochrome c synthase mediate interactions with heme.
abstract: C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS-heme-cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical "correction" supports the proposed role of heme binding for the corresponding residues.
authors: Department of Biology, Washington University , St. Louis, Missouri 63130, United States.
authors: Shalon E
authors: SE
authors: Babbitt
authors: Brian
authors: B
authors: San Francisco
authors: Eric C
authors: EC
authors: Bretsnyder
authors: Robert G
authors: RG
authors: Kranz
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D015971:Gene Expression Regulation, Enzymologic; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008190:Lyases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D006418:Heme; D008190:Lyases; C031581:cytochrome C synthetase