title: An investigation into a cardiolipin acyl chain insertion site in cytochrome c.
abstract: Mitochondrial cytochrome c associates with the phosphoplipid cardiolipin (CL) through a combination of electrostatic and hydrophobic interactions. The latter occurs by insertion into cytochrome c of an acyl chain, resulting in the dissociation of the axial Met-80 heme-iron ligand. The resulting five coordinate cytochrome c/CL complex has peroxidatic properties leading to peroxidation of CL and dissociation of the complex. These events are considered to be pre-apoptotic and culminate with release of cytochrome c from the mitochondria into the cytoplasm. Two distinct surface regions on cytochrome c have been suggested to mediate CL acyl chain insertion and this study has probed one of these regions. We have constructed a series of alanine mutants aimed at disrupting a surface cleft formed between residues 67-71 and 82-85. The physicochemical properties, peroxidase activity, CL binding, and kinetics of carbon monoxide (CO) binding to the ferrous cytochrome c/CL complex have been assessed for the individual mutants. Our findings reveal that the majority of mutants are capable of binding CL in the same apparent stoichiometry as the wild-type protein, with the extent to which the Met-80 ligand is bound in the ferrous cytochrome c/CL complex being mutant specific at neutral pH. Mutation of the species conserved Arg-91 residue, that anchors the cleft, results in the greatest changes to physicochemical properties of the protein leading to a change in the CL binding ratio required to effect structural changes and to the ligand-exchange properties of the ferrous cytochrome c/CL complex.
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK.
authors.forename: Badri S
authors.initials: BS
authors.lastname: Rajagopal
authors.forename: Gary G
authors.initials: GG
authors.lastname: Silkstone
authors.forename: Peter
authors.initials: P
authors.lastname: Nicholls
authors.forename: Michael T
authors.initials: MT
authors.lastname: Wilson
authors.forename: Jonathan A R
authors.initials: JA
authors.lastname: Worrall
mesh_terms: D000215:Acylation; D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D002308:Cardiolipins / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D045304:Cytochromes c / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism; D010782:Photolysis; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013057:Spectrum Analysis; D013997:Time Factors
chemical_list: D002308:Cardiolipins; D050505:Mutant Proteins; D002248:Carbon Monoxide; D045304:Cytochromes c; D007501:Iron; D009195:Peroxidase