An investigation into a cardiolipin acyl chain insertion site in cytochrome c

• Published in: Biochimica et biophysica acta Vol. 1817; no. 5; pp. 780 - 791
• Main Authors: Rajagopal, Badri S., Silkstone, Gary G., Nicholls, Peter, Wilson, Michael T., Worrall, Jonathan A.R.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2012
• Subjects: Acylation; alanine; Animals; Apoptosis; carbon monoxide; Carbon Monoxide - metabolism; Cardiolipin; Cardiolipins - metabolism; Cattle; Circular Dichroism; Cytochrome c; Cytochromes c - metabolism; dissociation; heme iron; Hydrogen-Ion Concentration; hydrophobic bonding; Iron - metabolism; Kinetics; Lasers; Ligand binding; mitochondria; Models, Biological; Mutant Proteins - metabolism; mutants; mutation; Oxidation-Reduction; Peroxidase; Peroxidase - metabolism; peroxidation; Phospholipids; Photolysis; Protein Binding; Saccharomyces cerevisiae - metabolism; Spectrum Analysis; stoichiometry; Time Factors; CD; Ligand binding; Amp; Em; CL; cyt c; TMPD; Phospholipids; ET; Tris; Cytochrome c; Peroxidase; Cardiolipin; WT; GuHCl; CAPS; Apoptosis
• ISSN: 0005-2728; 0006-3002; 1879-2650
• DOI: 10.1016/j.bbabio.2012.02.010

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. ► A putative cardiolipin insertion site on mitochondrial cytochrome c has been studied. ► A series of alanine mutations in cytochrome c have been constructed. ► Cardiolipin interaction has been assessed using carbon monoxide as a small ligand probe. ► The alanine mutations influence the extent of CO binding in the ferrous cytochrome c complex. ► Arg-91 alters the cardiolipin binding stoichiometry.