Site A‑Mediated Partial Unfolding of Cytochrome <i>c</i> on Cardiolipin Vesicles Is Species-Dependent and Does Not Require Lys72

Measurements at pH 8 allow evaluation of binding of 100% cardiolipin vesicles to site A of cytochrome <i>c</i> without interference from other known binding sites. Site A encompasses Lys72, Lys73, Lys86, and Lys87, located in or adjacent to Ω-loop D (residues 70–85), which positions Met80 for binding to the heme. Binding of cytochrome <i>c</i> to cardiolipin disrupts Met80 heme binding, permitting peroxidase activity. Binding of cardiolipin to yeast iso-1-cytochrome <i>c</i> versus human cytochrome <i>c</i> is compared to assess how binding of cardiolipin to site A has evolved for cytochrome <i>c</i> from species that do not have a complete intrinsic apoptotic pathway to species that do. Using a nondestructive method of quantifying cardiolipin concentration, highly reproducible binding curves are obtained. The results indicate two sequential structural rearrangements on the surface of 100% cardiolipin vesicles. The first, more modest, structural rearrangement occurs at an exposed (outer leaflet) lipid:protein ratio of 8–10 for both cytochromes <i>c</i>. The second, occurring at higher lipid:protein ratios, causes significant unfolding of cytochrome <i>c</i> and requires a much higher lipid:protein ratio for human versus yeast cytochrome <i>c</i>. Higher lipid:protein ratios enhance the peroxidase activity of cytochrome <i>c</i>, suggesting that human cytochrome <i>c</i> has evolved a more stringent on/off switch for cardiolipin peroxidation in the early stages of apoptosis. For both human and yeast cytochrome <i>c</i>, the K72A mutation has only minor effects on binding to site A, suggesting that other nearby lysines can compensate for the lack of Lys72