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Binding of cytochrome c (Cyt c) to membranes containing cardiolipin (CL) is of considerable interest because of the importance of this interaction in the early stages of apoptosis. The molecular-level determinants of this interaction are still not well defined and there appear to be species-specific differences in Cyt c affinity for CL-containing membranes. Many studies are carried out at low ionic strength far from the 100–150 mM ionic strength within mitochondria. Similarly, most binding studies are done at Cyt c concentrations of 10 μM or less, much lower that the estimated range of 0.1 to 5 mM Cyt c present in mitochondria. In this study, we evaluate binding of human and yeast Cyt c to CL nanodiscs using size exclusion chromatography at 25 μM Cyt c concentration and 100 mM ionic strength. We find that yeast Cyt c affinity for CL nanodiscs is much stronger than that of human Cyt c. Mutational analysis of the site A binding surface shows that lysines 86 and 87 are more important for yeast Cyt c binding to CL nanodiscs than lysines 72 and 73, counter to results at lower ionic strength. Analysis of the electrostatic surface potential of human versus yeast Cyt c shows that the positive potential due to lysines 86 and 87 and other nearby lysines (4, 5, 11, 89) is stronger than that due to lysines 72 and 73. In the case of human Cyt c the positive potential around site A is less uniform and likely weakens electrostatic binding to CL membranes through site A. Yeast iso-1-cytochrome c binds more strongly to cardiolipin nanodiscs than human cytochrome c at physiological ionic strength because the charge distribution near site A (lysines 72, 73, 86, 87) is more uniformly positive. Mutagenesis studies on site A lysines suggest that N- and C-terminal helix lysines contribute to electrostatic binding. [Display omitted] • Human cytochrome c does not bind to cardiolipin nanodiscs at 100 mM ionic strength. • Yeast iso-1-cytochrome c binds to cardiolipin nanodiscs at 100 mM ionic strength. • The electrostatic surface potential is stronger for yeast versus human cytochrome c. • Site A lysines 86 and 87 are most important for binding at 100 mM ionic strength. [ABSTRACT FROM AUTHOR]

Naturally-occurring variants of human cytochrome c (Cyt c) that induce thrombocytopenia IV occur within Ω-loop C (residues 40–57). These variants enhance the peroxidase activity of human Cyt c apparently by facilitating access to the heme by destabilizing Ω-loops C and D (residues 70–85). Given the importance of peroxidase activity in the early stages of apoptosis, we identified three sites with the EVmutation algorithm in or near Ω-loop C that coevolve and differ between yeast iso-1-Cyt c and human Cyt c. We prepared iso-1-Cyt c variants with all possible combinations of the S40T, V57I and N63T substitutions to determine if these residues decrease the peroxidase activity of iso-1-Cyt c to that of human Cyt c producing an effective off state for a peroxidase signaling switch. At pH 6 and above, all variants significantly decreased peroxidase activity. However, the correlation of peroxidase activity with local and global stability, expected if cooperative unfolding of Ω-loops C and D is required for peroxidase activity, was generally poor. The m -values derived from the guanidine hydrochloride dependence of the kinetics of imidazole binding to horse Cyt c , which is well-characterized by native-state hydrogen exchange methods, and K72A/K73A/K79A iso-1-Cyt c show that local structural fluctuations and not subglobal cooperative unfolding of Ω-loops C and D are sufficient to permit binding of a small molecule like peroxide to the heme. A 2.46 Å structure of N63T iso-1-Cyt c identifies a change to a hydrogen bond network linking Ω-loops C and D that could modulate the local fluctuations needed for the intrinsic peroxidase activity of Cyt c. Substitutions to yeast iso-1-cytochrome c at coevolved residues in Ω-loop C from human cytochrome c have compensatory effects on local and global stability but decrease intrinsic peroxidase activity. Imidazole binding to cytochrome c indicates that small dynamic fluctuations in Ω-loop C may control the on/off switch for apoptotic peroxidase signaling. [Display omitted] • Yeast iso-1-cytochrome c variants at three coevolved sites in Ω-loop C were made. • Effects on local and global stability are modest. • All variants reduce peroxidase activity at pH 6 and above. • Apoptotic peroxidase activity is not solely controlled by Ω-loop C/D stability. • Imidazole binding studies show that local dynamics may mediate peroxidase activity. [ABSTRACT FROM AUTHOR]