Binding of yeast and human cytochrome c to cardiolipin nanodiscs at physiological ionic strength.

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]