MULTIPLE CONFORMATIONS OF PHYSIOLOGICAL MEMBRANE-BOUND CYTOCHROME-C

One-tenth of cytochrome c (cyt c) remains bound to the inner mitochond rial membrane (IMM) at physiological ionic strength (I; i.e., I approx imate to 150 mM), exhibiting decreased electron transport (ET) activit y. We now show that this form of membrane-bound cyt c (MB-cyt c) can b e obtained in vitro and that binding to membranes at low I generates a n additional conformation with higher ET activity, This low I bound fo rm of MB-cyt c (MBL-cyt c) exhibited intrinsic ET rates similar to tho se of electrostatically bound cyt c (EB-cyt c). The ET activity of IMM -bound MB-cyt c approached slowly that of MBL-cyt c or EB-cyt c, sugge sting that MB-cyt c converts to MBL-cyt c while bound to IMM, When mai ntained at physiological I, both forms of MB-cyt c were released from the membrane, indicating that they convert to an EB-cyt c-like form. T his process may be very dynamic in cellular mitochondria, as binding a nd release for both MB-cyt c forms increased considerably with tempera ture. I-Dependent binding of MB-cyt c does not require IMM, and it can be reproduced using large or small unilamellar vesicles (SUV). Using SUV-cyt c complexes, we characterized the secondary structure of MB-cy t c and MBL-cyt c by circular dichroism. Conformational analysis revea led that cyt c binding as MB-cyt c decreases its alpha-helical content (70-79%) and increases its beta-sheet up to 135%. The secondary struc ture of MBL-cyt c was similar to that of EB-cyt c and soluble cyt c, w ith a modest increase in beta-sheet, Taken together, our experiments s uggest that physiological cyt c exists in soluble and membrane-bound c onformations with similar ET activity, which may exchange very rapidly , and that soluble hydrophilic proteins can bind transiently to biomem branes.