BINDING AND DISSOCIATION OF CYTOCHROME-C TO AND FROM MEMBRANES CONTAINING ACIDIC PHOSPHOLIPIDS

Membrane association and detachment of cytochrome c (cyt c) in millise cond to second time domain were investigated by stopped-flow fluoresce nce spectroscopy monitoring the efficiency of energy transfer from a p yrene-fatty acid containing phospholipid derivative, (pyren-1-yl)decan oyl]-sn-glycero-3-phosphoglycerol (PPDPG, mole fraction X = 0.01) to t he heme of the cyt c. Large unilamellar liposomes composed of egg phos phatidylcholine (eggPC) with varying content of the acidic phospholipi d phosphatidylglycerol (eggPG) were employed. Unexpectedly, the rate o f binding of cyt c to membranes was attenuated upon increasing the mol e fraction of the acidic phospholipid (X-PG). For example, at 50 mu M phospholipid and 5 mu M cyt c, when X-PG was increased from 0.20 to 0. 40 the half-time for the single-exponential decay in fluorescence incr eased from 4.7 to 8.6 ms. A similar observation was made for the membr ane binding of another cationic protein, histone H1. We suggest that t he formation of cooperative hydrogen-bonded networks by deprotonated a nd protonated PG in the vesicle surface retards the binding of cyt c t o the liposome surface. However, once formed, the complex of cyt c wit h acidic phospholipids is stabilized by increasing X-PG. Accordingly, significantly prolonged half-times of dissociation of cyt c from lipos omes by NaCl, ATP, and different cationic proteins are measured upon i ncreasing X-PG. Differences between the latter cationic membrane bindi ng ligands most likely reflect the varying relative contributions of h ydrophobicity and Coulombic forces to their attachment to liposomes. O ur data on the release and binding of cyt c to liposomes as a function of X-PG and in the presence of ATP also provide the first direct expe rimental evidence for multiple lipid binding sites in cyt c.