EVIDENCE FOR 2 DISTINCT ACIDIC PHOSPHOLIPID-BINDING SITES IN CYTOCHROME-C

Binding of cytochrome c (cyt c) to cardiolipin/phosphatidylcholine (CL /PC) and phosphatidylglycerol/PC (PG/PC) liposomes was studied at neut ral pH utilizing fluorescence resonance energy transfer from a membran e-incorporated pyrene phospholipid derivative to the heme of cyt c. AT P in millimolar concentrations displaced nearly quantitatively cyt c f rom membranes containing 17.5 mol % CL or 30 mol % PG. Notably, increa sing the acidic phospholipid/PC molar ratio in the liposomes progressi vely reduced the membrane detachment of cyt c by ATP, and practically no dissociation of cyt c from neat PG or CL liposomes was observed. Co mplete dissociation of cyt c from PG/PC liposomes was also produced by subsequently added NaCl. However, the concentration of salt required for half-maximal effect increased upon increasing the PG/PC molar rati o. At 0.1 m NaCl no binding of cyt c to neat PG liposomes was observed whereas the extent of membrane association of cyt c increased with in creasing CL/PC molar ratios also in the presence of salt. This differe nce between CL and PG is attributed to the complex electrostatics of t he former lipid resulting in its high affinity for protons. The above results can be rationalized in terms of two acidic phospholipid-bindin g sites in cyt c. The electrostatically interacting site is constitute d by basic residues in cyt c and could be identical to the ATP-binding site (Craig, D. B., and Wallace, C. J. A. (1993) Protein Sci. 2, 966- 976). In addition there should be another lipid-binding site in cyt c with a high affinity to protonated acidic phospholipids. Both sites ap pear to be available for lipid binding at neutral bulk pH.