Evidence for two 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 neutral pH utilizing fluorescence resonance energy transfer from a membrane-incorporated pyrene phospholipid derivative to the heme of cyt c. ATP in millimolar concentrations displaced nearly quantitatively cyt c from membranes containing 17.5 mol% CL or 30 mol% PG. Notably, increasing the acidic phospholipid/PC molar ratio in the liposomes progressively reduced the membrane detachment of cyt c by ATP, and practically no dissociation of cyt c from neat PG or CL liposomes was observed. Complete 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 ratio. 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 increasing CL/PC molar ratios also in the presence of salt. This difference between CL and PG is attributed to the complex electrostatics of the former lipid resulting in its high affinity for protons. The above results can be rationalized in terms of two acidic phospholipid-binding sites in cyt c. The electrostatically interacting site is constituted 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 appear to be available for lipid binding at neutral bulk pH.
Rytömaa
M
M
Department of Medical Chemistry, University of Helsinki, Finland.
Kinnunen
P K
PK
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002308:Cardiolipins / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers; D008081:Liposomes; D009994:Osmolar Concentration; D010713:Phosphatidylcholines / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D002308:Cardiolipins; D003574:Cytochrome c Group; D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010743:Phospholipids; D000255:Adenosine Triphosphate