Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. II. Binding of cytochrome c to oxidase-containing cardiolipin/phosphatidylcholine membranes.
Complex formation between horse heart cytochrome c (cyt c) and bovine cytochrome c oxidase (cco) incorporated into a supported planar egg phosphatidylcholine membrane containing varying amounts of cardiolipin (CL) (0-20 mol%) has been studied under low (10 mM) and medium (160 mM) ionic strength conditions by surface plasmon resonance (SPR) spectroscopy. Both specific and nonspecific modes of cyt c binding are observed. The dissociation constant of the specific interaction between cyt c and cco increases from approximately 6.5 microM at low ionic strength to 18 microM at medium ionic strength, whereas the final saturation level of bound protein is independent of salt concentration and corresponds to approximately 53% of the total cco molecules present in the membrane. This suggests a 1:1 binding stoichiometry between the two proteins. The nonspecific binding component is governed by electrostatic interactions between cyt c and the membrane lipids and results in a partially ionic strength-reversible protein-membrane association. Thus, hydrophobic interactions between cyt c and the membrane, which are the predominant mode of binding in the absence of cco, are greatly suppressed. Both the amount of nonspecifically bound protein and the binding affinity can be varied over a broad range by changing the ionic strength and the extent of CL incorporation into the membrane. Under conditions approximating the physiological state in the mitochondrion (i.e., 20 mol% CL and medium ionic strength), 1-1.5 cyt c molecules are bound to the lipid phase per molecule of cco, with a dissociation constant of 0.1 microM. The possible physiological significance of these observations is discussed.
Salamon
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Z
Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Tollin
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G
D000818:Animals; D002308:Cardiolipins; D002417:Cattle; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D006736:Horses; D007700:Kinetics; D008051:Lipid Bilayers; D008961:Models, Structural; D010713:Phosphatidylcholines; D011485:Protein Binding
D002308:Cardiolipins; D003574:Cytochrome c Group; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D003576:Electron Transport Complex IV