Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. I. Binding of cytochrome c to cardiolipin/phosphatidylcholine membranes in the absence of oxidase.
The mechanism of interaction between cytochrome c and a solid-supported planar phosphatidylcholine membrane containing varying amounts of cardiolipin (0-20 mol%) has been studied over a wide range of protein concentrations (0-450 microM) and ionic strength conditions (10-150 mM), by direct measurement of protein binding using surface plasmon resonance (SPR) spectroscopy. The results demonstrate that cytochrome c binds to such phospholipid membranes in two distinct phases characterized by very different (approximately one order of magnitude) affinity constants. The second phase is dependent upon the prior occurrence of the first binding process. Although the binding affinities for both modes of binding are highly sensitive to both the cardiolipin concentration and the ionic strength of the buffer solution, indicating that electrostatic forces are involved in these processes, binding cannot be reversed by salt addition or by dilution. Furthermore, the final saturation levels of adsorbed protein are independent of ionic strength and cardiolipin concentration. These observations suggest that binding involves more than a simple electrostatic interaction. Invariance in the shapes of the SPR spectra indicates that no major structural transitions occur in the proteolipid membrane due to cytochrome c binding, i.e., the bilayer character of the lipid phase appears to be preserved during these interactions. Based on these results, a model of the lipid membrane-cytochrome c interaction is proposed that involves varying degrees of protein unfolding and subsequent binding to the membrane interior via hydrophobic forces.
Salamon
Z
Z
Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Tollin
G
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; D007834:Lasers; D008051:Lipid Bilayers; D008961:Models, Structural; D008962:Models, Theoretical; D009994:Osmolar Concentration; D010713:Phosphatidylcholines; D017785:Photons; D011485:Protein Binding; D013057:Spectrum Analysis
D002308:Cardiolipins; D003574:Cytochrome c Group; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D003576:Electron Transport Complex IV