Laser flash photolysis as a probe of redox protein-membrane interactions: effect of binding of spinach plastocyanin and horse cytochrome c to lipid bilayer vesicles on the kinetics of reduction by flavin semiquinone.
Spinach plastocyanin binds to both electrically neutral and positively charged lipid bilayer vesicles, whereas cytochrome c only binds electrostatically to negatively charged vesicles. Laser flash photolysis using lumiflavin semiquinone as a reductant demonstrates that the reactivity of plastocyanin is increased as much as 6-fold when it is membrane bound whereas the rate constant for cytochrome c reduction is decreased by approximately a factor of 3. Membrane-bound plastocyanin reduction occurs via a two-step mechanism, probably involving prior association of lumiflavin semiquinone with the bilayer. In contrast, cytochrome c reduction in the membrane-bound state follows simple second-order kinetics, implying that the redox site in the bound state is still accessible to lumiflavin semiquinone in solution, although the rate constant is decreased by approximately 3-fold. These results are interpreted as indicating that the bilayer-protein interaction with plastocyanin leads to a steric blockage of the electron-transfer site from the aqueous phase. Little or no hindrance of the redox site occurs with cytochrome c, suggesting a high degree of mobility of this protein on the bilayer surface. Although the increase in plastocyanin reactivity upon binding to the bilayer is quite interesting, its cause remains unclear and requires further study. The results illustrate the utility of laser flash photolysis as a probe of membrane-protein interactions.
Senthilathipan
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V
Department of Biochemistry, University of Arizona, Tucson 85721.
Tollin
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G
D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D006736:Horses; D007700:Kinetics; D007834:Lasers; D008051:Lipid Bilayers; D009206:Myocardium / Q000378:metabolism; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines; D010782:Photolysis; D010940:Plant Proteins / Q000378:metabolism*; D010944:Plants / Q000378:metabolism; D010970:Plastocyanin / Q000378:metabolism*; D011485:Protein Binding
D003574:Cytochrome c Group; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010940:Plant Proteins; D005182:Flavin-Adenine Dinucleotide; C029276:flavin semiquinone; D010970:Plastocyanin