Interaction of cytochrome c with cytochrome c oxidase: an understanding of the high- to low-affinity transition.
The steady-state kinetics of high- and low-affinity electron transfer reactions between various cytochromes c and cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) preparations were studied spectrophotometrically and polarographically. The dissociation constants for the binding of the first and second molecules of horse cytochrome c (I = 15 mM) are 5.10(-8) M and 1.10(-5) M, respectively, close to the spectrophotometric Km values and consistent with the controlled binding model for the interaction between cytochrome c and cytochrome oxidase (Speck, S.H., Dye, D. and Margoliash, E. (1984) Proc. Natl. Acad. Sci. USA 81, 346-351) which postulates that the binding of a second molecule of cytochrome c weakens that of the first, resulting in low-affinity kinetics. While the Km of the polarographically assayed high-affinity reaction is comparable to that observed spectrophotometrically, the low-affinity Km is over an order of magnitude smaller and cannot be attributed to the binding of a second molecule of cytochrome c. Increasing the viscosity has no effect on the Vmax of the low-affinity reaction assayed polarographically, but increases the Km. Thus, the transition from high- to low-affinity kinetics is dependent on the frequency of productive collisions, as expected for a hysteresis model ascribing the transition to the trapping of the oxidase in a primed state for turnover. At ionic strengths above 150 mM, the rate of cytochrome c oxidation decreases without any correlation to the calculated net charge of the cytochrome c, indicating rate-limiting rearrangement of the two proteins in proximity to each other.
Garber
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EA
Northwestern University, Department of Biochemistry, Evanston, IL.
Margoliash
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D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003574:Cytochrome c Group / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism; D009994:Osmolar Concentration; D011048:Polarography; D011485:Protein Binding; D013057:Spectrum Analysis; D013367:Submitochondrial Particles / Q000201:enzymology; D013772:Tetramethylphenylenediamine / Q000494:pharmacology; D014588:Ustilago / Q000201:enzymology; D014783:Viscosity
D003574:Cytochrome c Group; D003576:Electron Transport Complex IV; D013772:Tetramethylphenylenediamine