Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism.
The long-known biphasic response of cytochrome c oxidase to the concentration of cytochrome c has been explained, alternatively, by the presence of a catalytic and a regulatory site on the oxidase, by negative cooperativity between adjacent active sites in dimeric oxidase, or by a transition of the enzyme molecule between different conformational states. The three mechanistic hypotheses allow testable predictions about the relationship between substrate binding and steady-state kinetics catalyzed by the monomeric and dimeric (or oligomeric) enzyme. We have tested these predictions on monomeric, dimeric, and oligomeric beef heart oxidase and on monomeric oxidase from Paracoccus denitrificans. The aggregation state of the oxidase was evaluated from the sedimentation equilibrium in the ultracentrifuge and by gel chromatography. The binding of cytochrome c to cytochrome c oxidase was measured by spectrophotometric titration of cytochrome c oxidase with cytochrome c. The procedure makes use of a small perturbation in the Soret band of the absorption spectrum of the cytochrome c-cytochrome c oxidase complex. The steady-state oxidation of cytochrome c was followed spectroscopically by an automated assay procedure, and the kinetic parameters were deduced by numerical analysis of several hundred initial rate assays in the substrate concentration range 0.15-30 microM. The following results were obtained: (1) The kinetics of cytochrome c oxidation are always biphasic at low ionic strength, independent of the aggregation state of the enzyme. (2) The kinetics become apparently monophasic at ionic strengths above 100 mM or at slightly acidic pH values.(ABSTRACT TRUNCATED AT 250 WORDS)
Michel
B
B
Biochemisches Institut, Universität Zürich, Switzerland.
Bosshard
H R
HR
D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011487:Protein Conformation
D003574:Cytochrome c Group; D003576:Electron Transport Complex IV