Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism

Abstract

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)

Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation

“…To determine the end point of the ET reaction from Cyt c to CcO, a small amount of potassium ferricyanide(III) was added to the reaction solution. A first-order oxidation of reduced Cyt c (Equation 1) was observed as reported in previous papers (21,22):…”

Molecular Basis of Coupled Protein and Electron Transfer Dynamics of Cytochrome c in Biomimetic Complexes

“…The predictions of MD simulations on Cyt/SAM biomimetic systems are also in agreement with previous experimental results indicating differential adsorption of Cyt +2 and Cyt +3 to CcO, as well as rearrangements of the complex upon interprotein ET. [44][45][46][47] Moreover, it has been shown that increasing transmembrane potentials slow down the catalytic activity of CcO toward Cyt. 48,49 In the same line, the reaction between Cyt and CcP is inhibited at low ionic strengths, presumably due to electrostatic freezing of the interprotein complex.…”

Cytochrome oxidase, ligands and electrons

“…This observation was originally interpreted to imply two cyt c binding sites on CcOX, but in view of the information now available, this interpretation is not wholly satisfactory. Alternative explanations have been offered [105][106][107][108], but this point remains as yet obscure; given that the biphacisity tends to vanish at ionic strengths 50 mM, this phenomenon may have limited physiological significance. Nevertheless, it must be explained for a more complete understanding of the in vitro properties of the enzyme.…”