Cytochrome c oxidase exhibits a rapid conformational change upon reduction of CuA: a tryptophan fluorescence study

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Abstract
When cytochrome c oxidase is reduced, it undergoes a conformational change that shifts its tryptophan fluorescence maximum from 329 to 345 nm. Studies of ligand-bound, mixed-valence forms of the enzyme show that this conformational change is dependent on the redox state of the low-potential metal centers, cytochrome a and CuA. The intrinsic fluorescence of oxidized cytochrome c oxidase is not effectively quenched by Cs+; however, marked quenching is observed for the reduced enzyme with a Stern-Volmer constant of 0.69. These observations, together with the significant red shift of the emission maximum, suggest that the emitting tryptophan residues are becoming more solvent accessible in the reduced enzyme. Stopped-flow spectra show that this conformational transition occurs rapidly upon reduction of the low-potential sites with a pseudo-first-order rate constant of 4.07 .+-. 0.40 s-1. The conformational change monitored by tryptophan fluorescence is suggested to be related to the previously proposed "open-closed" transition of cytochrome c oxidase. Reductive titration of the cyanide-inhibited enzyme with ferrocytochrome c shows a nonlinear response of the fluorescence shift to added electron equivalents. A theoretical treatment of the reduction of the two interacting sites of the cyanide-inhibited enzyme has been developed that gives the population of each redox state as a function of the total number of electrons accepted by the enzyme. This treatment depends on two parameters: the difference in redox potential between the two metals and the redox interaction between the redox centers. By use of literature values of these two parameters for the cyanide-inhibited enzyme, the expected behavior has been evaluated for three situations: (i) a conformational change induced by reduction of both cytochrome a and CuA (a two-electron process), (ii) a conformational change induced by reduction of cytochrome a (a one-electron process), and (iii) a conformational change induced by reduction of CuA (also a one-electron process). The results of this theoretical treatment strongly suggest that the current data, as well as independent data from other laboratories, are most consistent with a conformational change induced by reduction of the CuA center. The nature of this conformational change and its implication for proton pumping mechanisms of the enzyme are discussed.