Evidence for a Hydroxide Intermediate in Cytochrome c

A transient intermediate of cytochrome c oxidase has been generated by exposing the enzyme to a laser beam in the presence of oxygen. This intermediate develops when the enzyme is simultaneously reduced photoreductively and oxidized chemically, thereby forcing it to turn over. Under these conditions a form of the enzyme is generated with a line at 477 cm" in the resonance Raman spectrum, which we attribute to an Fe-OH stretching mode based on oxygen and hydrogen isotopic substitution. This hydroxide intermediate relaxes back to the resting state of the enzyme upon removal from the laser beam. Hydroxide intermediates have been postulated many times in the past in proposed catalytic mechanisms. The data reported here supply the first evidence for the existence of such an intermediate and a method for stabilizing it. Cytochrome c oxidase, the terminal enzyme in the electron transport chain, catalyzes the reduction of oxygen to water by the transfer of four electrons. Cytochrome c is the electron donor to the cytochrome c oxidase that contains four redox centers, two heme groups (cytochrome a and cytochrome as), and two copper atoms. The iron atom of cytochrome a3 is the oxygen binding site (1,2). The four-electron reduction process is very complex, and several intermediates have been proposed to be part of the catalytic mechanism (2-6). However, data on the structure and properties of each of the intermediates are difficult to obtain owing to their short lifetime during the catalytic function of the enzyme. In order to determine the complete catalytic mechanisms of cytochrome c oxidase, it is first necessary to establish the properties of as many intermediates as possible and subsequently determine which ones are present during the enzymatic activity. Resonance Raman scattering is a powerful technique with which to study cytochrome c oxidase because the spectra are rich in information about the heme groups (7). In addition, vibrational modes of ligands bound to the heme in cytochrome a3, which is the oxygen binding site, may be assigned by isotopic substitution measurements and the properties of these modes, which reflect ligand structure, may be studied (8). In this communication we use the Raman technique to identify a hydroxide intermediate of cytochrome c oxidase. The intermediate is generated by laser illumination, and its * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.