Evidence for a hydroxide intermediate in cytochrome c oxidase.

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-1 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.