CATALYTIC MECHANISM OF CYTOCHROME OXIDASE

Cytochrome c oxidase, the terminal enzyme in the electron transfer chain, is responsible for more than 90% of the O2 consumption by living organisms on the earth. It plays a vital role in physiology owing to the dependence of essentially all vital organs on aerobic metabolism. The molecular basis for its catalytic mechanism (the 4-electron reduction of O2 to H2O) and the associated proton translocation remain to be clearly understood despite intensive work on the enzyme. In part, this results from the difficulty of following a reaction which proceeds as fast as the cytochrome oxidase catalyzed reduction of O2. To overcome this limitation, the reaction can be initiated by photodissociating the CO- bound enzyme in the presence of O2 (flow-flash-probe method). Advances have been made in recent years by combining this method with Raman scattering analysis of intermediates in the reaction. In the present application new experiments are proposed which, by utilizing Raman scattering, optical absorption, and spin resonance, will fully characterize the catalytic intermediates, including the kinetics for their formation and decay. This will allow us to determine the full catalytic mechanism of the O2 reduction by the enzyme. The mechanism determined by this flow-flash-probe method will be compared to that determined at low temperature by freeze trapping the intermediates (triple trapping). With this latter technique, complete characterization of the intermediates will be achieved by applying Raman scattering, optical absorption, and spin resonance. Finally, the influence of CO on the reaction will be evaluated by comparing the reaction generated by CO- photolysis to that generated by direct mixing of the enzyme with O2. First, a series of experiments will be carried out studying the reaction by these two methods on the millisecond time scale. Second, new rapid mixing instrumentation will be developed with a dead time of only 10 micros compared to a dead time of a few milliseconds with conventional instrumentation. This shortening of the dead time by about two orders of magnitude will allow the reaction of O2 with oxidase to be studied without the influence of CO. Furthermore, the new instrumentation will allow for the study of a multitude of other enzymatic reactions which was never before possible.