Project: Research project

Project Details


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.
Effective start/end date1/1/9312/31/96


  • Catalysis