The long term goal of this research program is to attain an understanding of the mechanism and regulation of cytochrome P-450 catalyzed monooxygenation reactions. These enzymes are important because they are involved in the oxidation of drugs, synthesis of steroid hormones, activation of carcinogens and metabolism of polyunsaturated fatty acids. The research has focused on the bacterial cytochrome P-450 which catalyzes the oxidation of camphor because this enzyme system can be readily isolated in pure form in quantities which are sufficient to be used for this study. In the coming grant period the research will be focused on the identification of the transient intermediates which are formed during the reduction of cytochrome P-450 by reduced putidaredoxin, the physiological reductant. The kinetics of formation and decay of these intermediates will be measured with stopped-flow and temperature jump spectrophotometry. The intermediates will be trapped by freeze-quench techniques so that the EPR spectrum can be examined and the identity of the intermediates confirmed. The kinetics and control of the overall reaction of cytochrome P-450 will be evaluated with an appropriate computer model so that the precise control of this important enzyme system can be better appreciated.
