Studies on the Activation and Reduction of Dioxygen By Cytochrome P-450.

Abstract

The studies in this thesis are concerned with the mechanism of oxygen activation by cytochrome P-450, a biological catalyst with remarkable versatility in the oxygenation of steroids and other lipids as well as drugs, anesthetics, carcinogens, pesticides, and a variety of other environmental chemicals. The formation and autoxidation of the ferrous dioxygen form of rabbit liver microsomal cytochrome P-450 were studied. Formation of the ferrous dioxygen complex was monophasic while the autoxidation exhibited multiphasic first-order kinetics. Superoxide was not detected as a product of the autoxidation of ferrous dioxygen P-450 while hydrogen peroxide was produced in about 70% yield. P-450 aggregation was ruled out as an explanation for the multiphasic kinetics and lack of superoxide since the kinetics and products of the autoxidation did not change when the usually aggregated protein was converted to the monomeric state. An examination of the stoichiometry of the reactions catalyzed by P-450 isozymes 2, 3a, 3b, and 4 has revealed the occurrence of an NADPH oxidase reaction involving 2 molecules of NADPH/molecule of dioxygen as well as the known 2-electron oxidase reaction producing hydrogen peroxide. The occurrence of a peroxidase reaction in which free hydrogen peroxide is reduced to water by NADPH was ruled out. When the 4-electron oxidase activity was taken into account, measurements of NADPH oxidation and O(,2) consumption were in accord with the amounts of products formed. Cyclohexanol and benzene oxidation were examined in a reconstituted system containing phenobarbital-inducible P-450(,LM(,2)). Steady state kinetic studies with cyclohexanol yielded a K(,m) of 8.7 mM and V(,max) of 5.7 nmol of cyclohexanone formed/min/nmol P-450. Similar studies with benzene yielded a K(,m) of 105 mM and V(,max) of 22. These parameters were found to be highly dissimilar in the oxidations of cyclohexanol or benzene in a model hydroxyl radical-generating system. In addition, superoxide dismutase, desferrioxamine, and catalase had no effect on the P-450-catalyzed oxidations. It was concluded that the oxidations of these compounds by P-450 do not involve the participation of free hydroxyl radicals.