The oxidative half-reaction of liver microsomal FAD-containing monooxygenase.

Abstract

The reactions of reduced forms of liver microsomal FAD-containing monooxygenase both with oxygen and with several oxygenatable substrates have been investigated using rapid reaction techniques. It was first shown that the pH optimum was at about 8.8 and that at pH values below 8 the stoichiometry was very nearly 1:1:1 for NADPH/(cysteamine or methimazole)/oxygen consumption, with only traces of H2O2 forming. Low temperatures (approximately 4 degrees C) were employed to better resolve kinetic intermediates occurring in the reaction. It was shown that in the absence of NADP+, substrate has no effect on the rate of reoxidation of reduced enzyme by O2. However, in the presence of NADP+ and the absence of substrate, the reduced enzyme reacts with O2 to form a remarkably stable C(4a)-hydroperoxyflavin species. This occurs in a reaction that saturates with respect to O2, implying the formation of a reversible complex with oxygen prior to the formation of the hydroperoxyflavin. This has been predicted by the steady state kinetics (Poulsen, L. L., and Ziegler, D. M. (1979) J. Biol. Chem. 254, 6449-6455). In the presence of NADP+, substrate increases the rate of reoxidation (by destabilizing the hydroperoxyflavin), but the rate is much slower than the reaction of reduced enzyme with O2 in the absence of NADP+. The stability of the hydroperoxyflavin permitted its preparation on the stopped flow apparatus for subsequent reaction with various substrates. This reaction involved the rapid formation of a species with a new spectrum which was indicative of a C(4a)-substituted flavin. Since this new species converts to oxidized flavin at about 2 min-1, which is equivalent to Vmax, it must be the rate-limiting step in the catalytic reaction. This new spectrum is likely due either to a substrate-hydroperoxyflavin species or to a product-hydroxyflavin species.