Trp-676 facilitates nicotinamide coenzyme exchange in the reductive half-reaction of human cytochrome P450 reductase: Properties of the soluble W676Rand W676A mutant reductases

The kinetics of flavin reduction in two mutant forms of human cytochrome P4 50 reductase have been studied by stopped-flow spectroscopy with absorption and fluorescence detection. The mutant enzymes were altered at the positio n of Trp-676, which, by analogy with the structure of rat CPR, is close to the isoalloxazine ring of the enzyme-bound FAD. We show that mutant CPRs in which Trp-676 has been changed to histidine (W676H) and alanine (W676A) ca n be reduced by NADPH only to the two-electron level in single mixing stopp ed-flow experiments. The concentration dependence of the rate of hydride tr ansfer indicates that the second, noncatalytic NADPH-binding site present i n wild-type CPR is retained in the mutant enzymes. Detailed studies of W676 H CPR indicate that further reduction of the enzyme beyond the two electron level is prevented due to the slow release of NADP(+) from the active site following the first hydride transfer from NADPH, owing to the stability of a reduced enzyme-NADP(+) charge-transfer complex. Reduction to the four-el ectron level is achieved in a sequential mixing stopped-flow experiment. In this procedure, W676H CPR is reacted first with a stoichiometric amount of NADPH, and then, following a delay of 100 ms, with excess NADPH. The data indicate that occupancy of the noncatalytic coenzyme site also hinders NADP (+) release from reduced enzyme. Fluorescence stopped-flow studies of the W 676H and wild-type CPR enzymes reveal that the complex signals associated w ith reduction of wild-type CPR by NADPH are attributable to changes in the environment of residue W676, From these studies, a model is proposed for ni cotinamide binding in wild-type CPR. In this model W676 serves as a trigger to release NADP(+) from the active site following hydride transfer. In the W676H enzyme, the slow release of NADP(+) is a consequence of the combined effects of (i) removing W676 by mutagenesis (thus removing the trigger for displacement) and (ii) the binding of NADPH in the noncatalytic site, thus trapping NADP(+) in the catalytic site.