Role of Thr(66) in porcine NADH-cytochrome b5 reductase in catalysis and control of the rate-limiting step in electron transfer.

Site-directed mutagenesis of Thr(66) in porcine liver NADH-cytochrome b(5) reductase demonstrated that this residue modulates the semiquinone form of FAD and the rate-limiting step in the catalytic sequence of electron transfer. The absorption spectrum of the T66V mutant showed a typical neutral blue semiquinone intermediate during turnover in the electron transfer from NADH to ferricyanide but showed an anionic red semiquinone form during anaerobic photoreduction. The apparent k(cat) values of this mutant were approximately 10% of that of the wild type enzyme (WT). These data suggest that the T66V mutation stabilizes the neutral blue semiquinone and that the conversion of the neutral blue to the anionic red semiquinone form is the rate-limiting step. In the WT, the value of the rate constant of FAD reduction (k(red)) was consistent with the k(cat) values, and the oxidized enzyme-NADH complex was observed during the turnover with ferricyanide. This indicates that the reduction of FAD by NADH in the WT-NADH complex is the rate-limiting step. In the T66A mutant, the k(red) value was larger than the k(cat) values, but the k(red) value in the presence of NAD(+) was consistent with the k(cat) values. The spectral shape of this mutant observed during turnover was similar to that during the reduction with NADH in the presence of NAD(+). These data suggest that the oxidized T66A-NADH-NAD(+) ternary complex is a major intermediate in the turnover and that the release of NAD(+) from this complex is the rate-limiting step. These results substantiate the important role of Thr(66) in the one-electron transfer reaction catalyzed by this enzyme. On the basis of these data, we present a new kinetic scheme to explain the mechanism of electron transfer from NADH to one-electron acceptors including cytochrome b(5).