Purified Liver Microsomal NADPH-Cytochrome P-450 Reductase

NADPH-cytochrome P-450 reductase was isolated from liver microsomes of phenobarbital-induced rats. The enzyme exhibits an apparent minimal molecular weight of 76,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 molecule each of FMN and FAD. Trypsin treatment of the reductase yields an enzyme with an apparent minimal molecular weight of 69,000 which retains the ability to reduce cytochrome c but has no activity toward cytochrome P-450. Various spectrophotometric titrations were performed to examine the electron-accepting properties of the purified NADPH-cytochrome P-450 reductase and, in particular, to determine the oxidation state of the stable semiquinone form produced by air oxidation of NADPH-reduced enzyme. Titration of the air-stable semiquinone form of the reductase with ferricyanide indicated that 1 mol/Z mol of flavin was required for complete oxidation. Furthermore, a spectrum corresponding to that of the air-stable semiquinone form was produced by the addition of approximately 0.5 mol of reductanU2 mol of flavin when the oxidized enzyme was titrated with NADPH or dithionite under anaerobic conditions. The spectral changes which accompanied the overall reduction of oxidized enzyme to the reduced form with dithionite produced four sets of isosbestic points, and the spectrophotometric titration curve consisted of four approximately equal phases. In the titration with NADPH, no significant further reduction was observed after the addition of approximately 1.5 mo1/2 mol of flavin. However, the enzyme was fully reduced by NADPH when an NADPHgenerating system was used to prevent the accumulation of NADP. Our results establish that the air-stable semiquinone form is a l-electron-reduced form, rather thr n a half-re-