Identification of the high and low potential flavins of liver microsomal NADPH-cytochrome P-450 reductase.

NADPH-cytochrome P-450 reductase, which catalyzes electron transfer to cytochrome P-450 in liver microsomes in reactions leading to the hydroxylation of a variety of substrates, is known to contain 1 molecule each of FMN and FAD per polypeptide chain. The spectrophotometric changes accompanying stepwise reduction of the oxidized enzyme under anaerobic conditions have recently been described. In addition, the stable semiquinone form produced by air oxidation of the reduced enzyme has been shown to be the l-electron-reduced species with the flavin of higher potential in the semiquinone state and the other in the oxidized state. In the present study, FMN was selectively removed from the reductase and the properties of the modified protein were compared with those of the native enzyme. The FMN-depleted enzyme lost the ability to catalyze electron transfer to the phenobarbital-inducible form of liver microsomal cytochrome P-450 in the reconstituted hydroxylation system as well as to cytochrome c and some other artificial acceptors, but retained activity toward ferricyanide and 3-AcPyADP. All catalytic activities were restored when the depleted enzyme was incubated with FMN. From fluorescence measurements a value of 1.3 x lo-’ M was determined for the FMN dissociation constant under the conditions used. Riboflavin and FAD were also bound by the FMNdepleted enzyme, but less effectively than FMN. A series of spectrophotometric experiments were carried out to determine whether the properties of the FMN-depleted enzyme correspond to those of the high or low potential flavin of the native enzyme, which have h”o values of -0.190 and -0.328 V, respectively. Addition of NADP to the fully reduced, FMN-depleted reductase resulted in significant oxidation of flavin, indicating a midpoint potential for FAD near, rather than above, that of the pyridine nucleotide couple. The semiquinone form of the FMN-depleted reductase, which was produced during air oxidation of NADPHreduced enzyme or during stepwise photochemical reduction of oxidized enzyme under anaerobic conditions, had spectral characteristics similar to those of the semiquinone of the low potential flavin of the native enzyme and was readily oxidized under aerobic conditions. Addition of oxidized FMN to l-electron-reduced, FMN-depleted reductase under anaerobic conditions produced an enzyme species with properties similar to those of the l-electron-reduced form of the native en-