DPNH synergism of TPNH-dependent mixed function oxidase reactions.

The role of TPNH as the donor of reducing equivalents to microsomal mixed function oxidations of drugs, other foreign compounds, and steroids in the liver is well established (1-5). DPNH will not substitute for TPNH in the cyt#{243}chrome P-450-mediated oxidation of drugs, but reaction rates are increased when both nucleotides are present (61 1 ). Although this synergistic role of DPNH has been known since 1949 (6), the mechanism whereby DPNH contributes electrons to the system remained largely unexplored until the recent investigations of Estabrook and associates (12-16). The mixed function oxidase system involving cytochrome P-450 requires two electrons for the coupled reduction of oxygen and the oxygenation of substrate, but the reduction of cytochrome P-450 requires only one electron ( I 719). The contribution of the first electron from TPNH in the reduction of cytochrome P-450 is well documented (17, 20, 21). In their search for the origin of the second electron, Estabrook and associates considered the possibility of its being contributed by DPNH and transferred via cytochrome b5 to the oxygenated cytochrome P-450-substrate complex. They provided substantial evidence for the following sequence: a) substrate binds to oxidized cytochrome P-450; b) an electron is transferred from TPNH via TPNH-cytochrome c reductase (TPNH-cytochrome P-450 reductase) to the oxidized cytochrome P-450-substrate complex; c) the reduced cytochrome P-450-substrate complex combines with oxygen and this complex is reduced by an electron from cytochrome b5 which may