Interaction of NADP(H) with oxidized and reduced P450 reductase during catalysis, studies with nucleotide analogues

Previous studies have shown that the interaction of P450 reductase with bou nd NADP(H) is essential to ensure fast electron transfer through the two fl avin cofactors. In this study we investigated in detail the interaction of the house fly flavoprotein with NADP(H) and a number of nucleotide analogue s. 1,4,5,6-Tetrahydro-NADP, an analogue of NADPH, was used to characterize the interaction of P450 reductase with the reduced nucleotide. This analogu e is inactive as electron donor, but its binding affinity and rate constant of release are very close to those fur NADPH. The 2'-phosphate contributes about 5 kcal/mol of the binding energy of NADP(H). Oxidized nicotinamide d oes not interact with the oxidized flavoprotein, while reduced nicotinamide contributes 1.3 kcal/mol of the binding energy. Oxidized P450 reductase bi nds NADPH with a K-d Of 0.3 mu M, while the affinity of the reduced enzyme is considerably lower, K-d = 1.9 mu M P450 reductase catalyzes a transhydro genase reaction between NADPH and oxidized nucleotides, such as thionicotin amide-NADP(+). acetylpyridine-NADP(+), or [H-3]NADP(+). The reverse reactio n, reduction of [H-3]NADP(+) by the reduced analogues, is also catalyzed by P450 reductase. We define the mechanism of the transhydrogenase reaction a s follows: NADPH binding, hydride ion transfer, and release of the NADP(+) formed. An NADP(+) or its analogue binds to the two-electron-reduced flavop rotein, and the electron-transfer steps reverse to transfer hydride ion to the oxidized nucleotide, which is released. Measurements of the flavin semi quinone content, rate constant for NADPH release, and transhydrogenase turn over rates allowed us to estimate the steady-state distribution of P450 red uctase species during catalysis, and to calculate equilibrium constants for the interconversion of catalytic intermediates. Our results demonstrate th at equilibrium redox potentials of the flavin cofactors are not the sole fa ctor governing rapid electron transfer during catalysis, but conformational changes must be considered to understand P450 reductase catalysis.