Differential redox and electron-transfer properties of purified yeast, plant and human NADPH-cytochrome P-450 reductases highly modulate cytochrome P-450 activities

Saccharomyces human and two Arabidopsis (ATR1 and ATR2) NADPH-P-450 reducta ses were expressed in yeast, purified to homogeneity and used to raise anti bodies. Among the P-450-reductases, ATR2 contrasted by its very low FMN aff inity and required a thiol-reducing agent for efficient cofactor binding to the FMN-depletedd enzyme. Analysis of reductase kinetic properties using a rtificial accepters and different salt conditions suggested marked differen ces between reductases in their FAD and FMN environments and confirmed the unusual properties of the ATR2 FMN-binding domain. Courses of flavin reduct ions by NADPH were analysed by rapid kinetic studies. The human enzyme was characterized by a FAD reduction rate sixfold to tenfold slower than values for the three other reductases. Following the fast phase of reduction, exp ected accumulation of flavin semiquinone was observed for the human and ATR 1 but not for ATR2 and the yeast reductases. Consistently, redox potential for the FMN semiquinone/reduced couple in the yeast enzyme was found to be more positive than the value for the FMN oxidized/semiquinone couple. This situation was reminiscent of similar inversion observed in bacterial P-450 BM3 reductase. Affinities of reductases for rabbit P-450 2B4 and supported monooxygenase activities in reconstituted systems highly depended on the re ductase source. The human enzyme exhibited the highest affinity but support ed the lowest k(cat) whereas the yeast reductase gave the best k(cat) but w ith the lowest affinity. ATR1 exhibited both high affinity and efficiency. No simple relation was found between reductase activies with artificial and natural (P-450) accepters. Thus marked differences in kinetic and redox pa rameters between reductases dramatically affect their respective abilities to to support P-450 functions.