Role of active site loop in coenzyme binding and flavin reduction in cytochrome P450 reductase

•An active site loop in human CPR comprises the residues Asp634, Ala635 and Arg636.•Electrostatic repulsion between Asp634 and NADPH causes reorientation of Trp679.•Mutation of Asp634 decreases rate constant for NADPH-dependent flavin reduction.•Asp634 lowers the energy barrier for displacement of the FAD-shielding Trp679.•Arg634 gates interflavin electron transfer with hydrogen bonds to the FMN-domain.

Cytochrome P450 reductase (CPR) contains a loop within the active site (comprising Asp634, Ala635, Arg636 and Asn637; human CPR numbering) that relocates upon NADPH binding. Repositioning of the loop triggers the reorientation of an FAD-shielding tryptophan (Trp679) to a partially stacked conformer, reducing the energy barrier for displacement of the residue by the NADPH nicotinamide ring: an essential step for hydride transfer. We used site-directed mutagenesis and kinetic analysis to investigate if the amino acid composition of the loop influences the catalytic properties of CPR. The D634A and D634N variants elicited a modest increase in coenzyme binding affinity coupled with a 36- and 10-fold reduction in cytochrome c3+ turnover and a 17- and 3-fold decrease in the pre-steady state rate of flavin reduction. These results, in combination with a reduction in the kinetic isotope effect for hydride transfer, suggest that diminished activity is due to destabilization of the partially stacked conformer of Trp677 and slower release of NADP+. In contrast, R636A, R636S and an A635G/R636S double mutant led to a modest increase in cytochrome c3+ reduction, which is linked to weaker coenzyme binding and faster interflavin electron transfer. A potential mechanism by which Arg636 influences catalysis is discussed.

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