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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