Structural and Kinetic Studies of Asp632 Mutants and Fully Reduced NADPH-Cytochrome P450 Oxidoreductase Define the Role of Asp632 Loop Dynamics in the Control of NADPH Binding and Hydride Transfer

Abstract

Conformational changes of NADPH-cytochrome P450 oxidoreductase (CYPOR) associated with electron transfer from NADPH to electron acceptors via FAD and FMN have been investigated through structural studies of the 4-electron-reduced NADP+-bound enzyme and kinetic and structural studies of mutants affecting the conformation of the mobile Gly631-Asn635 loop (Asp632 loop). The structure of 4-electron-reduced, NADP+-bound wild type CYPOR shows the plane of the nicotinamide ring positioned perpendicular to the FAD isoalloxazine with its carboxamide group forming H-bonds with N1 of the flavin ring and the Thr535 hydroxyl group. In the reduced enzyme, the C8-C8 atoms of the two flavin rings are ~1 Å closer compared to the fully oxidized and 1-electron-reduced structures, which suggests that flavin reduction facilitates interflavin electron transfer. Structural and kinetic studies of mutants, Asp632Ala, Asp632Phe, Asp632Asn, and Asp632Glu demonstrate that the carboxyl group of Asp632 is important for stabilizing the Asp632 loop in a retracted position that is required for the binding of the NADPH ribityl-nicotinamide in a hydride-transfer-competent conformation. Structures of the mutants and reduced wild type CYPOR permit us to identify a possible pathway for NADP(H) binding/release to/from CYPOR. Asp632 mutants unable to form stable H-bonds with the backbone amides of Arg634, Asn635, and Met636, exhibit decreased catalytic activity and severely impaired hydride transfer from NADPH to FAD, but leave interflavin electron transfer intact. Intriguingly, the Arg634Ala mutation slightly increases the cytochrome P450 2B4 activity. We propose that Asp632 loop movement, in addition to facilitating NADP(H) binding and release, participates in domain movements modulating interflavin electron transfer.

Coupling of Redox and Structural States in Cytochrome P450 Reductase Studied by Molecular Dynamics Simulation

“…Then, the redox state change, which is accompanied by the net charge change, could affect the electrostatic interaction between the FAD and the FMN domains. Binding of NADP + , which carries net negative charge, could also affect the interaction between the two domains, triggered by local rearrangement of the electrostatic bonds; Actually, such rearrangement of the electrostatic bonds involving Asp632, which is located near the binding site of NADP + and was noticed in the recent structural studies 27,28 , was observed in our MD simulation (data not shown). In addition, the redox state change in FMN (and also in heme) should affect the electrostatic interaction between the FMN domain and monooxygenase 29 .…”

Riboflavin Directly Mediates the Dealkylation by Microbial Cytochrome P450 Monooxygeneses

“…As anticipated, in addition to con-served residues of Cys347 and Thr241, both Arg233 and Asp74 could remarkably affect the activity of HmtS-L340F from the mutagenesis. In the complexed model consisting of enzyme, NADPH and riboflavin, Arg233 is the site to bind riboflavin ( Figure S17 and S18), and Asp74, similar to the situation of Asp632 in NADPH-cytochrome P450 oxidoreductase, 29 is responsible for the binding with NADPH ( Figure S20). Not surprisingly, the significant reduction of activity from the mutations of Arg233 and Asp74 was in accordance with the results of MD simulation.…”

Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches

“…The areas of greatest deshielding are seen in (i) the hinge domain of the CPR, (ii) within the beta-sheets of the CPR FMN domain, and (iii) in a peripheral alpha helix within the FMN domain (L2). Studies have suggested that NADP + binding to the CPR domain induces a closed conformation in which the FAD and FMN cofactors are a short distance apart (ca 4 Å) (38). On reduction by NADPH, an open conformation is adopted as the FAD/NADPH-and FMNbinding domains move apart to enable FMN domain/heme domain interactions (38).…”

“…Studies have suggested that NADP + binding to the CPR domain induces a closed conformation in which the FAD and FMN cofactors are a short distance apart (ca 4 Å) (38). On reduction by NADPH, an open conformation is adopted as the FAD/NADPH-and FMNbinding domains move apart to enable FMN domain/heme domain interactions (38). An important residue for the dissociation of NADP + is Ser634.…”

“…An important residue for the dissociation of NADP + is Ser634. Unfortunately, as no full-length structure has been determined for the BM3 CPR domain, the Ser634 residue is not represented in the CPR model and so the HDX data for Ser634 and its associated loop region cannot be mapped accurately (38). The deuterium uptake for this residue showed very little change across different state comparisons.…”