Abstract
The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp(147) and Arg(514) in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.
Highlights
The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners
We report the crystal structure of a mutant cytochrome P450 oxidoreductase (CYPOR) with an engineered disulfide bond between the FAD and FMN domains that is essentially incapable of supporting P450 activity unless the disulfide bond is reduced, demonstrating that CYPOR must undergo a large conformational change in order for the FMN domain to interact with P450
Requirement for Domain Movements in Electron Transfer— The FMN domain of CYPOR is of particular interest as it is the domain that interacts with P450
Summary
Materials—Sodium dithionite, dithiothreitol (DTT), benzphetamine, superoxide dismutase (SOD), catalase (CAT), and horse heart cytochrome c were purchased from Sigma. To measure cyt c reductase activity of the DTT-treated protein, a solution containing 270 mM potassium phosphate buffer and a final concentration of 65 M cyt c and 50 M NADPH was incubated in a cuvette for 5 min at 30 °C in a Cary 300 UVvisible spectrophotometer (Varian). The final concentrations of the reagents in the reaction mixture were 65 M cyt c, 50 M NADPH, 10 nM CYPOR, 750 units of SOD/ml, 1000 units of CAT/ml, in 1 ml of 270 mM potassium phosphate buffer. The final concentrations of reagents in the assay mixture were as follows: P450, 0.2 M; CYPOR, 0.2 M; DLPC, 24 M; benzphetamine, 1 mM; NADPH, 300 M; 750 units of SOD/ ml; and 1000 units of CAT/ml in 100 mM potassium phosphate buffer, pH 7.4.
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