Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding

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.

Membrane Anchor of Cytochrome P450 Reductase Suppresses the Uncoupling of Cytochrome P450

“…The rates were estimated with various NADPH concentrations (Fig. 2) and analyzed with the Michaelis-Menten equation 40) to give the maximal activity and K m NADPH . WT-and Δ60-CPR were evaluated similarly, and the maximal activity of WT-CPR (2.43±0.02 µmol/min/nmol of CPR) was almost the same as that reported previously.…”

“…The rate of cyt c reduction was estimated by absorbance change at 550 nm, which is characteristic of ferrous cyt c. Binding affinity of CPR for NADPH was also evaluated using various amounts of NADPH (1, 3, 5, 10, and 20 µM) in the regenerating system according to a previously reported method. 16,40) Drug Metabolizing Activity of CYP2C19 With the purified WT-and Δ60-CPR, we evaluated the metabolizing activities of CYP2C19 for four drugs, amitriptyline (AMT), imipramine (IMP), omeprazole (OPZ), and lansoprazole (LPZ). We previously reported several kinetic parameters for the metabolism of AMT and IMP with purchased CPR (SigmaAldrich).…”

The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly

“…All other known examples of this type of flavoprotein exist in a conformationally dynamic, 1:1 partnership with its oxidase, either ␣ 1 ␤ 1 (CYP (10) and methionine synthase (12)) or ␣ 2 ␤ 2 (nitric-oxide synthase (11)). Significant modification to their protein architecture was necessary to constrain the molecules for structure determination (10,48). Monomeric SiRFP, formed by removing its N-terminal 60 amino acids, is also dynamic, evidenced by the disordered FMN-binding domain in multiple crystal forms (40); likewise, octomeric SiRFP is conformationally complex, evidenced by its broad band in native gel analysis, even when it is part of the holoenzyme (Fig.…”

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

“…Microsomal NADPH-cytochrome P450 oxidoreductase, which has an FMN and an FAD domain, provides two electrons for reduction of oxygen by microsomal P450s. The microsomal reductase has been crystallized in a closed form in which the flavodoxin-like FMN domain is positioned for reduction by the FAD domain (13) and in a more open form in which the FMN domain is more accessible for interaction with the proximal face of the P450 (8,14,15). Microsomal cytochrome b 5 can also serve as a donor of the second electron, and interactions between cytochrome b 5 and P450s can modulate rates and product profiles (16).…”