NADPH-cytochrome P450 oxidoreductase. Structural basis for hydride and electron transfer

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• NADPH-cytochrome P450 oxidoreductase is crucial for electron transfer to P450 enzymes.
• The enzyme utilizes two flavin cofactors (FAD and FMN) for catalysis.
• Crystal structures indicate Trp-677 blocks nicotinamide access to FAD in the wild-type enzyme.

Purpose of the Study:

• To investigate the role of Trp-677 in the interaction between nicotinamide and FAD during catalysis.
• To elucidate the mechanism of electron transfer in NADPH-cytochrome P450 oxidoreductase.
• To understand the conformational changes involved in the enzyme's catalytic cycle.

Main Methods:

• X-ray crystallography was used to determine the structures of three mutant NADPH-cytochrome P450 oxidoreductase proteins.
• Mutants included W677X (Trp-677 and Ser-678 removed), W677G (Trp-677 replaced with Glycine), and a triple mutant (S457A/C630A/D675N).
• Structural analysis involved superimposing mutant structures with the wild-type enzyme.

Main Results:

• In W677X and W677G mutants, the NADP(+) nicotinamide moiety tilted ~30 degrees relative to the FAD isoalloxazine ring.
• The S457A/C630A/D675N mutant structure provided further insights into catalytic residue interactions.
• Significant mobility was observed between the two flavin domains, and the FMN domain showed disorder in all mutants.

Conclusions:

• A mechanism for hydride transfer is proposed, regulated by altered hydrogen bonding and pi-pi interactions involving the nicotinamide, FAD, and Trp-677.
• Conformational changes, including domain mobility and FMN domain disorder, likely occur during the enzyme's catalytic cycle.
• These findings highlight the dynamic nature of NADPH-cytochrome P450 oxidoreductase and the structural basis for its electron transfer function.