FMN binding site of yeast NADPH-cytochrome P450 reductase exposed at the surface is highly specific.
NADPH-cytochrome P450 reductase (CPR) transfers two reducing equivalents derived from NADPH via FAD and FMN to microsomal P450 monooxygenases in one-electron transfer steps. The crystal structure of yeast CPR (yCPR) contains a surface-exposed FMN binding site (FMN2 site) at the interface of the FMN binding and connecting domains, in addition to the single buried site that has been observed in rat CPR. This finding provides a testable hypothesis of how intramolecular (between FAD and FMN) and intermolecular (between FMN and P450) electron transfer may occur in CPR. To verify that occupancy of the FMN2 site is not an artifact of crystallization, a surface plasmon resonance (SPR) biosensor technique has been applied to probe the selectivity of this site under functional conditions. A series of kinetic and equilibrium binding experiments involving yCPR immobilized on different sensor chip surfaces was performed using FMN and FAD, as well as FMN-derived compounds, including riboflavin, dimethylalloxazine, and alloxazine, and other molecules that resemble the planar isoalloxazine ring structure. Only FMN and FAD showed stoichiometric binding responses. Binding affinity for FMN was in the submicromolar range, 30 times higher than that for FAD. Association kinetic rates for the yCPR/FMN complex were up to 60-fold higher than for the yCPR/FAD complex. Taken together, these data indicate that (i) the surface-exposed site in yCPR is highly selective toward binding flavins, (ii) binding of FMN in this site is notably favored, and finally, (iii) both the phosphate group and the isoalloxazine ring of FMN are essential for binding.
Ivanov
Alexis S
AS
Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia.
Gnedenko
Oksana V
OV
Molnar
Andrey A
AA
Archakov
Alexander I
AI
Podust
Larissa M
LM
D001665:Binding Sites; D005486:Flavin Mononucleotide / Q000737:chemistry*; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D015003:Yeasts / Q000201:enzymology*
D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase