High resolution studies of hydride transfer in the ferredoxin: NADP+ reductase superfamily

Ferredoxin-NADP+ reductase (FNR) is an FAD-containing enzyme that catalyzes the transfer of electrons from photoreduced ferredoxin (Fd) to NADP+ during photosynthesis. It is also the prototype for a broad superfamily of enzymes, including nictric oxide synthase, cytochrome P450 reductase, and the NOX family of NADPH oxidases, that all catalyze hydride transfer between NADPH and FAD. The goal of our research is to define the mechanistic details of that hydride transfer reaction. Previous studies using a pea FNR active site mutant revealed a 1.8 Å resolution view of productive NADP(H) binding, and differences in the electron density around the C4 position of NADP+ and NADPH suggested that mobility at this carbon is important for efficient hydride transfer. In order to obtain higher resolution views of the nicotinamide-flavin interaction, we turned to the corn root FNR system for which crystals grown in the absence of NADP(H) diffracted to 1.05 Å resolution. We created the Y316A and Y316S mutants of corn root FNR and have now determined structures at better than 1.5 Å resolution of their complexes with NADP+, NADPH, and nicotinamide. The mutant protein crystallizations required nicotinamide, and the other complexes were generated by subsequent soaks with NADP+ or NADPH. Comparisons among the FNR complexes reveals systematic, but subtle shifts in the active site packing and covalent distorsion that allow us to conclude there is significant compression that pushes the reactant together, thereby enhancing catalysis. Furthermore, the anisotropic B-factors confirm a stricking increase in mobility of the nicotinamide C4 atom in NADP+ as compared to NADPH, and the directionality of the disorder matches the expected motions needed to adopt the boat-like conformation of the nicotinamide ring that is known to enhance hydride transfer.