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

The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyzes the transfer of electrons from photoreduced ferredoxin to NADP+ during photosynthesis and serves as a model for a broad superfamily of enzymes including NO synthase, cytochrome P450 reductase, and NADPH oxidases. Our goal is to define the mechanistic details of hydride transfer between FAD and NADPH using corn root FNR as a model.In initial studies of spinach FNR crystals, the nicotinamide binding site was seen to be blocked by an aromatic side chain (Tyr) lying close and parallel to the re-face of the flavin. Using a Tyr to Ser mutant of pea FNR, 1.8 Å resolution structures with NADP(H) bound were obtained,1 revealing an unexpected binding mode in which the nicotinamide ring laid against the FAD isoalloxazine at a ~30° angle. Although similar complexes have been seen in other FNR superfamily members, based on stopped-flow studies it has been claimed that these complexes are non-productive2. To resolve this question and better define the mechanistic details of hydride transfer, we carried out spectroscopic studies and determined higher resolution structures of FNR-NADP(H) complexes. Here, we present the structures of wild type corn root FNR at ~1 Å resolution, along with ~1.5 Å resolution Y316A and Y316S mutants in complex with nicotinamide, NADP+, and NADPH. These enzymes are active and spectra of the crystalline complexes match those from solution studies. Also a reinterpretation of the earlier stopped-flow studies supports the relevance of these complexes to catalysis. Furthermore, our structures reveal more detailed information about the hydride transfer reaction. In particular, the complexes show higher anisotropic mobility of the C4 atom of NADP+ compared to NADPH, very short contact distances between NADPH and FAD, and distortion of FAD geometry that implicate active site compression as a key factor enhancing hydride transfer in the FNR superfamily. References 1 Deng, Z. et al. (1999), A productive NADP+ binding mode of ferredoxin-NADP + reductase revealed by protein engineering and crystallographic studies, 6: 847-853. 2 Lans, I. et al. (2010), Mechanism of the hydride transfer between Anabaena Tyr303Ser FNR(rd)/FNR(ox) and NADP+/H. A combined pre-steady-state kinetic/ensemble-averaged transition-state theory with multidimensional tunneling study, 114: 3368-3379.