INVOLVEMENT OF SERINE-96 IN THE CATALYTIC MECHANISM OF FERREDOXIN-NADP(- STRUCTURE-FUNCTION RELATIONSHIP AS STUDIED BY SITE-DIRECTED MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY() REDUCTASE )

The crystal structure of ferredoxin-NADP(+) reductase (FNR) suggests t hat Ser96 is directly involved in hydride transfer between the isoallo xazine moiety of FAD and the nicotinamide ring of NADP(H). To probe it s role, Ser96 has been mutated to valine (S96V) and glycine (S96G). Th ese mutations primarily affected the interaction of the nicotinamide r ing with the flavin. Absorbance, fluorescence, and circular dichroism spectra and the crystal structure of FNR-S96V indicate that this mutan t folds properly. FNR-S96V shows only 0.05% of wild-type activity, whi le the affinities for both ferredoxin and NADP(+) are virtually unchan ged. However, spectral perturbations induced by NADP(+) binding to FNR -S96V strongly resemble those elicited by the binding of 2'-monophosph oadenosine-5'diphosphoribose, a substrate analog lacking the nicotinam ide ring, both to the mutant and wild-type enzymes. Rapid reaction stu dies on the valine mutant failed to detect charge-transfer intermediat es during flavin reduction by NADPH. In addition, no semiquinone forma tion was seen during photoreduction of FNR-S96V. The three-dimensional structure of the valine mutant shows small, albeit definite, changes only in the isoalloxazine microenvironment. The glycine mutant of FNR displays behavior intermediate between that of wild-type enzyme and th at of the valine mutant. It maintains ca. 2% of the wild-type activity as well as the ability to form the charge-transfer species between re duced FNR and NADP(+). In photoreduction experiments, the same degree of flavin semiquinone stabilization was observed with FNR-S96G and wit h the wild-type enzyme. NADP(+) binding to the glycine mutant was very similar to that observed in the case of the valine mutant. Thus, thes e mutations of Ser96 clearly interfere with the proper binding of the nicotinamide and with the stabilization of the transition state during hydride transfer between nicotinamide and FAD. Furthermore, both muta tions seem to alter the redox properties of FAD, leading to either des tabilization of semiquinone (FNR-S96V) or stabilization of the reduced flavin (FNR-S96G).