Involvement of Serine 96 in the Catalytic Mechanism of Ferredoxin-NADP+ Reductase: Structure-Function Relationship As Studied by Site-Directed Mutagenesis and X-ray Crystallography

Abstract

The crystal structure of ferredoxin-NADP+ reductase (FNR) suggests that Ser96 is directly involved in hydride transfer between the isoalloxazine moiety of FAD and the nicotinamide ring of NADP(H). To probe its role, Ser96 has been mutated to valine (S96V) and glycine (S96G). These mutations primarily affected the interaction of the nicotinamide ring with the flavin. Absorbance, fluorescence, and circular dichroism spectra and the crystal structure of FNR-S96V indicate that this mutant folds properly. FNR-S96V shows only 0.05% of wild-type activity, while the affinities for both ferredoxin and NADP+ are virtually unchanged. However, spectral perturbations induced by NADP+ binding to FNR-S96V strongly resemble those elicited by the binding of 2'-monophosphoadenosine-5'-diphosphoribose, a substrate analog lacking the nicotinamide ring, both to the mutant and wild-type enzymes. Rapid reaction studies on the valine mutant failed to detect charge-transfer intermediates during flavin reduction by NADPH. In addition, no semiquinone formation 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 that 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 reduced FNR and NADP+. In photoreduction experiments, the same degree of flavin semiquinone stabilization was observed with FNR-S96G and with the wild-type enzyme. NADP+ binding to the glycine mutant was very similar to that observed in the case of the valine mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of NADPH Oxidase Activity in Phagocytes

“…This serine residue is essential to modulate reactivity of the flavin ring. Indeed, its replacement by an alanine in several FNR members leads to enzymes with only 0.05 and 0.07% catalytic efficiency remaining for spinach FNR and E. coli flavin reductase, respectively (4,35). In addition, mutation of the cytochrome b 5 reductase, which possesses a threonine at this position, leads to comparable effects (36).…”

“…(highly homologous to FNR of S. oleacera) (32). In addition, Thr-341 is replaced by a Ser in the RXYS sequence in the two FNRs, which is the FAD binding domain homologous to HPFT in the Nox family members (4). The amino acids of the ⌬507-509 are similar in Nox1, Nox2, and Nox3 but different in Nox4 and not found in FNRs.…”

“…The catalytic core of phagocyte NADPH oxidase (Nox) is gp91 phox or Nox2, a glycosylated integral membrane protein that is one of the subunits of flavocytochrome b 558 (cytb), with p22 phox being the second one. Heterodimer formation is needed for maturation and targeting of cytb to the plasma membrane or to the membranes of specific granules of phagocytes (3,4). Nox2 is the first member of a large family containing seven Nox analogs to be described.…”

Role of Thr66 in Porcine NADH-cytochromeb 5 Reductase in Catalysis and Control of the Rate-limiting Step in Electron Transfer

“…The neutral blue semiquinone form of a one-electron transfer flavoenzyme may be less active in the one-electron oxidation than the anionic red semiquinone form. It is of interest that the rate-limiting step of the nonphysiological diaphorase activity of leaf FNR is the reductive half-reaction and that a stable neutral blue semiquinone form is produced by anaerobic photoreduction in the absence of NADP ϩ (30,31). It is reasonable that the less active neutral blue semiquinone form is required for leaf FNR, because during photosynthesis, the one-electron reduced form of leaf FNR must be protected from unfavorable oxidation to form the twoelectron reduced FAD (FADH Ϫ ), which is necessary for the two-electron reduction of NADP ϩ .…”

“…This position corresponds to threonine or serine residues in the other members of the ferredoxin reductase family (22, 24 -27). Ser 96 in spinach leaf FNR is critical to the reductive halfreaction of FAD (30), and Ser 90 in the C-terminal Tyr 208 mutant of pea leaf FNR forms a hydrogen bond with the amide moiety on the nicotinamide ring of the pyridine nucleotide in both the enzyme-NADP ϩ and enzyme-NADPH complexes (31). However, b5R and cytochrome b reductase domain of nitrate reductase do not have an aromatic ring corresponding to that of the C-terminal Tyr 208 in pea leaf FNR, which contacts with the re-side of the isoalloxazine ring of FAD and moves away accompanied with the binding of nicotinamide (31).…”