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.
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)
Aliverti
A
A
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Bruns
C M
CM
Pandini
V E
VE
Karplus
P A
PA
Vanoni
M A
MA
Curti
B
B
Zanetti
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D017931:DNA Primers; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005998:Glycine; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D014633:Valine
D017931:DNA Primers; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D012694:Serine; D009249:NADP; D005287:Ferredoxin-NADP Reductase; D014633:Valine; D005998:Glycine