Probing the function of the invariant glutamyl residue 312 in spinach ferredoxin-NADP+ reductase.
Ferredoxin-NADP+ reductase, the prototype of a large family of structurally related flavoenzymes, pairs single electrons carried by ferredoxin I and transfers them as a hydride to NADP+. Four mutants of the enzyme, in which Glu-312 was replaced with Asp, Gln, Leu, and Ala to probe the role of the residue charge, size, and polarity in the enzyme activity, have been heterologously expressed, purified, and characterized through steady-state, rapid kinetic studies, ligand-binding experiments, and three-dimensional structure determination by x-ray crystallography. The E312L mutant was the only one that was almost inactive (approximately 1%), whereas unexpectedly the E312A reductase was 10-100% active with the various acceptors tested. Rapid kinetic absorption spectroscopy studies demonstrated that flavin reduction by NADPH was impaired in the mutants. Furthermore, NADP(H) binding was partially perturbed. These functional and structural studies lead us to conclude that Glu-312 does not fulfil the role of proton donor during catalysis, but it is required for proper binding of the nicotinamide ring of NADP(H). In addition, its charge modulates the two one-electron redox potentials of the flavin to stabilize the semiquinone form.
Aliverti
A
A
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Via Celoria 26, I-20133 Milano, Italy.
Deng
Z
Z
Ravasi
D
D
Piubelli
L
L
Karplus
P A
PA
Zanetti
G
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D017931:DNA Primers; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D018698:Glutamic Acid; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D018698:Glutamic Acid; D009249:NADP; D005287:Ferredoxin-NADP Reductase