Spinach Ferredoxin-NADP+ Reductase: Structure-Function Relationship as Studied by Site-Directed Mutagenesis

Aliverti, A.; Pandini, V.; Sternieri, F. A.; Corrado, Mario E.; Karplus, P. A.; Zanetti, G.

doi:10.1007/978-94-009-0173-5_382

Cited by 3 publications

(2 citation statements)

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“…Replacement of Ser-96 with Gly or Val clearly interfered with the proper binding of the nicotinamide and with the stabilization of the transition state during hydride transfer between NADPH and FAD (8). Substitution of Phe for Tyr-95, which interacts with the si face of the isoalloxazine and makes a H-bond to the ribityl 4Ј-OH group, did not alter significantly the physico-chemical properties of the enzyme (14). 2 Mutation of the C-terminal Tyr-314, whose side chain covers the re face of the isoalloxazine, was done for * This work was supported by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica, from Consiglio Nazionale delle Ricerche, Italy, and from the National Science Foundation (NSF-MCB-9630474).…”

Section: Ferredoxin-nadpmentioning

confidence: 96%

Probing the Function of the Invariant Glutamyl Residue 312 in Spinach Ferredoxin-NADP+ Reductase

Aliverti

Deng

Ravasi

et al. 1998

Journal of Biological Chemistry

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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 steadystate, 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 (ϳ1%), 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. Ferredoxin-NADPϩ reductase (FNR) 1 from plants and cyanobacteria fulfils the role of electrical switch between one-and two-electron transfer processes during NADP ϩ photoreduction in the photosynthetic electron transport chain (1, 2). FNR became the structural prototype of a large family of structurally related flavoenzymes since the resolution of its three-dimensional structure, which highlighted a novel flavin binding fold (3). Indeed, FNR-like modules are building blocks for constructing both simple and complex flavoproteins with the most varied biological functions not only in prokaryotes and plants but also in animals (4, 5). Generally, the members of the family are highly specific either for NAD ϩ or NADP ϩ , whereas they are more permissive with respect to the electron acceptor.The non-physiological reactions catalyzed in vitro by FNR can be divided into two half-reactions corresponding to transfer of a hydride between NADPH and FAD, and transfer of single electrons between reduced FAD and electron carriers (A), such as Fd (cytochrome c), ferricyanide, and presumably INT, according to Scheme 1.The stoichiometric coefficient n can be 1 or 2, depending on the type of electron acceptor. The reductive half-reaction of FNR and other FNR family members occurs in discrete steps, which involve two Michaelis complexes (MC) and two chargetransfer complexes (CT) (6 -10), according to Scheme 2.In order to clarify the mechanism of action of hydride transfer mediated by the enzyme through flavin-nicotinamide rings interaction, for which there was no support from crystal structures (3, 11, 12), our group and others have carried out sitedirected mutagenesis of the five conserved residues surrounding the isoalloxazine ring in the active center of FNR: Tyr-95, Ser-96, Cys-272, Glu-312, and Tyr-314 (spinach numbering)...

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Section: Ferredoxin-nadpmentioning

confidence: 96%

Probing the Function of the Invariant Glutamyl Residue 312 in Spinach Ferredoxin-NADP+ Reductase

Aliverti

Deng

Ravasi

et al. 1998

Journal of Biological Chemistry

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show abstract

“…Previous studies have replaced the homologous Tyr-95 of spinach FNR by phenylalanine, observing changes essentially in k cat values of the mutant enzyme respect to the wild type form (25).…”

Section: Ferredoxin-nadpmentioning

confidence: 99%

Involvement of the Flavin si-Face Tyrosine on the Structure and Function of Ferredoxin-NADP+ Reductases

Arakaki¹,

Orellano²,

Calcaterra³

et al. 2001

Journal of Biological Chemistry

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Controlled distribution of electrons between acceptors in chloroplasts: a theoretical consideration

Fridlyand

Scheibe

1999

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Spinach Ferredoxin-NADP+ Reductase: Structure-Function Relationship as Studied by Site-Directed Mutagenesis

Cited by 3 publications

References 4 publications

Probing the Function of the Invariant Glutamyl Residue 312 in Spinach Ferredoxin-NADP+ Reductase

Probing the Function of the Invariant Glutamyl Residue 312 in Spinach Ferredoxin-NADP+ Reductase

Involvement of the Flavin si-Face Tyrosine on the Structure and Function of Ferredoxin-NADP+ Reductases

Controlled distribution of electrons between acceptors in chloroplasts: a theoretical consideration

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