Journal of Biological Chemistry
Volume 273, Issue 51, 18 December 1998, Pages 34008-34015
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PROTEIN CHEMISTRY AND STRUCTURE
Probing the Function of the Invariant Glutamyl Residue 312 in Spinach Ferredoxin-NADP+ Reductase*

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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 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 (∼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.

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*

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).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors codes 1BX1 (Gln), 1FRQ (Ala), and 1BX0 (Leu) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.