Involvement of Glutamic Acid 301 in the Catalytic Mechanism of Ferredoxin-NADP⁺ Reductase from Anabaena PCC 7119

Abstract

The crystal structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR) suggests that the carboxylate group of Glu301 may be directly involved in the catalytic process of electron and proton transfer between the isoalloxazine moiety of FAD and FNR substrates (NADPH, ferredoxin, and flavodoxin). To assess this possibility, the carboxylate of Glu301 was removed by mutating the residue to an alanine. Various spectroscopic techniques (UV-vis absorption, fluorescence, and CD) indicate that the mutant protein folded properly and that significant protein structural rearrangements did not occur. Additionally, complex formation of the mutant FNR with its substrates was almost unaltered. Nevertheless, no semiquinone formation was seen during photoreduction of Glu301Ala FNR. Furthermore, steady-state activities in which FNR semiquinone formation was required during the electron-transfer processes to ferredoxin were appreciably affected by the mutation. Fast transient kinetic studies corroborated that removal of the carboxylate at position 301 decreases the rate constant approximately 40-fold for the electron transfer process with ferredoxin without appreciably affecting complex formation, and thus interferes with the stabilization of the transition state during electron-transfer between the FAD and the iron-sulfur cluster. Moreover, the mutation also altered the nonspecific reaction of FNR with 5'-deazariboflavin semiquinone, the electron-transfer reactions with flavodoxin, and the reoxidation properties of the enzyme. These results clearly establish Glu301 as a critical residue for electron transfer in FNR.

A Conserved Aspartate (Asp-1393) Regulates NADPH Reduction of Neuronal Nitric-oxide Synthase

“…This is consistent with CYPR and FNR enzymes tolerating similar mutations made at the homologous residue (35,37). The specific nature of the Asp-1393 mutations allowed us to examine how they impact flavin reduction and catalytic events such as cytochrome c reduction, nNOS heme reduction, and NO synthesis.…”

“…These residues are represented in rat nNOS by Asp-1393, Ser-1176, and Cys-1349 (27). Mutagenic substitution studies on the analogously positioned residues in FNR enzymes, cytochrome P450 reductase, and other related enzymes have established that each of the three residues helps to enhance the rate of hydride transfer between NAD(P)H and FAD (32)(33)(34)(35)(36)(37)(38)(39)(40). Given the structural and regulatory properties of the NOSs, we sought to examine how one of these conserved residues (Asp-1393, Fig.…”

“…Possible Structural Basis for Asp-1393 Function-Asp-1393 is part of a triad of residues in nNOS (Ser-1176, Cys-1349, and Asp-1393) that is conserved throughout the FNR enzyme family and has been shown in some members to enable reduction of their bound flavin by NAD(P)H (33)(34)(35)(36)(37)(38). A crystal structure of the FNR subdomain of nNOS showed that Asp-1393 and the two other residues of the triad are positioned as in related flavoproteins to facilitate hydride transfer to FAD (27).…”

Fast Kinetic Methods with Photodiode Array Detection in the Study of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP+-Reductase

“…The different FNR and Fld variants were purified from Luria-Bertani IPTG-induced E. coli cultures containing, respectively, the pTrc99a-Fld and pET28a-FNR plasmids encoding Anabaena WT or E301A FNRs and WT, E16K/E61K or E16K/E61K/D126K/D150K Flds as previously described (Casaus et al, 2002;Goñi et al, 2009;Martínez-Júlvez et al, 2001;Medina et al, 1998;Tejero et al, 2003). UV/Vis spectra were recorded in a Cary 100 spectrophotometer at 25 ºC.…”

“…Unless otherwise stated, all measurements were recorded in 50 mM Tris/HCl, pH 8.0. Binding ability between WT FNR ox and WT Fld ox was determined using difference absorption spectroscopy as previously described (Goñi et al, 2009;Martínez-Júlvez et al, 2001;Medina et al, 1998).…”