A hydrogen bond network in the active site of Anabaena ferredoxin-NADP(+) reductase modulates its catalytic efficiency

Abstract

Ferredoxin-nicotinamide-adenine dinucleotide phosphate (NADP(+)) reductase (FNR) catalyses the production of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) in photosynthetic organisms, where its flavin adenine dinucleotide (FAD) cofactor takes two electrons from two reduced ferredoxin (Fd) molecules in two sequential steps, and transfers them to NADP(+) in a single hydride transfer (HT) step. Despite the good knowledge of this catalytic machinery, additional roles can still be envisaged for already reported key residues, and new features are added to residues not previously identified as having a particular role in the mechanism. Here, we analyse for the first time the role of Ser59 in Anabaena FNR, a residue suggested by recent theoretical simulations as putatively involved in competent binding of the coenzyme in the active site by cooperating with Ser80. We show that Ser59 indirectly modulates the geometry of the active site, the interaction with substrates and the electronic properties of the isoalloxazine ring, and in consequence the electron transfer (ET) and HT processes. Additionally, we revise the role of Tyr79 and Ser80, previously investigated in homologous enzymes from plants. Our results probe that the active site of FNR is tuned by a H-bond network that involves the side-chains of these residues and that results to critical optimal substrate binding, exchange of electrons and, particularly, competent disposition of the C4n (hydride acceptor/donor) of the nicotinamide moiety of the coenzyme during the reversible HT event.

Keywords: 2′-P; 2′-P-AMP; 2′-P-AMP moiety of NADP(+)/H; 2′-phosphate group of NADP(+)/H; 5-deazariboflavin; A(H), A(D); Arrhenius preexponential factors for hydrogen and deuteride, respectively; C4Hn, C4 hydride acceptor/donor of the NADP(+)/H nicotinamide ring; C4n; CTC; CTC-1; CTC-2; Charge-transfer complex; DT; E(aH), E(aD); ET; FAD; FNR(hq)-NADP(+) CTC; FNR(ox)-NADPH CTC; FNR, FNR(ox), FNR(hq), FNR(sq); Fd, Fd(rd); Ferredoxin-NADP(+) reductase; Flavoenzyme; HT; Hydride transfer; I; Isoalloxazine:nicotinamide interaction; K(d)(NADPH), K(d)(NADP(+)); KIE; Kinetic isotope effect.; N5Hi; N5i, N5 hydride donor/acceptor of the FADH(−)/FAD isoalloxazine ring of FNR; NADP(+); NADPH, nicotinamide–adenine dinucleotide phosphate in its oxidised and reduced forms; NMN; PP(i); WT; activation energies for hydride and deuteride transfer, respectively; apparent/observed rate constants obtained by global analysis of spectral kinetic data; charge-transfer complex; dRf; deuteride transfer; deuteride transfer first-order rate constants for the forward and reverse reactions, respectively; dissociation constants for the intermediate complexes in the reduction and reoxidation of FNR, respectively; electron transfer; ferredoxin and in its reduced state; ferredoxin-NADP(+) reductase and FNR in the fully oxidised, anionic hydroquinone (fully reduced) and neutral semiquinone (one-electron reduced) states, respectively; first-order electron transfer rate; flavin adenine dinucleotide; hydride transfer; hydride transfer first-order rate constants for the forward and reverse reactions, respectively; ionic strength; k(2); k(A→B), k(B→C); k(DT), k(DT-1); k(HT), k(HT-1); k(et); k(obsHT), k(obsHT-1), k(obsDT), k(obsDT-1); kinetic isotopic effect; nicotinamide nucleotide moiety of NADP(+)/H; observed conversion HT and DT rate constants for the forward and reverse reactions; pyrophosphate; second-order rate constant for bimolecular electron transfer; wild-type.