Role of specific residues in coenzyme binding, charge–transfer complex formation, and catalysis in Anabaena ferredoxin NADP+-reductase

Abstract

Two transient charge-transfer complexes (CTC) form prior and upon hydride transfer (HT) in the reversible reaction of the FAD-dependent ferredoxin-NADP+ reductase (FNR) with NADP+/H, FNR(ox)-NADPH (CTC-1), and FNR(rd)-NADP+ (CTC-2). Spectral properties of both CTCs, as well as the corresponding interconversion HT rates, are here reported for several Anabaena FNR site-directed mutants. The need for an adequate initial interaction between the 2'P-AMP portion of NADP+/H and FNR that provides subsequent conformational changes leading to CTC formation is further confirmed. Stronger interactions between the isoalloxazine and nicotinamide rings might relate with faster HT processes, but exceptions are found upon distortion of the active centre. Thus, within the analyzed FNR variants, there is no strict correlation between the stability of the transient CTCs formation and the rate of the subsequent HT. Kinetic isotope effects suggest that, while in the WT, vibrational enhanced modulation of the active site contributes to the tunnel probability of HT; complexes of some of the active site mutants with the coenzyme hardly allow the relative movement of isoalloxazine and nicotinamide rings along the HT reaction. The architecture of the WT FNR active site precisely contributes to reduce the stacking probability between the isoalloxazine and nicotinamide rings in the catalytically competent complex, modulating the angle and distance between the N5 of the FAD isoalloxazine and the C4 of the coenzyme nicotinamide to values that ensure efficient HT processes.

A Highly Stable Plastidic-Type Ferredoxin-NADP(H) Reductase in the Pathogenic Bacterium Leptospira interrogans

“…Accordingly, the LepFNR crystal structure reveals that parts of the NADP + binding domain is highly mobile, as indicated by the obtained B factors [9]. This mobility may be important for obtaining high catalytic efficiency in FNRs [42], [44].…”

ArabidopsisFNRL protein is an NADPH‐dependent chloroplast oxidoreductase resembling bacterial ferredoxin‐NADP+reductases

“…; Tejero et al , Bortolotti et al , Sánchez‐Azqueta et al , ). Moreover, the reduction of FNRL by NADPH differs with respect to the stabilisation of FNRL CTCs (Tejero et al , Peregrina et al , Sánchez‐Azqueta et al , , Bortolotti et al ). Notably, differences in CTCs stabilisation have also been reported between bacterial‐type FPRs relative to plastidic FNRs, as well as upon mutation of key residues in plastid FNRs (Lans et al , Peregrina et al , Bortolotti et al , Sánchez‐Azqueta et al ).…”

“…Photo. Ltd., Leatherhead, Surrey, UK) (Tejero et al , Peregrina et al ). After mixing, reaction samples contained 12 μ M FNRL ox and 12–200 μ M NADPH.…”

“…Ltd., Leatherhead, UK). Data were fitted to either two or three‐step models, A → B → C or A → B → C → D, estimating observed conversion rate constants ( k A→B , k B→C , k C→D ) (Tejero et al , Peregrina et al ). Errors in the determination of kinetic constants were ±15%.…”

Characterization of the mechanism of the NADH-dependent polysulfide reductase (Npsr) from Shewanella loihica PV-4: Formation of a productive NADH-enzyme complex and its role in the general mechanism of NADH and FAD-dependent enzymes

“…1C) [11] and NADH oxidase [12], as well as in all Coenzyme A-disulfide reductases studied to date [6,[13][14][15]. The FAD-dependent ferredoxin-NADP + reductase, while not a member of the glutathione reductase family, also utilizes a tyrosine residue in a similar fashion [16,17].…”