Towards a new interaction enzyme : coenzyme

Ferredoxin-NADP+ reductase catalyses NADP+ reduction, being specific for NADP(+)/H. To understand coenzyme specificity determinants and coenzyme specificity reversion, mutations at the NADP+/H pyrophosphate binding and of the C-terminal regions have been simultaneously introduced in Anabaena FNR. The T155G/A160T/L263P/Y303S mutant was produced. The mutated enzyme presents similar k(cat) values for NADPH and NADH, around 2.5 times slower than that reported for WT FNR with NADPH. Its K-m, value for NADH decreased 20-fold with regard to WT FNR, whereas the K, for NADPH remains similar. The combined effect is a much higher catalytic efficiency for NAD(+)/H, with a minor decrease of that for NADP+/H. In the mutated enzyme, the specificity for NADPH versus NADH has been decreased from 67,500 times to only 12 times, being unable to discriminate between both coenzymes. Additionally, giving the role stated for the C-terminal Tyr in FNR, its role in the energetics of the FAD binding has been analysed. (c) 2004 Elsevier B.V. All rights reserved.</p

Mechanism of the Hydride Transfer between Anabaena Tyr303Ser FNRrd/FNRox and NADP(+)/H. A Combined Pre-Steady-State Kinetic/Ensemble-Averaged Transition-State Theory with Multidimensional Tunneling Study

The flavoenzyme ferredoxin-NADP(+) reductase (FNR) catalyzes the production of NADPH during photosynthesis. The hydride-transfer reactions between the Anabaena mutant Tyr303Ser FNRrd/FNRox and NADP(+)/H have been Studied both experimentally and theoretically. Stopped-flow pre-steady-state kinetic measurements have shown that, in contrast to that observed for WT FNR, the physiological hydride transfer from Tyr303Ser FNRrd to NADP(+) does not Occur. Conversely, the reverse reaction does take place with a rate constant just slightly slower than for WT FNR. This latter process shows temperature-dependent rates, but essentially temperature independent kinetic isotope effects, Suggesting the reaction takes place following the vibration-driven tunneling model. In turn, ensemble-averaged variational transition-state theory with multidimensional tunneling calculations provide reaction rate Constant Values and kinetic isotope effects that agree with the experimental results, the experimental and the theoretical values for the reverse process being noticeably similar. The reaction mechanism behind these hydride transfers has been analyzed. The formation of a close contact ionic pair FADH(-):NADP(+) surrounded by the polar environment of the enzyme in the reactant complex of the mutant might be the cause of the huge difference between the direct and the reverse reaction.</p

Theoretical study of the mechanism of the hydride transfer between ferredoxin-NADP⁺ reductase and NADP⁺: the role of Tyr303

During photosynthesis, ferredoxin-NADP+ reductase (FNR) catalyzes the electron transfer from ferredoxin to NADP+ via its FAD cofactor. The final hydride transfer event between FNR and the nucleotide is a reversible process. Two different transient charge-transfer complexes form prior to and upon hydride transfer, FNR(rd)-NADP(+) and FNR(ox)-NADPH, regardless of the hydride transfer direction. Experimental structures of the FNR(ox):NADP+ interaction have suggested a series of conformational rearrangements that might contribute to attaining the catalytically competent complex, but to date, no direct experimental information about the structure of this complex is available. Recently, a molecular dynamics (MD) theoretical approach was used to provide a putative organization of the active site that might represent a structure close to the transient catalytically competent interaction of Anabaena FNR with its coenzyme, NADP+. Using this structure, we performed fully microscopic simulations of the hydride transfer processes between Anabaena FNR(rd)/FNR(ox) and NADP+/H, accounting also for the solvation. A dual-level QM/MM hybrid approach was used to describe the potential energy surface of the whole system. MD calculations using the finite-temperature string method combined with the WHAM method provided the potential of mean force for the hydride transfer processes. The results confirmed that the structural model of the reactants evolves to a catalytically competent transition state through very similar free energy barriers for both the forward and reverse reactions, in good agreement with the experimental hydride transfer rate constants reported for this system. This theoretical approach additionally provides subtle structural details of the mechanism in wild-type FNR and provides an explanation why Tyr303 makes possible the photosynthetic reaction, a process that cannot occur when this Tyr is replaced by a Ser