Mechanism of the Hydride Transfer between Anabaena Tyr303Ser FNRrd/FNRox and NADP+/H. A Combined Pre-Steady-State Kinetic/Ensemble-Averaged Transition-State The

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• 作者：Isaias Lans ; Jos Ramn Peregrina ; Milagros Medina ; Mireia Garcia-Viloca ; ngels Gonzlez-Lafont ; Jos M. Lluch
• 刊名：Journal of Physical Chemistry B
• 出版年：2010
• 出版时间：March 11, 2010
• 年：2010
• 卷：114
• 期：9
• 页码：3368-3379
• 全文大小：332K
• 年卷期：v.114,no.9(March 11, 2010)
• ISSN：1520-5207

文摘

The flavoenzyme ferredoxin-NADP⁺ reductase (FNR) catalyzes the production of NADPH during photosynthesis. The hydride-transfer reactions between the Anabaena mutant Tyr303Ser FNR_rd/FNR_ox 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 FNR_rd 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.