Sétif Pierre
iBiTec-S, CNRS UMR 8221, CEA Saclay, 91191 Gif-sur-Yvette, France.
Biochim Biophys Acta. 2015 Feb;1847(2):212-222. doi: 10.1016/j.bbabio.2014.10.008. Epub 2014 Nov 1.
The inhibitor methyl viologen (MV) has been widely used in photosynthesis to study oxidative stress. Its effects on electron transfer kinetics in Synechocystis sp. PCC6803 cells were studied to characterize its electron-accepting properties. For the first hundreds of flashes following MV addition at submillimolar concentrations, the kinetics of NADPH formation were hardly modified (less than 15% decrease in signal amplitude) with a significant signal decrease only observed after more flashes or continuous illumination. The dependence of the P700 photooxidation kinetics on the MV concentration exhibited a saturation effect at 0.3 mM MV, a concentration which inhibits the recombination reactions in photosystem I. The kinetics of NADPH formation and decay under continuous light with MV at 0.3 mM showed that MV induces the oxidation of the NADP pool in darkness and that the yield of linear electron transfer decreased by only 50% after 1.5-2 photosystem-I turnovers. The unexpectedly poor efficiency of MV in inhibiting NADPH formation was corroborated by in vitro flash-induced absorption experiments with purified photosystem-I, ferredoxin and ferredoxin-NADP(+)-oxidoreductase. These experiments showed that the second-order rate constants of MV reduction are 20 to 40-fold smaller than the competing rate constants involved in reduction of ferredoxin and ferredoxin-NADP(+)-oxidoreductase. The present study shows that MV, which accepts electrons in vivo both at the level of photosystem-I and ferredoxin, can be used at submillimolar concentrations to inhibit recombination reactions in photosystem-I with only a moderate decrease in the efficiency of fast reactions involved in linear electron transfer and possibly cyclic electron transfer.
抑制剂甲基紫精（MV）已广泛用于光合作用研究氧化应激。研究了其对集胞藻PCC6803细胞中电子转移动力学的影响，以表征其电子接受特性。在亚毫摩尔浓度下添加MV后的最初几百次闪光中，NADPH形成的动力学几乎没有改变（信号幅度下降不到15%），只有在更多闪光或连续光照后才观察到明显的信号下降。P700光氧化动力学对MV浓度的依赖性在0.3 mM MV时表现出饱和效应，该浓度抑制光系统I中的复合反应。在0.3 mM MV连续光照下NADPH形成和衰减的动力学表明，MV在黑暗中诱导NADP池的氧化，并且在1.5 - 2次光系统I周转后线性电子传递的产率仅下降50%。用纯化的光系统I、铁氧还蛋白和铁氧还蛋白 - NADP(+) - 氧化还原酶进行的体外闪光诱导吸收实验证实了MV抑制NADPH形成的效率出乎意料地低。这些实验表明，MV还原的二级速率常数比参与铁氧还蛋白和铁氧还蛋白 - NADP(+) - 氧化还原酶还原的竞争速率常数小20至40倍。本研究表明，MV虽然在体内能在光系统I和铁氧还蛋白水平接受电子，但在亚毫摩尔浓度下使用时，仅适度降低线性电子传递以及可能的循环电子传递中快速反应的效率，就能抑制光系统I中的复合反应。
