Anusevicius Zilvinas, Miseviciene Lina, Medina Milagros, Martinez-Julvez Marta, Gomez-Moreno Carlos, Cenas Narimantas
Institute of Biochemistry, Mokslininku 12, LT-08662 Vilnius, Lithuania.
Arch Biochem Biophys. 2005 May 15;437(2):144-50. doi: 10.1016/j.abb.2005.03.015. Epub 2005 Apr 5.
Flavoenzymes may reduce quinones in a single-electron, mixed single- and two-electron, and two-electron way. The mechanisms of two-electron reduction of quinones are insufficiently understood. To get an insight into the role of flavin semiquinone stability in the regulation of single- vs. two-electron reduction of quinones, we studied the reactions of wild type Anabaena ferredoxin:NADP(+)reductase (FNR) with 48% FAD semiquinone (FADH*) stabilized at the equilibrium (pH 7.0), and its Glu301Ala mutant (8% FADH* at the equilibrium). We found that Glu301Ala substitution does not change the quinone substrate specificity of FNR. However, it confers the mixed single- and two-electron mechanism of quinone reduction (50% single-electron flux), whereas the wild type FNR reduces quinones in a single-electron way. During the oxidation of fully reduced wild type FNR by tetramethyl-1,4-benzoquinone, the first electron transfer (formation of FADH*) is about 40 times faster than the second one (oxidation of FADH*). In contrast, the first and second electron transfer proceeded at similar rates in Glu301Ala FNR. Thus, the change in the quinone reduction mechanism may be explained by the relative increase in the rate of second electron transfer. This enabled us to propose the unified scheme of single-, two- and mixed single- and two-electron reduction of quinones by flavoenzymes with the central role of the stability of flavin/quinone ion-radical pair.
黄素酶可以通过单电子、单电子与双电子混合以及双电子方式还原醌类。醌类双电子还原的机制尚未得到充分理解。为了深入了解黄素半醌稳定性在醌类单电子与双电子还原调控中的作用，我们研究了野生型鱼腥藻铁氧还蛋白：NADP(+)还原酶（FNR）与在平衡状态（pH 7.0）稳定的48% FAD半醌（FADH*）及其Glu301Ala突变体（平衡时8% FADH*）的反应。我们发现Glu301Ala取代不会改变FNR的醌类底物特异性。然而，它赋予了醌类还原的单电子与双电子混合机制（50%单电子通量），而野生型FNR以单电子方式还原醌类。在用四甲基-1,4-苯醌氧化完全还原的野生型FNR过程中，第一次电子转移（形成FADH*）比第二次（FADH*氧化）快约40倍。相比之下，Glu301Ala FNR中的第一次和第二次电子转移速率相似。因此，醌类还原机制的变化可能是由于第二次电子转移速率的相对增加所致。这使我们能够提出黄素酶对醌类进行单电子、双电子以及单电子与双电子混合还原的统一方案，其中黄素/醌离子自由基对的稳定性起着核心作用。
