Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA.
Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA.
Biochim Biophys Acta Bioenerg. 2021 Jul 1;1862(7):148415. doi: 10.1016/j.bbabio.2021.148415. Epub 2021 Mar 13.
Electron transfer bifurcation allows production of a strongly reducing carrier at the expense of a weaker one, by redistributing energy among a pair of electrons. Thus, two weakly-reducing electrons from NADH are consumed to produce a strongly reducing ferredoxin or flavodoxin, paid for by reduction of an oxidizing acceptor. The prevailing mechanism calls for participation of a strongly reducing flavin semiquinone which has been difficult to observe with site-certainly in multi-flavin systems. Using blue light (450 nm) to photoexcite the flavins of bifurcating electron transfer flavoprotein (ETF), we demonstrate accumulation of anionic flavin semiquinone in excess of what is observed in equilibrium titrations, and establish its ability to reduce the low-potential electron acceptor benzyl viologen. This must occur at the bifurcating flavin because the midpoint potentials of the electron transfer (ET) flavin are not sufficiently negative. We show that bis-tris propane buffer is an effective electron donor to the flavin photoreduction, but that if the system is prepared with the ET flavin chemically reduced, so that only the bifurcating flavin is oxidized and photochemically active, flavin anionic semiquinone is formed more rapidly. Thus, excited bifurcating flavin is able to draw on an electron stored at the ET flavin. Flavin semiquinone photogenerated at the bifurcation site must therefore be accompanied by additional semiquinone formation by oxidation of the ET flavin. Consistent with the expected instability of bifurcating flavin semiquinone, it subsides immediately upon cessation of illumination. However comparison with yields of semiquinone in equilibrium titrations suggest that during continuous illumination at pH 9 a steady state population of 0.3 equivalents of bifurcating flavin semiquinone accumulates, and then undergoes further photoreduction to the hydroquinone. Although transient, the population of bifurcating flavin semiquinone explains the system's ability to conduct light-driven electron transfer from bis-tris propane to benzyl viologen, in effect trapping energy from light.
电子传递分支允许通过在一对电子之间重新分配能量，以牺牲较弱的电子为代价产生强还原剂。因此，两个来自 NADH 的弱还原电子被消耗以产生强还原剂铁氧还蛋白或黄素蛋白，而代价是还原氧化性受体。目前的机制需要参与强还原黄素半醌，这在多黄素系统中很难用位点确定法观察到。我们使用蓝光（450nm）光激发分支电子传递黄素蛋白（ETF）的黄素，证明了阴离子黄素半醌的积累超过了平衡滴定中观察到的量，并确定了它还原低电位电子受体苄基紫精的能力。这必须发生在分支黄素上，因为电子转移（ET）黄素的中点电位不够负。我们表明，双三丙基丙烷缓冲液是黄素光还原的有效电子供体，但如果系统用 ET 黄素化学还原制备，使得只有分支黄素被氧化且光化学活性，则黄素阴离子半醌更快形成。因此，激发的分支黄素能够从储存在 ET 黄素中的电子中汲取。因此，在分支部位产生的黄素半醌必须伴随着 ET 黄素氧化产生的额外半醌形成。与分支黄素半醌的预期不稳定性一致，它在停止光照后立即消退。然而，与平衡滴定中半醌产量的比较表明，在 pH9 下连续光照时，积累了 0.3 当量的分支黄素半醌的稳定态，然后进一步光还原为氢醌。尽管是瞬时的，但分支黄素半醌的存在解释了系统从双三丙基丙烷向苄基紫精进行光驱动电子转移的能力，实际上从光中捕获了能量。
