Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany.
Biochemistry. 2021 Apr 20;60(15):1148-1164. doi: 10.1021/acs.biochem.1c00064. Epub 2021 Mar 31.
Proton-coupled electron transfer reactions play critical roles in many aspects of sensory phototransduction. In the case of flavoprotein light sensors, reductive quenching of flavin excited states initiates chemical and conformational changes that ultimately transmit light signals to downstream targets. These reactions generally require neighboring aromatic residues and proton-donating side chains for rapid and coordinated electron and proton transfer to flavin. Although photoreduction of flavoproteins can produce either the anionic (ASQ) or neutral semiquinone (NSQ), the factors that favor one over the other are not well understood. Here we employ a biologically active variant of the light-oxygen-voltage (LOV) domain protein VVD devoid of the adduct-forming Cys residue (VVD-III) to probe the mechanism of flavin photoreduction and protonation. A series of isosteric and conservative residue replacements studied by rate measurements, fluorescence quantum yields, FTIR difference spectroscopy, and molecular dynamics simulations indicate that tyrosine residues facilitate charge recombination reactions that limit sustained flavin reduction, whereas methionine residues facilitate radical propagation and quenching and also gate solvent access for flavin protonation. Replacement of a single surface Met residue with Leu favors formation of the ASQ over the NSQ and desensitizes photoreduction to oxidants. In contrast, increasing site hydrophilicity by Gln substitution promotes rapid NSQ formation and weakens the influence of the redox environment. Overall, the photoreactivity of VVD-III can be understood in terms of redundant electron donors, internal hole quenching, and coupled proton transfer reactions that all depend upon protein conformation, dynamics, and solvent penetration.
质子偶联电子转移反应在许多感觉光转导方面起着关键作用。在黄素蛋白光传感器的情况下，黄素激发态的还原猝灭引发化学和构象变化，最终将光信号传递到下游靶标。这些反应通常需要相邻的芳香族残基和供质子侧链，以实现黄素的快速和协调的电子和质子转移。尽管黄素蛋白的光还原可以产生阴离子（ASQ）或中性半醌（NSQ），但有利于一种形式而不是另一种形式的因素尚不清楚。在这里，我们使用缺乏加合物形成半胱氨酸残基的光氧电压（LOV）结构域蛋白 VVD 的生物活性变体（VVD-III）来探测黄素光还原和质子化的机制。通过速率测量、荧光量子产率、FTIR 差谱和分子动力学模拟研究的一系列等排和保守残基取代表明，酪氨酸残基促进了限制持续黄素还原的电荷重组反应，而蛋氨酸残基促进了自由基传播和猝灭，并为黄素质子化打开了溶剂通道。用亮氨酸替换单个表面 Met 残基有利于 ASQ 的形成而不是 NSQ 的形成，并使光还原对氧化剂脱敏。相比之下，通过 Gln 取代增加位点亲水性可促进快速 NSQ 的形成，并减弱氧化还原环境的影响。总体而言，可以根据冗余电子供体、内部空穴猝灭和偶联质子转移反应来理解 VVD-III 的光反应性，所有这些反应都取决于蛋白质构象、动力学和溶剂渗透。
