Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University , Bangkok, 10400 Thailand.
J Am Chem Soc. 2014 Jan 8;136(1):241-53. doi: 10.1021/ja4088055. Epub 2013 Dec 24.
Determination of the mechanism of dioxygen activation by flavoenzymes remains one of the most challenging problems in flavoenzymology for which the underlying theoretical basis is not well understood. Here, the reaction of reduced flavin and dioxygen catalyzed by pyranose 2-oxidase (P2O), a flavoenzyme oxidase that is unique in its formation of C4a-hydroperoxyflavin, was investigated by density functional calculations, transient kinetics, and site-directed mutagenesis. Based on work from the 1970s-1980s, the current understanding of the dioxygen activation process in flavoenzymes is believed to involve electron transfer from flavin to dioxygen and subsequent proton transfer to form C4a-hydroperoxyflavin. Our findings suggest that the first step of the P2O reaction is a single electron transfer coupled with a proton transfer from the conserved residue, His548. In fact, proton transfer enhances the electron acceptor ability of dioxygen. The resulting ·OOH of the open-shell diradical pair is placed in an optimal position for the formation of C4a-hydroperoxyflavin. Furthermore, the C4a-hydroperoxyflavin is stabilized by the side chains of Thr169, His548, and Asn593 in a "face-on" configuration where it can undergo a unimolecular reaction to generate H2O2 and oxidized flavin. The computational results are consistent with kinetic studies of variant forms of P2O altered at residues Thr169, His548, and Asn593, and kinetic isotope effects and pH-dependence studies of the wild-type enzyme. In addition, the calculated energy barrier is in agreement with the experimental enthalpy barrier obtained from Eyring plots. This work revealed new insights into the reaction of reduced flavin with dioxygen, demonstrating that the positively charged residue (His548) plays a significant role in catalysis by providing a proton for a proton-coupled electron transfer in dioxygen activation. The interaction around the N5-position of the C4a-hydroperoxyflavin is important for dictating the stability of the intermediate.
黄素酶催化的氧分子活化机制仍然是黄素酶学中最具挑战性的问题之一，其基础理论尚未得到很好的理解。在这里，通过密度泛函计算、瞬态动力学和定点突变研究了吡喃糖 2-氧化酶（P2O）催化的还原黄素与氧分子的反应，P2O 是一种独特的黄素氧化酶，它能形成 C4a-过氧黄素。基于 20 世纪 70 年代至 80 年代的研究工作，目前人们认为黄素酶中氧分子活化过程涉及黄素向氧分子的电子转移以及随后的质子转移以形成 C4a-过氧黄素。我们的研究结果表明，P2O 反应的第一步是单电子转移与保守残基 His548 的质子转移相偶联。事实上，质子转移增强了氧分子的电子接受能力。开壳自由基对的·OOH 被置于形成 C4a-过氧黄素的最佳位置。此外，C4a-过氧黄素被 Thr169、His548 和 Asn593 的侧链稳定在“面朝上”构象，在这种构象中，它可以进行单分子反应生成 H2O2 和氧化黄素。计算结果与 Thr169、His548 和 Asn593 残基改变的 P2O 变体的动力学研究、野生型酶的动力学同位素效应和 pH 依赖性研究一致。此外，计算出的能量垒与 Eyring 图获得的实验焓垒一致。这项工作揭示了还原黄素与氧分子反应的新见解，表明带正电荷的残基（His548）在催化中起着重要作用，通过为氧分子活化中的质子耦合电子转移提供质子，从而提供质子。C4a-过氧黄素的 N5 位周围的相互作用对于决定中间产物的稳定性很重要。
