MauG-preMADH intermediate structures: Insight into long range electron transfer

描述（由申请人提供）：一种不寻常的二血红素酶mag与单羟基化的2Trp57 （preMADH）催化前体甲胺脱氢酶的6电子氧化，形成成熟的色氨酸色氨酸醌（TTQ）辅助因子。反应通过三个双电子氧化进行，包括将第二个氧原子插入2Trp57，在2Trp57和2Trp108之间形成交联，氧化成醌。这些修改的顺序是未知的。MauG可以用3mol H2O2或O2加还原物氧化preMADH。在MauG中加入化学计量的H2O2，形成具有催化活性的双铁（IV），其中一个血红素处于Fe(IV)=O状态，而另一个是由His和Tyr配体连接的铁（IV）。这种中间体展示了一种前所未有的稳定高氧化性物质的方法，相当于铁(V)。preMADH- mag配合物的晶体结构表明，氧结合和活化的位点距离TTQ位点超过30 z。在晶体中加入过量的H2O2会导致TTQ的形成，这表明晶体具有催化活性，每一步都是在不解离络合物的情况下进行的，并且氧化是通过远距离蛋白质间电子转移发生的。本项目的目的之一是对mag的双铁（IV）催化中间体进行结构表征。另一个目的是表征发生在TTQ位点的每一个2电子氧化反应的顺序和性质。这些生物合成中间体将在晶体中生成并进行结构表征。质谱还将用于确认晶体学实验的结果，并表征涉及质子转移或自由基形成的步骤，这涉及到第一个氧化步骤。这些实验有望对自由基和高价氧化剂在蛋白质内的稳定方法以及蛋白质间电子转移的机制提供重要的见解。这些过程是有氧代谢的基础，与各种疾病状态和衰老有关，对人类健康具有重要意义。

DESCRIPTION (provided by applicant): An unusual di-heme enzyme MauG catalyzes the 6-electron oxidation of a precursor methylamine dehydrogenase with a monohydroxylated 2Trp57 (preMADH) to form the mature tryptophan tryptophyl quinone (TTQ) cofactor. The reaction proceeds via three, two-electron oxidations involving the insertion of the second oxygen atom into 2Trp57, formation of the crosslink between 2Trp57 and 2Trp108, and oxidation to the quinone. The order of these modifications is unknown. MauG can use 3 moles of either H2O2 or O2 plus reducing equivalents to oxidize preMADH. Addition of stoichiometric H2O2 to MauG results in the formation of a catalytically competent bis-Fe(IV) species with one of the hemes in a Fe(IV)=O state while the other is a Fe(IV) species ligated by His and Tyr ligands. This intermediate demonstrates an unprecedented method for stabilizing a highly oxidizing species equivalent to an Fe(V). A crystal structure of the preMADH- MauG complex shows that the site of oxygen binding and activation is over 30 z from the TTQ site. Addition of excess H2O2 to the crystals results in formation of TTQ, demonstrating that the crystals are catalytically active, that each step occurs processively without dissociation of the complex, and that oxidation occurs via long range inter-protein electron transfer. One goal of this project is to structurally characterize the bis-Fe(IV) catalytic intermediate of MauG. The other objective is to characterize the order and nature of each of the 2-electron oxidation reactions occurring at the TTQ site. These biosynthetic intermediates will be generated in crystallo and structurally characterized. Mass spectrometry will also be used to confirm the results of crystallography experiments and to characterize steps involving proton transfer or radical formation, which has been implicated in the first oxidation step. These experiments promise to provide significant insight into methods of radical and high- valent oxidant stabilization within proteins as well as mechanisms of inter-protein electron transfer. These processes underpin aerobic metabolism and have been implicated in various disease states and aging, making them significant to human health. PUBLIC HEALTH RELEVANCE: Cell damage due to reactive oxygen species and free radicals has been linked to aging as well as certain cancers and a host of other disease states. The unusual properties of MauG provide an excellent opportunity to enhance our understanding of the biological control of radicals and reactive oxygen species as well as mechanisms of oxygen activation and long-range inter-protein electron transfer.