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

描述（由申请方提供）：一种不常见的二血红素酶MauG催化前体甲胺脱氢酶与单羟基化2 Trp 57（preMADH）的6电子氧化，形成成熟的色氨酸羟醌（TTQ）辅因子。该反应通过三个两电子氧化进行，包括将第二个氧原子插入2 Trp 57，在2 Trp 57和2 Trp 108之间形成交联，以及氧化成醌。这些修改的顺序是未知的。MauG可以使用3摩尔的H2 O2或O2加上还原当量来氧化preMADH。此外化学计量的H2 O2的MauG的结果在形成的催化活性的bis-Fe（IV）的物种与亚铁（IV）=O状态的血红素之一，而另一个是由His和Tyr配体连接的Fe（IV）物种。该中间体证明了一种前所未有的方法，用于稳定相当于Fe（V）的高度氧化物质。preMADH-MauG复合物的晶体结构显示氧结合和活化的位点距离TTQ位点超过30 z。向晶体中加入过量的H2 O2导致TTQ的形成，表明晶体具有催化活性，每个步骤在没有络合物解离的情况下发生，并且氧化通过长距离蛋白质间电子转移发生。该项目的一个目标是结构表征MauG的bis-Fe（IV）催化中间体。另一个目标是表征在TTQ位点发生的每个2-电子氧化反应的顺序和性质。这些生物合成中间体将以晶体形式产生并进行结构表征。质谱也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，这在第一个氧化步骤中有牵连。这些实验有望为蛋白质内自由基和高价氧化剂的稳定方法以及蛋白质间电子转移机制提供重要的见解。这些过程是有氧代谢的基础，并与各种疾病状态和衰老有关，使其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.