STRUCTURAL STUDIES OF REDOX STATES OF MAUG

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个合作项目的目标是从结构上表征MAUG的不同氧化还原状态。MAUG是一种非常不寻常的双血红素酶，它完成了甲胺脱氢酶(MADH)中发现的新型氨基酸衍生催化辅因子色氨酸色氨酸对苯二酚(TTQ)的合成。TTQ是通过对MADHβ多肽链上的两个Trp残基进行翻译后修饰而形成的，在此过程中，两个氧原子结合到BetaTrp57的吲哚环上，并在BetaTrp57和BetaTrp108的吲哚环之间形成共价键。MAUG的天然底物(PreMADH)是MADH的119 kDa蛋白质前体，βTrp57单羟化，无交联物。MAUG催化六电子氧化完成TTQ的生物合成。它可以在依赖于过氧化氢或依赖于O2/还原当量的反应中做到这一点。该酶已被证明形成了一种史无前例的双血红素双铁(IV)物种，具有催化活性。这种中间体在电子上相当于Fe(V)，由Fe(IV)=O组成，第二个氧化当量位于第二个亚铁血红素的铁上。我们解决了MAUG与前MADH形成的络合物的晶体结构。这已经达到了2.1°的分辨率，并且这些晶体可以在不损失衍射的情况下支持催化周转来形成TTQ。我们预期的5配位P-血红素是Fe(IV)=O形成的位置，来自TTQ为32°。6坐标E-血红素位于P-血红素和TTQ之间的等距离，具有非常不寻常的His/Tyr轴向配体构型，我们推测这是稳定Fe(IV)所必需的。在溶液和晶体中使用XAS，我们希望表征能够支持和稳定MAUG不寻常的氧化还原物种的分子环境，并使我们能够对一种史无前例的双血红素双铁(IV)中间体的氧活化做出根本性的发现。这项工作将是这种不寻常的酶的第一次XAS研究。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objective of this collaborative project is to structurally characterize different redox states of MauG. MauG is a highly unusual di-heme enzyme that completes the synthesis of the novel amino acid derived catalytic cofactor, tryptophan tryptophylquinone (TTQ) found in the enzyme methylamine dehydrogenase (MADH). TTQ is formed by post-translational modification of two Trp residues in the MADH beta polypeptide chain during which two atoms of oxygen are incorporated into the indole ring of betaTrp57 and a covalent bond is formed between the indole rings of betaTrp57 and betaTrp108. The natural substrate for MauG (preMADH) is a 119 kDa protein precursor of MADH with mono-hydroxylated betaTrp57 and no cross-link. MauG catalyzes a six-electron oxidation to complete TTQ biosynthesis. It can do this in a H2O2-dependent or O2/reducing equivalents-dependent reaction. The enzyme has been shown to form a unprecedented di-heme bis-Fe(IV) species that is catalytically competent. This intermediate is electronically equivalent to Fe(V), and is composed of an Fe(IV)=O with the second oxidizing equivalent residing on the Fe of the second heme. We have solved the crystal structure of MauG in complex with preMADH. This has been achieved to a resolution of 2.1 ¿, and these crystals can support catalytic turnover to form TTQ without loss of diffraction. The 5-coordinate P-heme that we expect to be the site of Fe(IV)=O formation is 32 ¿ from TTQ. The 6-coordinate E-heme lies equidistant between the P-heme and TTQ, and has a highly unusual His/Tyr axial ligand configuration that we hypothesize is required for stabilizing Fe(IV). Using XAS both in solution and in the crystal, we wish to characterize the molecular environments that can support and stabilize the unusual redox species of MauG, and enable us to make fundamental discoveries about oxygen activation by an unprecedented di-heme bis-Fe(IV) intermediate. This work would be the first XAS study of this unusual enzyme.