Metal Cluster Active Sites in Proteins

具有羧酸桥接二铁中心的活性位点的蛋白质构成了一个新的金属蛋白亚类。这类生长的蛋白质包括各种功能的蛋白质，如二氧载体氰菊酯、一些碳氢化合物单加氧酶、核糖核苷酸还原酶、脂肪酸去饱和酶、紫酸磷酸酶和丝氨酸/苏氨酸蛋白磷酸酶。该项目主要研究不同蛋白质的活性位点是如何相互关联的，以及金属中心如何发挥各自的功能。参与2e-氧化的酶的光谱研究重点是通过EXAFS获得双铁位点在其各种氧化状态下的度量参数，包括催化相关的高价中间态。这些酶包括可溶性酶、甲烷单加氧酶、硬脂酰酰基载体蛋白去饱和酶、甲苯4单加氧酶以及膜结合烷烃羟化酶。共振拉曼研究将用于表征双铁(III)-过氧、双铁(IV)-氧和双铁(III)-生成物态的中间体。对于核糖核苷酸还原酶，研究了二铁簇的氧化还原电位的变构效应，酪氨酸自由基形成所需的“额外电子”的来源，以及高价中间体X的推定二铁(III)-过氧前体。这些研究中使用的物理技术包括UV-vis，共振拉曼，EPR, M÷ssbauer， EXAFS和光谱电化学，以及使用突变蛋白来提高捕获某些中间体和探测远程电子转移途径的前景。来自猪子宫的紫色酸性磷酸酶，子宫铁蛋白，将被用作研究双核水解酶的一般机制，特别是参与细胞信号传导的丝氨酸/苏氨酸蛋白磷酸酶的模型。非竞争性抑制剂氟可以作为一种非常有用的催化机制探针，因为氟很可能取代负责磷酸酯水解的亲核试剂。氟与酶-底物复合物的相互作用将通过EPR、EXAFS和共振拉曼技术进行研究，以深入了解酶-底物复合物水解前的性质。钙调磷酸酶和λ蛋白磷酸酶也将进行类似的实验。该项目旨在了解由两个金属中心组成的活性位点的金属酶如何催化代谢重要反应。例如，甲烷单加氧酶在温和的条件下将甲烷转化为甲醇，这与目前的能源密集型工业过程形成鲜明对比。核糖核苷酸还原酶在细胞生长过程中负责制造脱氧核糖核苷酸，脱氧核糖核苷酸是DNA的组成部分。了解如何控制这种酶可以提供抑制肿瘤细胞失控生长的策略。细胞信号磷酸酶是另一类使用两个金属中心的酶；它们对调节细胞过程很重要。利用各种光谱方法作为探针，对这些酶在不同形式下的活性位点进行比较，了解它们的结构关系，它们如何催化各自的反应，以及单个金属中心在进行反应中的作用。

Que 9808350 Proteins with active sites consisting of carboxylate-bridged diiron centers comprise a new subclass of metalloproteins. This growing class includes proteins of various functions such as the dioxygen carrier hemerythrin, a number of hydrocarbon monooxygenases, ribonucleotide reductase, fatty acid desaturases, purple acid phosphatases, and Ser/Thr protein phosphatases. This project concentrates on how the active sites of the different proteins are interrelated and how the metal centers perform their respective functions. Spectroscopic studies on enzymes involved in 2e- oxidation focuses on obtaining the metric parameters for the diiron site in its various oxidation states by EXAFS including catalytically relevant high valent intermediate states. These enzymes include the soluble enzymes, methane monooxygenase, stearoyl acyl carrier protein desaturase, and toluene 4-monooxygenase, as well as the membrane-bound alkane hydroxylase. Resonance Raman studies will be used to characterize intermediates at the diiron(III)-peroxo, diiron(IV)-oxo, and diiron(III)-product states. For ribonucleotide reductase, allosteric effects of the redox potential of the diiron cluster, the source of the "extra electron" required for tyrosyl radical formation, and the putative diiron(III)-peroxo precursor to the high-valent intermediate X are studied. Physical techniques used in these studies include UV-vis, resonance Raman, EPR, M÷ssbauer, EXAFS, and spectro-electrochemistry, together with the use of mutant proteins to enhance prospects for trapping certain intermediates and for probing long range electron transfer pathways. The purple acid phosphatase from porcine uterus, uteroferrin, will be used as a model for studying the mechanism of dinuclear hydrolases in general and the Ser/Thr protein phosphatases involved in cell signaling in particular. The uncompetitive inhibitor fluoride will serve as a very useful mechanistic probe for the catalytic mechanism, since fluoride very likely substitutes for the nucleophile responsible for phosphate ester hydrolysis. The interaction of fluoride with the enzyme-substrate complex will be investigated by EPR, EXAFS, and resonance Raman techniques to gain insight into the nature of the enzyme-substrate complex prior to hydrolysis. Similar experiments will be carried out on calcineurin and lambda protein phosphatase.This project is aimed at understanding how metalloenzymes with active sites consisting of two metal centers catalyze metabolically important reactions. For example, methane monooxygenase converts methane to methanol under mild conditions, in contrast to the current energy-intensive industrial process. Ribonucleotide reductase is responsible for making deoxyribonucleotides, the building blocks of DNA, during cell growth. Understanding how to control this enzyme can provide strategies for inhibiting the runaway growth of tumor cells. Cell signaling phosphatases are yet another group of enzymes that use two metal centers; they are important for regulating cellular processes. Using a variety of spectroscopic methods as probes, the active sites of these enzymes in their various forms will be compared to understand their structural relationships, how they catalyze their respective reactions, and the roles of the individual metal centers in carrying out the reactions.