Metal Cluster Active Sites in Proteins

QUE 9808350蛋白的活性中心由羧酸桥联的双铁中心组成，构成了金属蛋白的一个新亚类。这一不断增长的类别包括具有各种功能的蛋白质，如氧气载体杂色菊酯、一些碳氢单加氧酶、核苷酸还原酶、脂肪酸去饱和酶、紫色酸性磷酸酶和丝氨酸/苏氨酸蛋白磷酸酶。这个项目集中在不同蛋白质的活性部位是如何相互关联的，以及金属中心如何发挥各自的功能。涉及2E氧化的酶的光谱研究主要集中在用EXAFS获得二铁中心在其各种氧化状态下的计量参数，包括催化相关的高价中间状态。这些酶包括可溶酶、甲烷单加氧酶、硬脂酰基载体蛋白去饱和酶、甲苯4-单加氧酶以及膜结合的烷烃羟基酶。共振拉曼研究将被用来表征二铁(III)-过氧基、二铁(IV)-氧代和二铁(III)-产物状态的中间体。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，二铁簇是形成酪氨酰自由基所需的“额外电子”的来源，以及高价中间体X的可能的二铁(III)-过氧基前体。这些研究中使用的物理技术包括UV-Vis、共振拉曼、EPR、穆斯堡尔谱、EXAFS和光谱电化学，以及使用突变蛋白质来增强捕获某些中间体和探测远程电子转移途径的前景。从猪子宫中提取的紫色酸性磷酸酶--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转导的丝氨酸/苏氨酸蛋白磷酸酶的模型。非竞争性抑制剂氟化物将作为催化机理的一个非常有用的机械探针，因为氟化物很可能替代负责磷酸酯水解的亲核试剂。氟与酶-底物复合体的相互作用将通过EPR、EXAFS和共振拉曼技术进行研究，以在水解前了解酶-底物复合体的性质。类似的实验将在钙调神经磷酸酶和lambda蛋白磷酸酶上进行。这个项目的目的是了解具有两个金属中心的活性部位的金属酶如何催化代谢重要的反应。例如，甲烷单加氧酶在温和的条件下将甲烷转化为甲醇，这与目前能源密集型工业过程形成了鲜明对比。核糖核苷酸还原酶负责在细胞生长过程中合成脱氧核糖核苷酸，这是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组使用两个金属中心的酶；它们对调节细胞过程很重要。使用各种光谱方法作为探针，将比较这些酶的各种形式的活性部位，以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.