Structure's Influence on Reactivity in Metalloenzymes

描述（由申请人提供）：我们的研究项目旨在确定半胱氨酸如何影响非血红素铁酶的功能。含有半胱氨酸连接的非血红素铁SOR和nase分别参与超氧自由基的解毒和腈废物的解毒。超氧化物与许多疾病状态有关，包括癌症、阿尔茨海默氏症、帕金森症和心脏病发作后的心脏损伤。SOR的活性位点与氧化非活性碳氢化合物的血红素酶P450的活性位点非常相似。在过去的资助期间，我们报道了SOR的第一个功能模型，第一个硫酸盐连接的fil - ooh的例子，以及一个未修饰形式的nase的模型。无论是SOR还原超氧化物的机制，还是翻译后半胱氨酸酶修饰的功能和机制，目前都还不清楚。在此资助期内，我们计划：-研究顺式和反式的富尔过氧化物的形成机制，以了解这些反应的质子依赖性，并确定硫酸盐的位置（顺式和反式）如何影响可用的机制途径。-比较顺式和反式硫代酸连接的菲尔ooh与亲电和亲核底物以及h原子给体的反应活性，以观察SOR模型是否可以促进P450化学反应。-确定硫代酸盐配体是否产生有利的反应途径，以提供FelV=O，甚至FeV=O的物种。并且，通过产生更基本的高价FelV=0（如最近提出的P450），确定反式硫酸盐是否比顺式硫酸盐在促进氧化化学方面更有效。-合成一种新的硫代酸连接（NSSPy）铁过氧化物，与广泛表征的氮连接N4Py铁过氧化物结构相关，从而确定硫代酸盐如何影响功能。-合成一种新的反式硫代酸配体，该配体包含空间体和h键残基，旨在稳定fall - ooh或FelV=O，和/或直接将质子传递到远端过氧。-探索SOR的其他功能，包括SO42-、NO3-或NO2-还原。-研究nase半胱氨酸的翻译后修饰通过涉及fell - ooh的机制发生的可能性。-通过检查未修改的NHase模型与氧原子和质子供体的反应性，确定两种顺式NHase半胱氨酸的翻译后氧合如何影响功能。

DESCRIPTION (provided by applicant): Our research program is aimed at determining how cysteinates influence function in non-heme iron enzymes. Cysteinate-ligated non-heme iron containing SOR and NHase are involved in the detoxification of superoxide radicals, and the detoxification of nitrile wastes, respectively. Superoxide has been implicated in a number of disease states, including cancer, Alzheimers, Parkinsons, and cardiac damage following a heart attack. The SOR active site closely resembles that of the heme enzyme P450 which oxidizes unactivated hydrocarbons. During this past funding period, we reported the first functional model for SOR, the first example of a thiolate-ligated Felll-OOH, and a model for the unmodified form of NHase. Neither the mechanism of superoxide reduction by SOR, nor the function and mechanism of post-translational NHase cysteinate modification, are well understood. During this funding period we plan to: - investigate the mechanism of formation of our cis and trans Felll-peroxos in order to understand the proton-dependence of these reactions, and determine how the positioning of the thiolate (cis vs trans) influences the available mechanistic pathways. - compare the reactivity of our cis vs. trans thiolate-ligated Felll-OOH with electrophilic and nucleophilic substrates, and H-atom donors, in order to see if an SOR model can promote P450 chemistry. - determine whether thiolate ligands create favorable reaction pathways affording FelV=O, or perhaps even FeV=O species. And, determine whether a trans-thiolate is more efficient than a cis-thiolate at promoting oxidation chemistry by creating a more basic high valent FelV=0 (as was recently proposed for P450). - synthesize a new thiolate-ligated (NSSPy) Fe-peroxide structurally-related to the extensively characterized nitrogen-ligated N4Py Fe-peroxides so that we can determine how thiolates influence function. - synthesize a new trans thiolate-ligand that incorporates steric bulk and H-bonding residues designed to stabilize an Felll-OOH or FelV=O, and/or direct proton delivery to the distal peroxo oxygen. - explore alternative functions of SOR involving SO42-, NO3-, or NO2- reduction. - examine the possibility that post-translational modification of the NHase cysteinates occurs via a mechanism involving an Felll-OOH. -determine how the post-translational oxygenation of two cis NHase cysteinates influences function by examining the reactivity of our unmodified NHase model with oxo-atom and proton donors.