Structure's Influence on Reactivity in Metalloenzymes

我们的研究计划旨在确定半胱氨酸如何影响非血红素铁的功能， 内切酶半胱氨酸连接的含非血红素铁的SOR和NHase参与了 超氧自由基，和解毒的腈废物，分别。超氧化物与 许多疾病状态，包括癌症、老年痴呆症、帕金森病和心脏病后的心脏损伤。 攻击SOR的活性部位与血红素酶P450的活性部位非常相似， 芳香烃.在过去的资助期间，我们报告了SOR的第一个功能模型， 硫醇盐连接的Felll-OOH的实例，以及未修饰形式的NHase的模型。无论是 SOR还原超氧化物的机制，也不翻译后的NHase的功能和机制 半胱氨酸修饰是众所周知的。在此期间，我们计划： 我们的顺式和反式Felll-过氧化物的形成机制，以了解质子依赖性 这些反应，并确定如何定位硫醇盐（顺式与反式）影响可用的 机械路径。将我们的顺式与反式硫醇盐连接的Felll-OOH的反应性与 亲电和亲核底物，以及H原子供体，以观察SOR模型是否可以促进 P450化学确定硫醇盐配体是否产生提供FelV=O的有利反应途径， 或者甚至可能是FeV=O物种。并且，确定反式硫醇盐是否比 顺式硫醇盐通过产生更碱性的高价FelV=0（如最近 P450）。合成一种新的硫醇盐连接的（NSSPy）Fe-过氧化物，其结构与 广泛表征氮连接的N4 Py Fe-过氧化物，以便我们可以确定硫醇化 影响函数合成一种新的反式硫醇配体，它结合了空间位阻和氢键 设计用于稳定FellI-OOH或FellV =O的残基，和/或将质子直接递送至远端过氧 氧气探索涉及SO 42-、NO3-或NO2-还原的SOR的替代功能。检查 腈水合酶半胱氨酸的翻译后修饰可能通过一种涉及 哇确定两个顺式NHS半胱氨酸的翻译后氧化如何影响 通过检查我们的未修饰的NHase模型与氧原子和质子供体的反应性来实现功能。

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.