Structure's Influence on Reactivity in Metalloenzymes

描述（由申请人提供）：我们的研究计划旨在确定半胱氨酸如何影响非血红素铁酶的功能。半胱氨酸连接的含非血红素铁的SOR和NHase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、老年痴呆症、帕金森病和心脏病发作后的心脏损伤。SOR的活性部位与氧化未活化烃的血红素酶P450的活性部位非常相似。在过去的资助期间，我们报道了SOR的第一个功能模型，硫醇连接的Felll-OOH的第一个例子，以及未修饰形式的NHase的模型。SOR还原超氧化物的机制，以及翻译后半胱氨酸修饰的功能和机制都没有很好的理解。在此资助期间，我们计划：-研究我们的顺式和反式Felll-过氧化物的形成机制，以了解这些反应的质子依赖性，并确定硫醇盐的位置（顺式与反式）如何影响可用的机制途径。- 比较我们的顺式与反式硫醇盐连接的Felll-OOH与亲电和亲核底物以及H原子供体的反应性，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有利的反应途径，提供FeV =O，或者甚至FeV=O物种。并且，确定反式硫醇盐是否比顺式硫醇盐更有效地促进氧化化学，通过产生更碱性的高价FelV=0（如最近针对P450提出的）。- 合成一种新的硫醇盐连接的（NSSPy）铁过氧化物结构相关的广泛表征的氮连接的N4 Py铁过氧化物，使我们可以确定硫醇盐如何影响功能。- 合成一种新的反式硫醇盐配体，其结合了空间体和H-键合残基，设计用于稳定FellI-OOH或FellV =O，和/或将质子直接传递到远端过氧氧。- 探索SOR涉及SO 42-、NO3-或NO2-还原的替代功能。 - 检查通过涉及Felll-OOH的机制发生腈水合酶半胱氨酸的翻译后修饰的可能性。- 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确定两个顺式NHase半胱氨酸的翻译后氧化如何影响功能。

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.