Structure's Influence on Reactivity in Metalloenzymes

本提案中描述的研究将旨在：* 了解结构如何影响金属酶活性位点的功能。* 确定结构、性质（例如自旋状态）和反应性之间是否存在相关性。我们将试图解决的问题包括：* 需要什么样的配体环境来稳定亚硝酸盐水合酶（NHases）的低自旋状态？如果有的话，自旋态对反应性有什么影响？硫磺是如何影响这些特性的？酰胺化物在腈水合酶中起什么作用？腈水合酶水合亚硝酸盐的机理是什么？保守的Ser-OH（或Co-NHase中的Thr-OH）在NHase的作用机制中起什么作用？如果有的话，保守的丝氨酸在腈水合酶的作用机制中起什么作用？超氧化物还原酶（SORs）的超氧化物还原是否通过Fe（III）-OOH中间体进行？保守的谷氨酸在SOR机制中起什么作用？抑制剂N3-和CN-如何抑制SOR活性？半胱氨酸在SOR中的位置，相对于开放结合位点的陷阱，在促进功能中重要吗？我们计划通过以下方式探索这些问题：* 检查合成模型复合物的反应性，并试图通过系统地改变配体的结构将其与结构和性质（如自旋态和电子结构）联系起来。* 在某些情况下，配体将掺入侧链醇或胍臂，作为NH ase中附近丝氨酸和丝氨酸的模型。* 将检测这些模型与底物RCN（NHase）、OH-（NHase）、O2-（SOR）和抑制剂（N3-、NO、CN-、RCO 2-）的反应性。* 这些模型的反应性将在动力学和热力学研究的基础上进行比较。* 反应性将在低温下通过电子顺磁共振（EPR）进行监测。* 使用停流技术和NMR线形分析获得动力学数据。

Research described in this proposal will be aimed at: * understanding how structure influences the function of metalloenzyme active sites. * determining if there is a correlation between structure, properties (such as spin-state), and reactivity. Questions we will be attempting to address include: * What ligand environment is needed to stabilize the low-spin state of nitrite hydratases (NHases)? What influence, if any, does spin-state have on reactivity? How do the sulfurs influence these properties? What role do the amidates play in NHase? What is the mechanism by which NHase hydrates nitrites? What role, if any, does the conserved Ser-OH (or Thr-OH in Co-NHases) play in the mechanism of NHase? What role, if any, do the conserved arginines play in the mechanism of NHase? Does superoxide reduction by superoxide reductases (SORs) proceed via an Fe(III)-OOH intermediate? What role, if any, does the conserved glutamate play in the mechanism of SOR? How do inhibitors N3- and CN- inhibit SOR activity? Is the positioning of the Cys in SOR, traps with respect to the open binding site, important in promoting function? We plan to explore these questions by: * examining the reactivity of synthetic model complexes, and attempting to correlate this with structure and properties, such as spin- state and electronic structure, by systematically altering the structure of our ligands. * ligands will, in some cases, incorporate pendent alcohol or guanidinium arms, as models for the nearby serine and arginines in NHase. * reactivity of these models with subtrates RCN (NHase), OH- (NHase), O2- (SOR) and inhibitors (N3- , NO, CN-, RC02-) will be examined. * reactivity of these models will be compared on the basis of kinetic and thermodynamic studies. * reactivity will be monitored spectrophotometrically, at low temperature, and by EPR. * kinetics data will be obtained using stopped-flow techniques, and NMR line-shape analysis.