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（如自旋态）和反应性之间是否存在相关性。我们将尝试解决的问题包括：*需要什么样的配体环境来稳定亚硝酸盐水合酶（nases）的低自旋状态？如果有的话，自旋态对反应性有什么影响？硫是如何影响这些性质的？中间体在nase中起什么作用？NHase与亚硝酸盐水合的机制是什么？如果有的话，保守的Ser-OH（或co - nase中的Ser-OH）在nase的机制中起什么作用？如果有的话，保守的精氨酸在nase的机制中起什么作用？超氧化物还原酶（sor）的超氧化物还原是否通过Fe(III)-OOH中间体进行？如果有的话，保守的谷氨酸在SOR的机制中起什么作用？抑制剂N3-和CN-如何抑制SOR活性？相对于开放结合位点，Cys在SOR陷阱中的位置对促进功能是否重要？我们计划通过研究合成模型配合物的反应性来探索这些问题，并试图通过系统地改变配体的结构来将其与结构和性质（如自旋态和电子结构）联系起来。在某些情况下，配体将包含悬垂的醇臂或胍臂，作为nase中附近丝氨酸和精氨酸的模型。*将检测这些模型与底物RCN （NHase）、OH- (NHase)、O2- (SOR)和抑制剂（N3-、NO、CN-、RC02-）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。*反应性将用分光光度法、低温和EPR法监测。*动力学数据将通过停止流动技术和核磁共振线形分析获得。

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.