CAREER: Experimental and Theoretical Studies of Thiolate-Heme Enzymes

细胞色素P450、一氧化氮合酶和氯过氧化物酶是硫酸盐连接的血红素酶，在许多重要的生理过程(如外源物质的代谢)中发挥关键作用。它们在氧化型血红素蛋白中的独特之处在于，它们催化来自分子氧或过氧化氢的氧原子插入到各种有机底物中，通常具有高度的区域和立体选择性。这三种酶都被认为是通过一种活性很高的铁氧中间体发挥作用的，称为化合物I。众所周知，就氧转移化学而言，金属氧的反应活性通常与氧化还原电位有关：金属氧的氧化程度越高，它的活性就越强。因此，自然界在其氧化性最强的血红素酶中使用捐赠硫酸盐配体，这有点令人惊讶，因为硫酸盐连接可以使血红素活性部位的还原电位降低数百毫伏。这个项目考察了大自然在氧化血红素酶时使用硫酸盐配体的情况。光谱方法(EXAFS、EPR、穆斯堡尔谱、拉曼光谱和紫外可见光谱)将与理论相结合，研究天然存在的和同位素标记的氯过氧化物酶中的活性中间体。其目的是为了更好地了解硫酸盐连接酶催化循环中发现的高价中间体的电子结构和几何结构。该项目还涉及细胞色素P450的Cys_Sec突变体的制备和光谱表征。了解反应性的变化如何与硒的不同大小和氧化还原性质有关，将使我们进一步深入了解轴向配体在这些体系中的作用。更广泛的影响：该奖项将支持课程和课程的开发，以及研究生和本科生在化学和生物方面的研究培训。该项目将扩大代表不足的群体对科学的参与，并通过增加共享仪器来加强宾夕法尼亚州立大学的研究基础设施。这项研究的社会效益包括更好地了解硫酸盐连接的血红素蛋白中的氧转移化学，这可以用于工业应用的改进催化剂。被研究的酶只使用电子、质子和氧气(或过氧化氢)来氧化底物。唯一的副产品是水。因此，这些酶是特别“绿色”的催化剂，能够模拟它们的化学作用的合成系统将具有明显的价值。

Cytochrome P450, nitric oxide synthase, and chloroperoxidase are thiolate-ligated heme-enzymes that play critical roles in a number of important physiological processes (e.g. the metabolism of xenobiotics). They are unique among oxidative heme-proteins in that they catalyze the insertion of an oxygen atom, derived either from molecular oxygen or peroxide, into a variety of organic substrates, often with high degrees of regio- and stereo-selectivity. All three of these enzymes are thought to function through a highly reactive iron-oxo intermediate, called compound I. It is known that the reactivity of metal-oxos, with respect to oxygen transfer chemistry, generally scales with redox potential: The more oxidizing the metal-oxo the more reactive it is. Thus, it is somewhat surprising that Nature uses donating thiolate-ligands in its most oxidizing heme-enzymes, as thiolate-ligation can drop the reduction potential of a heme active site by several hundred millivolts. This project examines Nature's use of thiolate ligands in oxidizing heme-enzymes. Spectroscopic methods (EXAFS, EPR, Mossbauer, Raman, and UV/visible) will be coupled with theory to study reactive intermediates in naturally-occurring and isotopically-labeled chloroperoxidase. The aim is to obtain a better understanding of the electronic and geometrical structures of the high-valent intermediates found in the catalytic cycles of thiolate-ligated enzymes. The project also involves the preparation and spectroscopic characterization of Cys_Sec mutants of cytochrome P450. Understanding how changes in reactivity can be related to the different size and redox properties of selenium will give us further insight into the role of the axial-ligand in these systems. Broader Impacts: This award will support the course and curriculum development and the research training of graduate and undergraduate students at the interface of chemistry and biology. The project will broaden the participation of underrepresented groups in science and enhance the infrastructure for research at Penn State University through an increase in shared instrumentation. The societal benefits of the research include a better understanding of oxygen transfer chemistry in thiolate-ligated heme-proteins, which could be parlayed into improved catalysts for industrial applications. The enzymes to be studied use only electrons, protons, and dioxygen (or peroxide) to oxidize substrates. The only byproduct is water. Thus, these enzymes are particularly 'green' catalysts, and synthetic systems that could mimic their chemistry would be of obvious value.