Probing Metalloenzymes with Sensitizer-Tethered Substrates

该奖项在无机、生物无机和有机金属化学项目中支持金属酶的研究。加州理工学院贝克曼研究所的Harry B. Gray和John H. Richards说。本工作的目的是开发一种新的方法来探测氧化还原金属酶的性质和反应性。血红素加氧酶的原型，细胞色素P450，将是主要的系统研究。这种酶利用二氧作为氧化剂立体特异性地羟基化碳氢化合物。在许多金属酶中，限速电子转移（ET）步骤阻碍了其他关键激活步骤的观察。为了解决这个问题，天然氧化还原伙伴将被能够以更快的速度交换电子的新试剂所取代。激光引发的电子转移反应将用于驱动酶促反应。为了提供激光生成的氧化还原等价物，直接共价或氢键途径已被证明是必不可少的。在这里，氧化还原敏化剂和酶活性位点之间的直接连接将通过利用酶对其活性位点的亲和力形成。钌-二亚胺增敏剂将共价连接到细胞色素P450底物上，为传递血红素辅助因子的氧化和还原等价物提供管道。最初的目标是制备和表征氧-铁（铁基）中间体，已被建议作为活性羟基化剂。这项工作的酶将在大肠杆菌中表达，位点定向诱变将用于研究特定残基在铁基形成反应中的重要性。反应中间体将使用各种光化学方法进行研究。金属酶催化许多涉及小分子活化的反应。例如，酶促进氧气还原为水，氮气还原为氨，并利用氧气将氧原子添加到碳链上。这些反应不仅在生物学上而且在商业上都很重要。这项研究旨在了解促进氧气向碳氢化合物中添加氧气的酶。活性酶中间体从未被观察到，因此一种用更容易反应的试剂取代自然系统中的试剂的策略将被用于在活性位点进行反应的光化学观察。

This award in the Inorganic, Bioinorganic, and Organometallic Chemistry Program supports metalloenzyme research by Drs. Harry B. Gray and John H. Richards of the Beckman Institute, California Institute of Technology. The goal of the work is to develop a new method for probing the properties and reactivities of redox metalloenzymes. The prototypical heme oxygenase, cytochrome P450, will be the main system studied. This enzyme stereospecifically hydroxlates hydrocarbons using dioxygen as the oxidant. In many metalloenzymes, rate-limiting electron transfer (ET) steps preclude observation of other critical activation steps. To attack this problem, natural redox partners will be replaced with new reagents that are able to exchange electrons at much faster rates. Laser-initiated electron transfer reactions will be used to drive enzymatic reactions. In order to deliver the laser-generated redox equivalents, a direct covalent or hydrogen-bonded pathway has been shown to be essential. Here a direct link between a redox sensitizer and the enzyme active site will be formed by taking advantage of the affinity of the enzyme for its active site. Ruthenium-diimine sensitizers will be covalently tethered to cytochrome P450 substrates to provide a conduit for delivering oxiding and reducing equivalents to the heme cofactor. The initial goal is to prepare and characterize the oxo-iron (ferryl) intermediates that have been suggested as the active hydroxylating agents. Enzyme for this work will be expressed in E. coli, and site-directed mutagenesis will be employed to study the importance of specific residues in ferryl-formation reactions. The reactive intermediates will be studied using a variety of photochemical methods. Metalloenzymes catalyze many reactions involving small-molecule activation. For example, enzymes promote reduction of oxygen gas to water, reduction of nitrogen gas to ammonia, and use oxygen gas to add oxygen atoms to carbon chains. These reactions are important not only biologically but also commercially. This research aims at understanding the enzyme that promotes addition of oxygen to hydrocarbons by oxygen gas. The reactive enzyme intermediate has never been observed, so a stategy for replacing the reagents from the natural system with ones that can react more readily will be used to enable photochemical observation of reactions at the active site.