EPR Studies of Biological PCET Elements

生物PCET元素的EPR研究

• 批准号: 7195691
• 负责人: R David Britt
• 金额: $ 25.49万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-05 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7195691
• 关键词: Anabolism; Arginine; Bilin; Biochemical Reaction; Biological; Biopterin; Cell Nucleus; Citrulline; Complex; Conversion disorder; Coupled; Electron Nuclear Double Resonance; Electron Transport; Electrons; Elements; Engineering; Enzymes; Family; Ferredoxin; Goals; Hydrogen; Hydrogen Bonding; Learning; Location; Magnetism; Methods; Modeling; Molecular Biology; Movement; Nitric Oxide Synthase; Oxidation-Reduction; Oxidoreductase; Photons; Play; Prosthesis; Proteins; Protons; Reaction; Ribonucleotide Reductase; Role; Spectrum Analysis; Synthesis Chemistry; System; Techniques; Tetrapyrroles; Tyrosine; base; electron donor; interest; member; oxidation; photosystem; photosystem II; phycobilin; protonation; research study; tyrosine radical; yeast two hybrid system

DESCRIPTION (provided by applicant): Organic radicals play crucial roles in many enzymes, where they are typically formed as electron transfer intermediates in redox reactions. In many cases the radical formation and elimination steps are coupled to the movement of protons, in what is generally termed proton-coupled electron transfer (PCET). The suite of modern EPR techniques provide an ideal basis for probing radical-centered biochemical reactions. In particular, if one is targeting PCET reactions, double resonance techniques such as ENDOR and ESEEM can provide crucial information about the location of magnetic hydrogen nuclei during the radical reaction. For example, the protonation state of the radical and the identity of key hydrogen bond donors or acceptors can be obtained with these methods. We propose multifrequency EPR/ENDOR experiments to target several interesting enzymatic radical intermediates where the electron transfers are coupled (or possibly coupled) to proton transfers. Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is a ferredoxin-dependent bilin reductase involved in the biosynthesis of linear tetrapyrrole prosthetic groups. PcyA carries out two regiospecific vinyl reductions, and we have identified two substrate radical intermediates formed during the course of the reaction. Nitric oxide synthases.(NOS) catalyzes the conversion of L-arginine to citrulline and NO, with N-hydroxy-L-arginine (NHA) as an intermediate. A biopterin radical is transiently formed in the conversion of arginine to NHA, and a similar radical intermediate likely forms in the oxidation of NHA to the final products. Photosystem II contains two redox active tyrosines, YD and YZ, that function as electron donors to the photooxidized Chl+ formed by the initial photon-driven electron transfer. These tyrosines are both involved in PCET reactions. Methods of molecular biology, synthetic chemistry, and hybrids of the two can provide newly engineered motifs for exploring PCET in tyrosyl radical systems. We will study bacterial reactions centers engineered to generate tyrosine radicals, synthetic tyrosine model compounds with pendant bases to facilitate intramolecular proton transfer, and protein complexes (PSII and ribonucleotide reductase) incorporating chemically modified tyrosines to probe details of tyrosine-based PCET reactions.

描述（由申请人提供）：有机自由基在许多酶中发挥着至关重要的作用，它们通常在氧化还原反应中作为电子转移中间体形成。在许多情况下，自由基的形成和消除步骤与质子的运动耦合，通常称为质子耦合电子转移（PCET）。这套现代 EPR 技术为探测以自由基为中心的生化反应提供了理想的基础。特别是，如果目标是 PCET 反应，ENDOR 和 ESEEM 等双共振技术可以提供有关自由基反应过程中磁性氢核位置的重要信息。例如，自由基的质子化状态和关键氢键供体或受体的身份可以通过这些方法获得。我们提出多频率 EPR/ENDOR 实验来针对几种有趣的酶自由基中间体，其中电子转移与质子转移耦合（或可能耦合）。 藻蓝蛋白：铁氧还蛋白氧化还原酶 (PcyA) 是一种铁氧还蛋白依赖性胆蛋白还原酶，参与线性四吡咯辅基的生物合成。 PcyA 进行两次区域特异性乙烯基还原，我们已经确定了反应过程中形成的两种底物自由基中间体。 一氧化氮合酶（NOS）催化L-精氨酸转化为瓜氨酸和NO，并以N-羟基-L-精氨酸（NHA）为中间体。生物蝶呤自由基在精氨酸转化为 NHA 的过程中瞬时形成，并且类似的自由基中间体可能在 NHA 氧化成最终产物的过程中形成。 光系统 II 包含两个氧化还原活性酪氨酸 YD 和 YZ，它们充当由初始光子驱动电子转移形成的光氧化 Chl+ 的电子供体。这些酪氨酸都参与 PCET 反应。 分子生物学、合成化学以及两者的混合方法可以为探索酪氨酰自由基系统中的 PCET 提供新的工程基序。我们将研究设计用于产生酪氨酸自由基的细菌反应中心、具有侧链碱基以促进分子内质子转移的合成酪氨酸模型化合物，以及结合化学修饰酪氨酸的蛋白质复合物（PSII和核糖核苷酸还原酶）以探究基于酪氨酸的PCET反应的细节。