EPR Studies of Biological PCET Elements

描述（由申请人提供）：有机自由基在许多酶中起关键作用，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相耦合，通常称为质子耦合电子转移（PCET）。这套现代EPR技术为探索以自由基为中心的生化反应提供了理想的基础。特别是，如果一个目标是PCET反应，双共振技术，如ENDOR和ESEEM可以提供有关的位置磁氢核在自由基反应的关键信息。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用这些方法获得。我们提出多频EPR/ENDOR实验，以针对几个有趣的酶自由基中间体的电子转移耦合（或可能耦合）质子转移。 藻蓝胆素：铁氧还蛋白氧化还原酶（Phycocyanobilin：ferredoxin oxidoreductase，PcyA）是一种铁氧还蛋白依赖性的胆色素还原酶，参与线性四吡咯辅基的生物合成。PcyA进行两个区域特定的乙烯基还原，我们已经确定了两个底物自由基中间体在反应过程中形成的。 一氧化氮合酶。(NOS)催化L-精氨酸转化为瓜氨酸和NO，N-羟基-L-精氨酸（NHA）作为中间体。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氧化为最终产物的过程中形成。 光系统II含有两个氧化还原活性酪氨酸，YD和YZ，作为电子供体的光氧化的Chl+形成的初始光子驱动的电子转移。这些酪氨酸都参与PCET反应。 分子生物学、合成化学和两者的杂交方法可以为探索酪氨酰自由基系统中的PCET提供新的工程图案。我们将研究细菌反应中心工程产生酪氨酸自由基，合成酪氨酸模型化合物与悬垂碱，以促进分子内质子转移，和蛋白质复合物（PSII和核糖核苷酸还原酶）纳入化学修饰的酪氨酸探测酪氨酸为基础的PCET反应的细节。

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.