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技术为探究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可以提供关于自由基反应中磁性氢核位置的关键信息。例如，自由基的质子化状态和关键氢键给体或受体的身份可以通过这些方法获得。我们提出了多频EPR/ENDOR实验，以几种有趣的酶自由基中间体为目标，其中电子转移与质子转移耦合（或可能耦合）。

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.