High-Field Electron Paramagnetic Resonance (EPR) and Double Resonance Methods for Mechanistic Studies of Ribonucleotide Reductase and Heterodisulfide Reductase

微波技术的进步使脉冲和高频EPR成为研究基于自由基化学的酶催化作用的独特工具。本课题拟将180ghz脉冲EPR、电子核双共振（ENDOR）和脉冲电子双共振（PELDOR）等先进的EPR方法应用于核糖核苷酸还原酶（RNR）和杂二硫还原酶（Hdr）研究中的代表性问题。我们建议使用PELDOR方法来监测在催化循环过程中产生的自由基之间的纳米范围内的距离。在I类RNRs的研究中，该方法可以检测到大肠杆菌酶的一个同型二聚体R2亚基上的两个酪氨酸自由基。我们将研究范围扩大到R1:R2活化复合物，以获得两个亚基在翻转过程中的对接位点信息，翻转对R1中自由基起始的电子转移机制起着至关重要的作用。此外，我们使用高频脉冲EPR和ENDOR来表征自由基中间体的结构。在产甲烷古菌的Hdr中，酶与底物的反应产生了一个基于fes簇的中间体，具有非常不寻常的EPR光谱特征。高频ENDOR结合选择性同位素标记将允许详细研究底物与提议的4Fe4S簇的相互作用。

Progress in microwave technology has rendered pulsed and high frequency EPR a unique tool for research directed at the understanding of enzymatic catalysis based on radical chemistry. This research project is for the application of advanced EPR methods such as 180 GHz pulse EPR, Electron Nuclear Double Resonance (ENDOR) and Pulsed Electron Double Resonance (PELDOR), to representative problems as found in the investigation of ribonucleotide reductase (RNR) and heterodisulfide reductase (Hdr). We propose the use of PELDOR methodology to monitor distances in the nanometer range between radicals generated during the catalytic cycle. In studies of class I RNRs, the method has allowed to detect two tyrosyl radicals on one homodimeric R2 subunit of the enzyme from E.coli. We are extending the investigation to the R1:R2 activated complex to obtain information about the docking sites of the two subunits during turnover, which plays an essential role for the postulated electron transfer mechanism of radical initiation in R1. Furthermore, we employ high frequency pulse EPR amd ENDOR to characterise the structure of radical intermediates. In Hdr from methanognic archaea, reaction of the enzyme with the substrate gives rise to a FeS-cluster based intermediate with very unusual EPR spectroscopic features. High frequency ENDOR in combination with selective isotopic labelling will allow to investigate in detail the interaction of the substrate with the proposed 4Fe4S cluster.