Biophysical Investigations on the Coupled Electron/Proton-Transfer in recombinant Ribonucleotide Reductase from Mouse Englisch:

核糖核苷酸还原酶（RNR）是脱氧核糖核苷酸和DNA的唯一来源。I类rnr进行两个有趣的反应：(I)由双铁中心（重组）形成稳定的酪氨酸自由基的氧活化，这对于（ii）酶促反应至关重要。它们的动力学可以通过快速冷冻淬火电子顺磁共振（EPR）和光学止流技术来跟踪。(i)小鼠和大肠杆菌RNR的重构动力学不同，可以研究反应途径上的不同步骤和小鼠RNR中新中间体的积累。初步结果显示，小鼠RNR中存在相当大的H/D动力学同位素效应，表明存在一种高价铁中间体的积累。详细的动力学测量将揭示这种质子转移是否与电子转移相耦合。此外，将研究小鼠RNR中假定的氢传递途径的位点定向突变。（ii）酶促反应机制是基于自由基的，可能包括质子/电子在35 Å以上的耦合转移。我们将尝试通过在H2O和D2O中的单次周转测量来捕获这种转移途径上和活性位点上的新自由基中间体。(i)和（ii）两个反应将通过使用低温保护剂和在零下（-30°C）温度下测量而减慢。主要目标是在(i)和（ii）过程中捕获具有催化活性的自由基中间体，并通过多频EPR、ENDOR、ESEEM和其他合适的光谱方法（Mössbauer、共振拉曼等）对它们进行表征。

Ribonucleotide reductases (RNR) are the only source for deoxyribonucleotides and thus DNA. Class I RNRs perform two interesting reactions: (i) oxygen activation by a diiron center (reconstitution) forming a stable tyrosyl radical, which is essential for (ii) the enzymatic reaction. Their kinetics may be followed by rapid freeze quench electron paramagnetic resonance (EPR) and optical stopped-flow technique. (i) The reconstitution kinetics of mouse and E. coli RNR are different, allowing the investigation of different steps on the reaction pathway and the accumulation of new intermediate(s) in mouse RNR. Preliminary results show a considerable H/D kinetic isotope effect in mouse RNR suggesting accumulation of a high-valent iron intermediate. Detailed kinetic measurements will reveal whether this proton transfer is coupled to an electron transfer. Further, site directed mutants on the putative hydrogen delivery pathway in mouse RNR will be investigated. (ii) The enzymatic reaction mechanism is radical-based and might comprise a coupled proton/electron transfer over 35 Å. We will try to trap novel radical intermediates on this transfer pathway and in the active site by single turnover measurements in H2O and D2O. Both reactions, (i) and (ii), will be slowed down by use of cryoprotectants and measurements at sub-zero temperatures (-30°C). The main goal is to trap catalytically competent radical intermediates during (i) and (ii) and to characterize them by multi-frequency EPR, ENDOR, ESEEM, and other suitable spectroscopic methods (Mössbauer, resonance Raman, etc.).