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

小鼠重组核糖核苷酸还原酶中耦合电子/质子转移的生物物理研究：

• 批准号: 5390454
• 负责人: Dr. Peter Paul Schmidt
• 金额: --
• 依托单位: Max-Planck-Institut für Chemische Energiekonversion (CEC)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2002
• 资助国家: 德国
• 起止时间: 2001-12-31 至 2005-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5390454?language=en
• 关键词: Biophysical; Investigations; Coupled; Electron; Proton

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.).

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）通过二铁中心的氧活化（重构），形成稳定的酪氨酰基自由基，这对于（ii）酶促反应是必需的。它们的动力学可以通过快速冷冻淬灭电子顺磁共振（EPR）和光学停流技术来跟踪。(i)小鼠和E.大肠杆菌RNR是不同的，允许研究反应途径上的不同步骤和小鼠RNR中新中间体的积累。初步结果表明，相当大的H/D动力学同位素效应在小鼠RNR建议积累的高价铁中间体。详细的动力学测量将揭示这种质子转移是否与电子转移耦合。此外，将研究小鼠RNR中假定的氢递送途径上的定点突变体。(ii)酶促反应机制是基于自由基的，可能包括超过35 ℃的耦合质子/电子转移。我们将尝试捕获新的自由基中间体在此转移途径和活性位点的单营业额测量H2O和D2 O。通过使用冷冻保护剂和在零度以下（-30 ° C）进行测量，反应（i）和（ii）都将减慢。主要目标是在（i）和（ii）过程中捕获催化活性自由基中间体，并通过多频EPR，ENDOR，ESEEM和其他合适的光谱方法（穆斯堡尔，共振拉曼等）对其进行表征。