High-field EPR studies on reaction intermediates of NO Synthase

NO合酶反应中间体的高场EPR研究

• 批准号: 5381321
• 负责人: Dr. Friedhelm Lendzian
• 金额: --
• 依托单位: Arbeitsgruppe Physikalische Chemie/ Biophysikalische Chemie
• 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 2004-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5381321?language=en
• 关键词: field; EPR; studies; reaction; intermediates

The key intermediate in the reaction cycle of the heme enzyme nitric oxide synthase (NOS) which catalyses the synthesis of the signal molecule NO in biological systems is generally believed to be a Fe(IV)=O porphyrin-p-cation radical. This so called Compound I species in the NOS reaction cycle has been proposed from analogy with other enzymes but not prepared and spectroscopically characterized so far. Our first X-band EPR studies on rapid-freeze quenched iNOS samples which have been prepared by reaction with peroxy acetic acid (peroxide shunt) for 8 ms indicate the presence of a protein side chain radical as well as a ferryl (Fe(IV)) porphyrin-p-cation radical. We propose to investigate the nature of the protein radical by multi-frequency EPR (in particular by W-band EPR) of NOS intermediates prepared by the rapid-mixing/freeze-quench technique for reaction times ranging from the millisecond up to the minute time scale. We will improve the preparation procedure of small freeze-quenched W-band (NOS) samples, a technique which has been recently developed in the laboratory of the Institute of Physics, Medical University of Lübeck. This technique will then be also available for other projects in this priority programme, where short-lived intermediates must be trapped.

血红素一氧化氮合酶（NOS）催化生物系统中信号分子NO的合成，其反应循环中的关键中间体通常被认为是Fe(IV)=O卟啉-p-阳离子自由基。 NOS 反应循环中的这种所谓的化合物 I 物种是通过与其他酶的类比而提出的，但迄今为止尚未制备和光谱表征。我们对快速冷冻猝灭的 iNOS 样品进行的第一个 X 波段 EPR 研究表明，该样品是通过与过氧乙酸（过氧化物分流器）反应 8 毫秒而制备的，表明存在蛋白质侧链自由基以及 Ferri1 (Fe(IV)) 卟啉-p-阳离子自由基。我们建议通过快速混合/冷冻猝灭技术制备的 NOS 中间体的多频率 EPR（特别是 W 波段 EPR）来研究蛋白质自由基的性质，反应时间范围从毫秒到分钟。我们将改进小型冷冻淬火W波段（NOS）样品的制备程序，这是吕贝克医科大学物理研究所实验室最近开发的一项技术。该技术也将可用于该优先计划中必须捕获短寿命中间体的其他项目。