High-field EPR studies on reaction intermediates of NO Synthase

血红素酶一氧化氮合酶（NOS）催化生物系统中信号分子NO的合成的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物质是通过与其他酶类比而提出的，但迄今为止还没有制备和光谱表征。我们的第一个X-波段EPR研究快速冷冻淬灭的iNOS样品，已通过与过氧乙酸（过氧化物分流）反应8毫秒表明存在的蛋白质侧链自由基以及铁（Fe（IV））卟啉-p-阳离子自由基。我们建议调查的性质的蛋白质自由基的多频EPR（特别是W-波段EPR）的NOS中间体制备的快速混合/冷冻淬火技术的反应时间范围从毫秒到分钟的时间尺度。我们将改进小的冷冻淬火W带（NOS）样品的制备过程，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。然后，这一技术也将用于这一优先方案中的其他项目，在这些项目中，必须捕获寿命短的中间体。

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.