Nitric Oxide Reactions in Metalloenzymes

描述(申请人提供)：这项研究旨在阐明二铁蛋白中NO-还原酶活性的反应中间体，最终目的是了解金属簇如何催化这一反应。我们的研究将集中在三个方面：1)脱氮副球藻的NO还原酶CNOR和固氮芽孢杆菌的qCuANOR；2)嗜热嗜热杆菌的[血红素-铜]Ba3末端氧化酶；3)解毒热拟青霉的NO还原酶黄素蛋白A(FprA)。考虑到这些酶反应对哺乳动物的免疫反应具有抵抗力，更好地了解微生物的NO还原酶是非常必要的。虽然这些酶中的一些存在晶体结构，但它们的非络合物的结构和反应活性尚不清楚。DiIron蛋白参与解毒和反硝化NO还原酶反应，并被认为与NO反应形成[FeNO]2中间体。在该方案中，考虑并测试了可供选择的机械模型。共振拉曼光谱和傅里叶变换红外光谱具有识别亚硝基中间体和确定它们与这两种金属离子的非结合几何构型的独特能力。新型快速冷冻-淬火(RFQ)仪器可以在亚毫微秒的时间尺度内捕获中间体，这为表征以前光谱方法无法获得的反应中间体提供了新的机会。FTIR光谱结合N3~和CO的低温光解，作为NO替代物，提供了与这些NO还原酶机制相关的结合几何结构、潜在的氢键相互作用和质子转移的信息。

DESCRIPTION (provided by applicant): This research aims to elucidate reaction intermediates of NO-reductase activity in diiron proteins, with the ultimate goal of understanding how the metal clusters catalyze this reaction. Our studies will focus on three enzymes: 1) denitrifying NO reductases cNOR from Paracoccus denitrificans and qCuANOR from Bacillus azotoformans, 2) the [heme-copper] ba3 terminal oxidase from Thermus thermophilus, and 3) detoxifying NO reductase flavoprotein A (FprA) from Moorella thermoacetica. A better understanding of microbial NO reductases is highly desirable considering that these enzymatic reactions provide a resistance to the mammalian immune response. Although crystal structures exist for some of these enzymes, the structure and reactivity of their NO-complexes are not known. Diiron proteins participate in both detoxifying and denitrifying NO reductase reactions and are thought to react with NO to form [FeNO]2 intermediates. Alternative mechanistic models are considered and tested in this proposal. Resonance Raman and FTIR spectroscopies have the unique capability to identify nitrosyl intermediates and to define their NO-binding geometries with regard to the two metal ions. Novel rapid freeze-quench (RFQ) instrumentation that can trap intermediates within a sub-ms timescale provides new opportunities to characterize reaction intermediates that were previously inaccessible to spectroscopic methods. FTIR spectroscopy, in conjunction with low temperature photolysis of N3~ and CO, acting as NO surrogates, offers insight into binding geometries, potential hydrogen bond interactions and proton transfers relevant to these NO reductase mechanisms.