Nitric Oxide Reactions in Metalloenzymes

描述（由申请人提供）：本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该反应。我们的研究将集中在三种酶上：1)反硝化副球菌的NO还原酶cNOR和偶氮样芽孢杆菌的qCuANOR， 2)嗜热菌的[血红素铜]ba3末端氧化酶，以及3)热酵母菌的解毒NO还原酶黄蛋白A （FprA）。考虑到这些酶促反应提供了对哺乳动物免疫反应的抗性，更好地了解微生物NO还原酶是非常可取的。虽然这些酶存在晶体结构，但它们的no复合物的结构和反应性尚不清楚。二铁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。在本建议中考虑并测试了其他机制模型。共振拉曼光谱和傅里叶红外光谱具有识别亚硝基中间体和确定其no结合几何形状的两种金属离子的独特能力。新型快速冻灭（RFQ）仪器可以在亚毫秒的时间尺度内捕获中间体，这为表征以前光谱方法无法实现的反应中间体提供了新的机会。FTIR光谱，结合低温光解N3~和CO作为NO替代品，提供了与这些NO还原酶机制相关的结合几何形状，潜在的氢键相互作用和质子转移的见解。

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.