Mossbauer Studies of Nitrite Reductase and Vanadium-containing Nitrogenase

生物依靠能量和化学元素的大规模循环来维持生命，而生物材料中最基本的元素之一是氮。因此，研究控制生物圈中氮状态的许多不同的生物过程是至关重要的。氮酶和亚硝酸盐还原酶是催化氮循环关键步骤的两种酶。氮酶催化大气中的二氮转化为氨（固氮），许多微生物利用它将氮吸收到生命系统中。硝酸还原酶将亚硝酸盐还原为氨，各种硝酸还原细菌以及一些硫酸盐还原细菌利用它进行终末呼吸，将氮异化回物质世界。因此，对这些酶的结构和功能的研究具有根本的重要性，并应提供与了解我们的生活环境有关的信息。在这个申请中，Huynh博士建议对以下酶进行莫斯堡尔研究：氮化酶系统中的钒铁蛋白。2. 从脱硫弧菌中提取亚硝酸盐还原酶。钒铁蛋白和亚硝酸盐还原酶都是含有多个铁中心的复合酶，可以用穆斯堡尔光谱进行最有效的研究。拟议的实验包括(i)天然酶的表征，（ii）再沉淀法研究，（iii） pH依赖性研究和（iv）底物和配体结合效应的检查。穆斯堡尔结果将与其他物理生化方法获得的数据相关联，包括光学和电子顺磁共振技术。Huynh博士希望充分表征这些酶中的铁中心，获得物理和生化特性，揭示这些铁中心在酶功能中的作用，并增强我们对生物固氮和亚硝酸盐还原机制的理解。

Living matters are sustained by large-scale cycles of energy and chemical elements, and one of the most essential elements in biological materials is nitrogen. To study the many different biological processes which govern the nitrogen status in the biosphere is, therefore, of vital importance. Nitrogenase and nitrite reductase are two enzymes which catalyze key steps in the nitrogen cycle. Nitrogenase catalyzes the conversion of atmospheric dinitrogen into ammonia (nitrogen fixation) and many microorganisms use it to assimilate nitrogen into the living system. Nitrate reductase reduces nitrite to ammonia and a variety of nitrate-reducing bacteria, as well as some sulfate reducing bacteria, use it for terminal respiration and dissimilates nitrogen back to the physical world. Structural and functional studies of these enzymes are therefore of fundamental importance and should provide information pertinent to the understanding of our living environment. In this application, Dr. Huynh proposes to perform Mossbauaer studies of the following enzymes: 1. the vanadium-iron protein of the nitrogenase enzyme system from Azotobacter vinelandii. 2. the nitrite reductase from Desulfovibrio desulfuricans. Both the vanadium-iron protein and nitrite reductase are complex enzymes containing multiple iron centers which can be most effectively studied by Mossbauer spectroscopy. The proposed experiments include (i)characterizations of the native enzymes, (ii)redoxtitration studies, (iii) pH- dependence investigations and (iv)examinations of substrate- and ligand-binding effects. The Mossbauer results will be correlated with data obtained form other physico-biochemical methods, including optical and electron paramagnetic resonance techniques. Dr. Huynh hopes to fully characterize the iron centers in these enzymes, to obtain physical and biochemical properties, to reveal the roles these iron centers play in their enzymatic functions and to enhance our understanding of the mechanisms of biological nitrogen fixation and nitrite reduction.