Nitrogenase Assembly and Mechanism

描述（由申请人提供）：还原态氮是核酸和蛋白质的重要组分。因此，所有生物都需要这种营养物质来生长。不幸的是，尽管元素二氮（N2）占地球大气的79%，但这种丰富的来源是惰性的，只能在转化为可用形式（如氨）后才能用于生物合成。在自然界中，这种N2固定能力仅限于一小部分不同的固氮微生物。固氮生物共同具有固氮酶，这是本提案的主题，并且催化N2到氨的MgATP依赖性还原。固氮酶是一种复杂的金属蛋白，由两种可分离纯化的蛋白质组分组成，即铁蛋白（Fe）和钼-铁蛋白（MoFe），两者都含有金属簇。虽然它是公认的，金属蛋白在分解代谢和代谢调节以及金属储存中发挥各种重要作用，很少有人知道它们的金属中心的生物合成和这些金属簇通过何种机制被纳入蛋白质。固氮酶也不例外，可能是迄今为止分离到的最有趣和最复杂的金属蛋白。固氮酶研究的另一个方面引起了相当大的关注，涉及其催化机制。能量传递是一个非常重要的过程，如何与蛋白质的构象变化相关，使其能够发挥催化功能，迄今为止还没有完全了解。 在这里，我们建议大大扩展我们的理解固氮酶组装和催化 通过结合遗传、生物化学和生物物理学方法研究其机制。感兴趣的生物体是棕色固氮菌（Azotobacter vinelandii），它是产生钼固氮酶的固氮菌之一。拟研究的主要焦点将是固氮酶MoFe蛋白的组装过程，该蛋白包含两个复杂且独特的金属簇，P-簇和FeMoco。同时，本研究还将探讨固氮酶的催化机制，重点研究固氮酶Fe蛋白及其与核苷酸的相互作用。我们的研究将奋进拓宽我们对Fe蛋白催化机制的认识，并提高我们对生物学中的一个基本问题的理解：能量传递，这涉及到蛋白质在核苷酸结合及其随后的水解时的构象状态之间的转换。

DESCRIPTION (provided by applicant): Reduced nitrogen is an essential component of nucleic acids and proteins. Therefore, all organisms require this nutrient for growth. Unfortunately, even though elemental dinitrogen (N2) comprises 79% of the earth's atmosphere, this abundant source is inert and can only be mobilized for biosynthesis following its conversion to a usable form like ammonia. In nature, this N2 fixation ability is restricted to a small but diverse group of diazotrophic microorganisms. Diazotrophs have in common the enzyme nitrogenase, which is the subject of this proposal, and which catalyses the MgATP-dependent reduction of N2 to ammonia. Nitrogenase is a complex metalloprotein composed of two separately purifiable protein components, the iron (Fe) protein and the molybdenum-iron (MoFe) protein, both of which containing metal cluster(s). Although it is well established that metalloproteins play a variety of essential roles in the catabolic and metabolic regulation as well as metal storage, very little is known about the biosynthesis of their metal centers and the mechanism through which these metal clusters are incorporated into proteins. Nitrogenase is no exception, being perhaps the most intriguing and complicated metalloprotein isolated so far. Another aspect of nitrogenase study that draws considerable attention involves its catalytic mechanism. How energy transduction, a fundamentally important process, is correlated with conformational changes of the protein, allowing it to carry out its catalytic function is not fully understood so far. Here we propose to greatly expand our understanding of the nitrogenase assembly and catalytic mechanism by combined genetic, biochemical and biophysical approaches. The organism of interest is Azotobacter vinelandii, one of the diazotrophs producing the molybdenum nitrogenase. The main focus of the proposed investigation will be the assembly process of nitrogenase MoFe protein, which contains two complex and unique metal clusters, P-cluster and FeMoco. Meanwhile, questions regarding the catalytic mechanism of nitrogenase will also be addressed in this study, with the nitrogenase Fe protein and its interaction with nucleotides as the center of attention. Our proposed studies will endeavor to greatly broaden our knowledge on the catalytic mechanism of Fe protein and improve our understanding on one of the fundamental issues in biology: energy transduction, which involves the switching of protein between conformational states upon nucleotide binding and its subsequent hydrolysis.