Nitrogenase Assembly and Mechanism

描述(申请人提供)：还原氮是核酸和蛋白质的重要组成部分。因此，所有的生物体都需要这种营养来生长。不幸的是，尽管元素氮(N2)占地球大气的79%，但这种丰富的来源是惰性的，只有在它转化为可用的形式(如氨)后才能动员起来进行生物合成。在自然界中，这种固定氮气的能力仅限于一小群不同的重氮菌。重氮菌有一个共同的酶-固氮酶，这是本提案的主题，它催化依赖于镁三磷酸腺苷的氮气还原为氨。固氮酶是一种复杂的金属蛋白，由铁(Fe)蛋白和钼铁(MoFe)蛋白两种可分离纯化的蛋白质组分组成，均含有金属簇(S)。虽然金属蛋白在分解代谢调节和金属储存中起着重要的作用，但人们对其金属中心的生物合成以及这些金属簇结合到蛋白质中的机制知之甚少。固氮酶也不例外，它可能是迄今为止分离到的最耐人寻味和最复杂的金属蛋白。固氮酶研究的另一个重要方面涉及其催化机理。能量转导是一个基本的重要过程，它如何与蛋白质的构象变化相关，从而使其发挥催化功能，目前还不完全清楚。 在这里，我们建议大大扩展我们对固氮酶组装和催化的理解 遗传、生物化学和生物物理相结合的方法。令人感兴趣的生物是棕色固氮细菌，它是一种产生钼固氮酶的重氮菌。拟议的研究重点将是固氮酶MoFe蛋白的组装过程，该蛋白包含两个复杂而独特的金属簇，P-簇和FeMoco。同时，本研究还将探讨固氮酶的催化机理问题，其中固氮酶铁蛋白及其与核苷酸的相互作用是本研究关注的焦点。我们提出的研究将极大地拓宽我们对铁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：能量转导的理解。能量转导涉及蛋白质在核苷酸结合时在构象状态之间的转换以及随后的水解。

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.