Nitrogenase Assembly and Mechanism

固氮酶组装和机制

• 批准号: 6743792
• 负责人: Markus W Ribbe
• 金额: $ 22.73万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6743792
• 关键词: Azotobacter vinelandii; atomic absorption spectrometry; bacterial proteins; bioenergetics; catalyst; circular dichroism; conformation; electron spin resonance spectroscopy; enzyme activity; enzyme biosynthesis; gel electrophoresis; intermolecular interaction; iron; metal complex; metalloproteins; molecular assembly /self assembly; molybdenum; nitrogenase; nucleotides; protein purification; protein structure; site directed mutagenesis

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.

描述（由申请人提供）：还原氮是核酸和蛋白质的重要组成部分。因此，所有生物的生长都需要这种养分。不幸的是，尽管单质二氮（N2）占地球大气的79%，但这种丰富的来源是惰性的，只能在转化为氨等可用形式后进行生物合成。在自然界中，这种固氮能力仅限于一组小而多样的重氮营养微生物。重氮营养体有共同的酶氮酶，这是本提案的主题，并催化mgatp依赖的N2还原为氨。氮酶是一种复杂的金属蛋白，由两个单独纯化的蛋白质组分组成，铁（Fe）蛋白和钼铁（MoFe）蛋白，两者都含有金属簇。虽然金属蛋白在分解代谢调节和金属储存中发挥着多种重要作用，但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。氮酶也不例外，它可能是迄今为止分离出的最有趣、最复杂的金属蛋白。氮酶研究的另一个备受关注的方面是其催化机理。能量转导是一个非常重要的过程，它是如何与蛋白质的构象变化相关联的，从而使其发挥催化功能，目前还不完全清楚。