Nitrogenase Assembly and Mechanism

固氮酶组装和机制

• 批准号: 7058830
• 负责人: Markus W Ribbe
• 金额: $ 22.34万
• 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/7058830
• 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，所有生物体的生长都需要这种营养。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，只有在转化为可用形式（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群但多样化的固氮微生物。固氮生物具有共同的固氮酶，这是本提案的主题，它催化 MgATP 依赖性的 N2 还原为氨。固氮酶是一种复杂的金属蛋白，由两种可单独纯化的蛋白质成分组成，即铁 (Fe) 蛋白质和钼铁 (MoFe) 蛋白质，两者都含有金属簇。尽管金属蛋白在分解代谢和代谢调节以及金属储存中发挥着多种重要作用，但人们对其金属中心的生物合成以及这些金属簇掺入蛋白质的机制知之甚少。固氮酶也不例外，它可能是迄今为止分离出的最有趣、最复杂的金属蛋白。固氮酶研究引起广泛关注的另一个方面涉及其催化机制。能量转导是一个非常重要的过程，它如何与蛋白质的构象变化相关，从而使其发挥催化功能，目前尚不完全清楚。 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 结合遗传、生物化学和生物物理方法的机制。感兴趣的生物体是固氮菌，它是产生钼固氮酶的固氮菌之一。拟议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，本研究还将解决有关固氮酶催化机制的问题，其中固氮酶Fe蛋白及其与核苷酸的相互作用是关注的焦点。我们提出的研究将努力大大拓宽我们对铁蛋白催化机制的认识，并提高我们对生物学基本问题之一的理解：能量转导，其中涉及核苷酸结合及其随后的水解时蛋白质在构象状态之间的转换。