Nitrogenase Assembly and Mechanism

固氮酶组装和机制

• 批准号: 6888041
• 负责人: Markus W Ribbe
• 金额: $ 22.85万
• 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/6888041
• 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）：减少氮是核酸和蛋白质的重要​​组成部分。因此，所有生物都需要这种养分才能生长。不幸的是，即使元素二氮（N2）占地球大气的79％，但这种丰富的来源是惰性的，并且只能在转换为氨（如氨（如氨）之后，才能动员起来进行生物合成。在自然界中，这种N2固定能力仅限于一小群但多样化的重生营养微生物。重18酶具有共同的氮氮酶，这是该提案的主题，并催化了N2对氨的MGATP依赖性降低。硝化剂是一种复杂的金属蛋白，由两个单独纯化的蛋白质成分组成，即铁（Fe）蛋白和钼铁（MOFE）蛋白，两者都包含金属簇（S）。尽管可以很好地确定金属蛋白在分解代谢和代谢调节以及金属储存中起多种基本作用，但对其金属中心的生物合成以及将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，也许是到目前为止分离的最有趣和最复杂的金属蛋白。吸引大量注意的氮酶研究的另一个方面涉及其催化机制。到目前为止，尚未完全了解能量转导（从根本上重要的过程）与蛋白质的构象变化相关，从而使其能够执行其催化功能。 在这里，我们建议大大扩展我们对氮酶总成和催化的理解 结合遗传，生化和生物物理方法的机制。感兴趣的生物是氮杂杆菌葡萄菌，这是产生钼氮酶的重18zotrophs之一。拟议的研究的主要重点是氮酶MOFE蛋白的组装过程，其中包含两个复杂而独特的金属簇，P-Cluster和Femoco。同时，有关氮酶Fe蛋白及其与核苷酸的相互作用作为关注的中心，有关氮酶催化机制的问题也将解决。我们提出的研究将努力大大扩大我们对Fe蛋白催化机制的了解，并提高我们对生物学的基本问题之一的理解：能量转导，涉及核苷酸结合构象状态之间蛋白质在构型结合及其随后的水解上的转换。