Nitrogenase Mechanism

固氮酶机制

• 批准号: 7036615
• 负责人: Lance C. Seefeldt
• 金额: $ 24.14万
• 依托单位: UTAH STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7036615
• 关键词: X ray crystallography; active sites; adenosine triphosphate; aminoacid; binding sites; cofactor; electrochemistry; electron transport; enzyme mechanism; enzyme substrate; ferredoxin; hemoprotein; hydrolysis; metal complex; nitrogenase; protein purification; site directed mutagenesis; spectrometry; X ray crystallography; active sites; adenosine triphosphate; aminoacid; binding sites; cofactor; electrochemistry; electron transport; enzyme mechanism; enzyme substrate; ferredoxin; hemoprotein; hydrolysis; metal complex; nitrogenase; protein purification; site directed mutagenesis; spectrometry

DESCRIPTION (provided by applicant): The element nitrogen (N) is essential to all living organisms, and the availability of fixed forms of nitrogen is often a limiting factor in food production. Biological nitrogen fixation, which occurs in a diverse group of microbes, represents the single largest input of fixed nitrogen in the reductive phase of the global nitrogen cycle. A molecular understanding of the enzyme responsible for biological nitrogen fixation, nitrogenase, could contribute to enhanced food production worldwide, and thus to the overall health of the human population. In addition, as a representative of the diverse family of metalloproteins and nucleotide-dependent enzymes, understanding the mechanism of nitrogenase will contribute to a broader understanding of these important enzymes. Our goal is to use a comprehensive approach to address some of the significant remaining questions about the nitrogenase mechanism. The related mechanistic issues that will be examined include the following: (i) understanding how MgATP hydrolysis is coupled to substrate reduction; (ii) defining how electron transfer through nitrogenase is controlled; (iii) understanding the roles of the associated metal clusters in this process; and (iv) defining how substrates access and products exit the active site. These goals will be accomplished by use of a wide array of methods including genetic selections, site-directed mutagenesis, purification of nitrogenase proteins with individual and multiple amino acid substitutions, steady state and pre-steady-state kinetics, electrochemistry, and spectroscopic methods including EPR, ENDOR, and X-ray crystallography.

描述（由申请人提供）：元素氮（N）对于所有生物体都是必不可少的，固定形式的氮的可用性通常是食品生产中的限制因素。生物氮固定发生在多种微生物组中，代表了在全球氮循环的还原相中，固定氮的最大输入。对负责生物氮固定氮的酶的分子理解，氮酶可能会导致全球增强的粮食生产，从而有助于人口的整体健康。此外，作为多元化金属蛋白和核苷酸依赖性酶的代表，了解氮酶的机制将有助于对这些重要酶有更广泛的了解。我们的目标是使用一种全面的方法来解决有关氮酶机制的一些重要剩余问题。将要检查的相关机制问题包括以下内容：（i）了解MGATP水解如何与底物减少耦合； （ii）定义如何控制电子通过氮气转移； （iii）了解相关金属簇在此过程中的作用； （iv）定义底物如何访问和产品退出活动站点。这些目标将通过使用多种方法来实现，包括遗传选择，定位的诱变，具有个体和多个氨基酸取代的氮酶蛋白的纯化，稳态和稳态动力学，电化学和光谱方法，以及光谱方法，包括EPR，EPR，EPR，senor，nator，endor和X-Ray Crysallography。