Nitrogenase Mechanism

固氮酶机制

• 批准号: 6770737
• 负责人: Lance C. Seefeldt
• 金额: $ 25.55万
• 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/6770737
• 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

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需的，固定形式氮的可用性通常是食品生产的限制因素。生物固氮发生在多种微生物中，代表了全球氮循环还原阶段固定氮的最大单一输入。对负责生物固氮的酶（固氮酶）的分子理解可能有助于提高全世界的粮食产量，从而促进人类的整体健康。此外，作为金属蛋白和核苷酸依赖性酶的不同家族的代表，了解固氮酶的机制将有助于更广泛地了解这些重要的酶。我们的目标是使用综合方法来解决有关固氮酶机制的一些重要的剩余问题。将要研究的相关机制问题包括以下内容：（i）了解 MgATP 水解如何与底物还原耦合； (ii) 定义如何控制通过固氮酶的电子转移； (iii) 了解相关金属簇在此过程中的作用； (iv) 定义底物如何进入活性位点以及产物如何离开活性位点。这些目标将通过使用多种方法来实现，包括遗传选择、定点诱变、用单个和多个氨基酸取代纯化固氮酶蛋白、稳态和稳态前动力学、电化学和光谱方法，包括EPR、ENDOR 和 X 射线晶体学。