Mechanism and Function in Nitrogenase

固氮酶的机制和功能

• 批准号: 9513512
• 负责人: James Howard
• 金额: $ 30万
• 依托单位: University of Minnesota-Twin Cities
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9513512&HistoricalAwards=false
• 关键词: Mechanism; Function; Nitrogenase

9513512 Howard Nitrogenase is a two-protein component enzyme which mediates both electron transfer between the two proteins (Fe-protein and Mo-Fe protein) and ATP hydrolysis, which provides the energy for the electron transfer. Fe-protein has a strong secondary structural motif similar to G-proteins and Ras which suggests that Fe-protein functions as a nucleotide-dependent switch. The important distinction is that Fe-protein is competent for electron transfer to MoFe-protein only in the complex and during ATP hydrolysis. The objective of this proposal is to develop methods to stabilize the complex by trapping the transition from ATP to ADP states. Mutagenesis and kinetic analyses with nucleotide analogues provide a rational approach based upon the nucleotide switch model. Specific questions to be addressed are: l. What is the location of the electron in the AlF4- ADP complex of Avl and Av2? 2. Can electron transfer be trapped in one of the redox clusters of the complex? 3. What are the amino acid residues that stabilize the transition state during the electron transfer step? %%% Nitrogen fixation is the process whereby atmospheric nitrogen gas is converted to the biologically important form, ammonia. The latter is incorporated into all major classes of biomolecules and, hence, is a required component of all living systems. How this process is performed by the nitrogenase enzyme has both practical and general biological importance. For example, nitrogenase is able to catalyze the reaction at 30 C and normal atmospheric pressure while the industrial process requires a hundred times the normal atmospheric pressure and elevated temperature. Understanding details of the enzyme catalysis portends more efficient and therefore more cheaply abundant ammonia for fertilizer. The nitrogenase reaction involves the transfer, one at a time, of electrons between the component proteins. Concomitant with the electron transfer, 2 ATP are hydrolyzed (broken down) to provide the energy for the transfer. It is the coupling of the two events that controls the overall chemical reaction. Based upon the elegant x-ray crystal structures of the two nitrogenase proteins, we noted that the electron donor component (Fe-protein) had a striking structural similarity to other nucleotide-dependent switch proteins such as those involved in muscle contraction, metabolic regulation associated with tumor initiation and others. In all of these molecules, the role of the nucleotide is to stabilize specific protein conformations; the conformation switches as the nucleotide is hydrolyzed. The hypothesis that a conformational "gate" in nitrogenase is opened during ATP hydrolysis that allows the electron to move from one site to another will be tested by trapping the proteins in the conformation where the gate is momentarily open. Because of the similarity between the nitrogenase protein and the proteins involved in the other biological processes, these results should help to elucidate the mechanisms of these other systems as well. ***

9513512霍华德固氮酶是一种双蛋白质组分的酶，它既调节两种蛋白质(铁蛋白和钼-铁蛋白)之间的电子传递，又调节三磷酸腺苷的水解，为电子传递提供能量。Fe-蛋白具有与G-蛋白和RAS相似的强二级结构基序，表明Fe-蛋白具有核苷酸依赖开关的功能。重要的区别在于，只有在络合物中和在ATP水解过程中，铁蛋白才能将电子传递给MoFe蛋白。这个提议的目的是开发通过捕捉从ATP到ADP状态的转变来稳定络合物的方法。核苷酸类似物的诱变和动力学分析提供了一种基于核苷酸开关模型的合理方法。具体要解决的问题是：L。Av1和Av2的AlF4-ADP络合物中的电子位置是什么？2.电子转移能捕获到络合物的一个氧化还原簇团中吗？3.在电子转移步骤中稳定过渡态的氨基酸残基是什么？%%固氮是大气中氮气转化为生物重要形式氨的过程。后者被合并到所有主要的生物分子类别中，因此是所有生命系统的必备组件。固氮酶是如何完成这一过程的，具有实用和普遍的生物学意义。例如，固氮酶能够在30℃和常压下催化反应，而工业过程需要100倍的常压和高温。了解酶催化的细节预示着更有效的氨，因此更便宜地丰富的化肥。固氮酶反应涉及组成蛋白质之间的电子转移，一次一个。伴随着电子转移，2个三磷酸腺苷被水解(分解)，为电子转移提供能量。正是这两个事件的耦合控制了整个化学反应。基于这两个固氮酶蛋白优雅的X射线晶体结构，我们注意到电子供体成分(铁蛋白)与其他核苷酸依赖的开关蛋白有惊人的结构相似之处，如参与肌肉收缩、与肿瘤启动相关的代谢调节等。在所有这些分子中，核苷酸的作用是稳定特定的蛋白质构象；当核苷酸被水解时，构象发生变化。在ATP水解过程中，固氮酶中的构象“门”被打开，允许电子从一个位置移动到另一个位置，这一假设将通过捕获构象中的蛋白质来检验，在构象中，门是暂时打开的。由于固氮酶蛋白与其他生物过程中涉及的蛋白质之间的相似性，这些结果也应该有助于阐明这些其他系统的机制。***