Mechanism and Function in Nitrogenase

9513512霍华德固氮酶是一种双蛋白组分酶，它既介导两种蛋白质（Fe蛋白和Mo-Fe蛋白）之间的电子转移，又介导ATP水解，ATP水解为电子转移提供能量。 铁蛋白具有与G蛋白和Ras相似的二级结构基序，这表明铁蛋白具有核苷酸依赖性开关的功能。 重要的区别是，铁蛋白是主管电子转移到钼铁蛋白只有在复杂的和ATP水解。该提案的目的是开发通过捕获从ATP到ADP状态的转变来稳定复合物的方法。核苷酸类似物的突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。 要解决的具体问题是：l。 电子在Av 1和Av 2的AlF 4- ADP复合物中的位置是什么？ 2.电子转移能被捕获在络合物的氧化还原簇中吗？3.在电子转移步骤中，稳定过渡态的氨基酸残基是什么？ 固氮是将大气中的氮气转化为生物学上重要的形式氨的过程。后者被纳入所有主要类别的生物分子中，因此是所有生命系统的必要组成部分。固氮酶如何完成这一过程既有实际意义，也有普遍的生物学意义。例如，固氮酶能够在30 ℃和正常大气压下催化反应，而工业过程需要一百倍的正常大气压和高温。了解酶催化的细节预示着更有效，因此更便宜的丰富的氨肥料。 固氮酶反应涉及电子在组成蛋白质之间的转移，每次一个。伴随着电子转移，2 ATP被水解（分解）以提供转移所需的能量。 正是这两个事件的耦合控制了整个化学反应。基于这两种固氮酶蛋白的优雅的X射线晶体结构，我们注意到电子供体组分（Fe蛋白）与其他核苷酸依赖性开关蛋白（例如参与肌肉收缩、与肿瘤起始相关的代谢调节等）具有惊人的结构相似性。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;当核苷酸水解时，构象发生变化。假设固氮酶中的构象“门”在ATP水解过程中打开，允许电子从一个位点移动到另一个位点，将通过将蛋白质捕获在门暂时打开的构象中来测试。由于固氮酶蛋白与其他生物过程中所涉及的蛋白质之间的相似性，这些结果应该有助于阐明这些其他系统的机制。 ***

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. ***