Atomic Models of Nitrogen Fixation by Nitrogenase from CryoEM Structures

项目总结： 固氮酶复合体是产生代谢有用形式的唯一生物途径。 来自大气氮素的氮。这个由专性还原酶组成的两组分蛋白质系统， 铁蛋白和催化蛋白MoFe-蛋白只存在于少数微生物中，但 产生近50%的生物可利用氮。因此，生物固氮机制具有极强的 对全球农作物生产和人类健康的重要影响。在过去的半个世纪里，开创性的作品 不仅揭示了固氮酶复杂的动力学途径，而且还揭示了在 自然界中的钼铁蛋白活性部位。这种簇被称为FEMO辅因子，其组成如下 [7Fe：9S：1C：1Mo]-R-高柠檬酸，只与活性中心内的两个残基配位。电子是 穿梭通过铁蛋白中的[4Fe：4S]簇到达MoFe蛋白中的[8Fe：7S]中心，称为P- 集群。然后，这些还原当量被传递到底物还原的活性部位FeMo-辅因子 发生。结合氮素需要四个连续的电子转移循环，还需要四个循环 将一个氮素分子完全转化为两个氨分子。目前尚不清楚FEMO辅因数是如何 为底物结合做好准备，FEMO辅助因子如何将电子结合或穿梭到底物上，或扮演什么角色 周围的活性中心残基在底物还原中起作用。这项提议的主要假设是 FEMO辅因子和周围蛋白质中原子的物理重排 底物还原是底物进入和与辅因子结合所必需的，并可能进一步发挥作用 在减排机制中发挥作用。这项工作的目标是确定在 固氮酶通过利用低温EM的能量和进行实验来完成底物的周转 概括起来有两个目标。目的1：固氮酶静止状态的结构特征：单独的MoFe蛋白和 用单颗粒低温电子显微镜制备了ADP-AlF4稳定的铁蛋白络合物。目标2：结构特征 用单颗粒低温电子显微镜研究固氮酶周转相关形式。通过追求目标1和目标2，我将获得 在单粒子低温EM领域的培训，每个目标都将提供金属酶化学方面的专业知识。 这项研究将在加州理工学院进行，主要是在众所周知的低温EM 中心有充足的显微镜和专业知识。这些目标将为瞬变现象提供亟需的信息 固氮酶周转途径中的中间体，从而为合理的 高效合成固氮平台的设计。此外，还开发了洞察力和方法 这一建议也可应用于其他知之甚少的与人类健康相关的金属酶。

PROJECT SUMMARY: The nitrogenase enzyme complex is the only biological pathway for producing metabolically useful forms of nitrogen from atmospheric dinitrogen. This two component protein system made up of an obligate reductase, Fe-protein, and the catalytic protein, MoFe-protein, is only present in a small number of microorganisms, but produces nearly 50% of all bioavailable nitrogen. Thus, the biological nitrogen fixation mechanism has extremely important effects on global crop production and human health. Seminal works in the past half century have not only revealed an intricate kinetic pathway for nitrogenase, but also the most complex metallocluster found in nature within the MoFe-protein active site. This cluster, known as the FeMo-cofactor, has the composition [7Fe:9S:1C:1Mo]-R-homocitrate and is only coordinated by two residues within the active site. Electrons are shuttled through a [4Fe:4S] cluster in the Fe-protein to an [8Fe:7S] center in the MoFe-protein, known as the P- cluster. These reducing equivalents are then delivered to the active site FeMo-cofactor where substrate reduction occurs. Four consecutive cycles of electron transfer are required to bind dinitrogen, and four more are required to fully convert one dinitrogen molecule to two ammonia molecules. It is unknown how the FeMo-cofactor is primed for substrate binding, how the FeMo-cofactor binds or shuttles electrons to the substrate, or what role the surrounding active site residues play in substrate reduction. The primary hypothesis of this proposal is that physical rearrangements of the atoms in the FeMo-cofactor and in the surrounding protein during substrate reduction are necessary for substrate access and binding to the cofactor, and may further play a role in the reduction mechanism. The goal of this work is to determine what rearrangements occur in nitrogenase throughout turnover of substrate by harnessing the power of cryoEM and performing experiments outlined in two Aims. Aim 1: Structural characterization of resting states of nitrogenase: MoFe protein alone and the ADP-AlF4- stabilized complex with Fe-protein by single particle cryoEM. Aim 2: Structural characterization of nitrogenase turnover-related forms by single particle cryoEM. Through the pursual of Aims 1 and 2, I will obtain training in the field of single particle cryoEM, and each Aim will provide expertise in metalloenzyme chemistry. The research will be performed at the California Institute of Technology primarily in the well-known CryoEM Center with ample microscopes and expertise. These Aims will shed much needed light on transient intermediates within the nitrogenase turnover pathway thereby providing a better foundation for the rational design of efficient synthetic nitrogen fixation platforms. In addition, the insights and the methodology developed in this proposal can be applied to other poorly understood metalloenzymes related to human health.