Atomic Models of Nitrogen Fixation by Nitrogenase from CryoEM Structures

项目概要： 固氮酶复合物是产生代谢上有用的形式的唯一生物途径。 氮来自大气中的二氮。这种双组分蛋白质系统由专性还原酶组成， Fe-蛋白和催化蛋白MoFe-蛋白仅存在于少数微生物中， 产生近50%的生物可利用氮。因此，生物固氮机制具有极其重要的意义。 对全球作物生产和人类健康产生重大影响。在过去的半个世纪， 不仅揭示了固氮酶复杂的动力学途径，而且还揭示了 在MoFe-蛋白质活性位点内的性质。这个簇，被称为FeMo-辅因子，具有以下组成： [7Fe：9 S：1C：1 Mo]-R-高柠檬酸盐，并且仅由活性位点内的两个残基配位。电子被 穿梭通过Fe蛋白中的[4Fe：4S]簇到MoFe蛋白中的[8 Fe：7S]中心，称为P- 集群这些还原当量然后被递送到活性位点FeMo-辅因子，在那里底物还原 发生。需要四个连续的电子转移循环来结合双氮，还需要四个 将一个二氮分子完全转化为两个氨分子。目前还不清楚FeMo辅因子是如何 为底物结合做好准备，FeMo-辅因子如何结合或穿梭电子到底物，或起什么作用 周围的活性位点残基在底物还原中起作用。该提案的主要假设是 FeMo辅因子和周围蛋白质中原子的物理重排， 底物还原对于底物进入和与辅因子结合是必需的，并且可以进一步发挥作用。 减少机制中的作用。这项工作的目标是确定什么重排发生在 固氮酶通过利用cryoEM的能量和进行实验， 有两个目标。目的1：固氮酶静息态的结构表征：单独的MoFe蛋白和 通过单颗粒cryoEM研究ADP-AlF 4稳定的铁蛋白复合物。目的2：结构表征 固氮酶周转相关的形式通过单颗粒cryoEM。通过追求目标1和2，我将获得 在单粒子cryoEM领域的培训，每个目标将提供金属酶化学的专业知识。 这项研究将在加州理工学院进行，主要是在著名的CryoEM 拥有大量的显微镜和专业知识。这些目标将阐明瞬态 中间体的固氮酶周转途径，从而提供了更好的基础，合理的 设计高效的合成固氮平台。此外， 在这个建议可以适用于其他了解较少的金属酶与人类健康有关。

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.