VANADIUM-CONTAINING NITROGENASE

描述：固氮作用，从土壤中转化二氮。 在固氮酶的催化下，空气转化为氨。 固氮酶 使用两个独特的金属簇来分裂氮三键。 的 这些金属簇的不寻常结构引起了人们的想象， 生物化学家和无机化学家。 最近，两个X射线结构 含有这些金属簇的组分1蛋白质已经被 测定 尽管已经有大量的研究 固氮酶在过去的二十五年中，氮的机制 固定是未知的。 X射线结构的可用性打开了 为固氮酶的结构机制研究打开了大门。 我们 实验室具有使用两种酶形式的优势，常规的 该菌的钼固氮酶和替代性钒固氮酶 棕色固氮菌，研究金属簇的各种作用 和蛋白质成分的酶功能。 提出了一种研究酶作用机理的创新实验方法 基于固氮酶的杂交和突变形式的使用。 混合 形式将通过将Vfe-辅因子并入Mo-中来产生。 缺乏辅因子的固氮酶蛋白，以及相互掺入 将钼铁辅因子转化为V-固氮酶蛋白。 初步研究 显示后一种混合物改变了底物还原模式。 突变蛋白将通过使用定点诱变来产生， 系统地替换V-中辅因子周围的关键氨基酸， 固氮酶与Mo-固氮酶的相应氨基酸。 这些蛋白质的酶学将被充分表征（即，公里， 产物分布和对变化的电子通量的响应 固定、乙炔还原和析氢。 CO抑制 外源底物减少和H2抑制固氮 也将被定性。 在后一系统中，D2和H2O（或H2和 D2 O）将被用于研究这些新蛋白质的能力， 在固氮过程中支持HD形成。 最后，这些蛋白质 将进行调查，以确定突变是否产生了 MgATP水解与电子传递的解偶联。 EPR光谱将用于探测电子的变化， 新蛋白质中辅因子的结构。 该技术还将 用于监测辅因子是否被分量2减少， 当蛋白质被激活时，是否产生CO诱导的S = 1/2信号 最后，一些蛋白质 （后来根据其独特的表型选择）将是 结晶并获得它们的X射线衍射光谱， 更多的结构信息 这些提出的杂交和突变酶的实验将奠定 为今后设计固氮酶奠定基础。

DESCRIPTION: Nitrogen fixation, the conversion of dinitrogen from the air into ammonia, is catalyzed by the enzyme nitrogenase. Nitrogenase uses two unique metal clusters to split the nitrogen triple bond. The unusual structures of these metal clusters have captured the imagination of biochemists and inorganic chemists. Recently, two X-ray structures of component 1 proteins containing these metal clusters have been determined. Despite the large volume of research that has been done on nitrogenase during the past twenty-five years, the mechanism of nitrogen fixation is not known. The availability of the X-ray structure opens the door to structure-based mechanistic studies of nitrogenase. Our laboratory has the advantage of using two enzyme forms, the conventional Mo-nitrogenase and the alternative V-nitrogenase from the bacterium Azotobacter vinelandii, to study the various roles of the metal clusters and protein components in enzyme function. An innovative experimental approach to the enzyme mechanism is proposed based on the use of hybrid and mutant forms of nitrogenase. The hybrid forms will be produced by incorporation of the Vfe-cofactor into the Mo- nitrogenase protein lacking cofactor, and the reciprocal incorporation of the MoFe-cofactor into the V-nitrogenase protein. Preliminary studies show that the latter hybrid has altered substrate reduction patterns. Mutant proteins will be produced by using site-directed mutagenesis to systematically replace key amino acids surrounding the cofactor in V- nitrogenase with the corresponding amino acid of Mo-nitrogenase. The enzymology of these proteins will be fully characterized (i.e., Km, product distribution and response to varying electron flux) for nitrogen fixation, acetylene reduction and dihydrogen evolution. CO inhibition of exogenous substrate reduction and H2 inhibition of nitrogen fixation will also be characterized. In the latter system, D2 and H2O (or H2 and D2O) will be used to investigate the ability of these new protein to support HD formation during nitrogen fixation. Finally, these proteins will be investigated to determine whether the mutation has produced an uncoupling of MgATP hydrolysis from electron transport. EPR spectroscopy will be used to probe changes in the electronic structure of the cofactor in the new proteins. This technique will also be used to monitor whether the cofactor is reduced by component 2 and whether CO-induced S = 1/2 signals are generated when the proteins are in turnover-competent medium with CO. Finally, some of the proteins (later selected according to their unique phenotype) will be crystallized and their x-ray diffraction spectra taken in order to gain greater structural information. These proposed experiments with hybrid and mutant enzymes will lay the foundation for future work on designed nitrogenases.