STUDY OF VANADIUM-CONTAINING NITROGENASE

固氮作用，将空气中的二氮转化为 氨是由固氮酶催化的。 尽管有 对这种酶的大量研究 在过去的二十年里，固氮机制 对目前仍不清楚. 本研究的目的是为了获得一个更好的 通过研究一种新的替代方法来了解这种机制 含钒固氮酶最近分离在我们的 棕色固氮菌（Azotobacter vinelandii）。 具体而言，拟议的研究将包括以下项目： V-固氮酶酶学 底物（Km）和抑制剂 (Ki)N2、C2 H2、HCN、CH 3 NC、环丙烯和CO的性质 将研究它们对温度和pH值的依赖性。 酶的不同多肽的氨基酸序列 将与分离的抗体一起沿着测定和使用， 合成对应于以下基因nif基因的寡核苷酸探针 替代酶。 ESR、ENDOR、MCD、 穆斯堡尔和X射线吸收光谱将用于 阐明不同的物理和化学性质， 金属簇的替代酶。 在这些实验中， 酶将在不同的氧化状态下进行研究， 存在和不存在不同的底物和抑制剂。 此外，使用已建立的程序， 集群将被隔离，并根据 元素分析和电子和磁性。 这 辅因子将用于重组载脂蛋白组分1中的活性 传统酶和替代酶。 最后， 将对常规的和 含有混合金属簇的替代固氮酶。

Nitrogen fixation, the conversion of dinitrogen from the air into ammonia, is catalyzed by the enzyme nitrogenase. In spite of the large volume of research that has been done on this enzyme during the past twenty years, the mechanism of nitrogen fixation is still unclear. The purpose of this research is to gain a better understanding of this mechanism by studying a new alternative vanadium-containing nitrogenase enzyme recently isolated in our laboratory from the bacterium Azotobacter vinelandii. Specifically, the proposed research will include projects on the enzymology of V-nitrogenase. The substrate (Km) and inhibitor (Ki) properties of N2, C2H2, HCN, CH3NC, cyclopropene and CO will be studies as will their dependency on temperature and pH. The amino acid sequences of the enzyme's different polypeptides will be determined and used, along with isolated antibodies, to synthesize oligolnucleotide probes for nif genes corresponding to the alternative enzyme. The techniques of ESR, ENDOR, MCD, Mossbauer and x-ray absorption spectroscopy will be used to elucidate the physical and chemical properties of the different metal clusters in the alternative enzyme. In these experiments, the enzyme will be studied in different oxidation states and in the presence and absence of different substrates and inhibitors. Furthermore, using established procedures, the VFe cofactor cluster will be isolated and characterized with regard to elemental analysis and electronic and magnetic properties. This cofactor will be used to reconstitute activity in apo-component 1 of both the conventional and alternative enzymes. Finally, purification will be undertaken of both conventional and alternative nitrogenase containing mixed metal clusters.