STUDY OF VANADIUM-CONTAINING NITROGENASE

固氮，将空气中的氮素转化为 氨是由固氮酶催化的。尽管有 已经对这种酶进行了大量研究 在过去的二十年里，固氮的机理 目前仍不清楚。这项研究的目的是为了更好地 通过研究一种新的替代品来理解这一机制 我厂新近分离的含钒固氮酶 实验室里的细菌是棕色固氮杆菌。 具体地说，拟议的研究将包括关于 V-固氮酶的酶学研究。底物(Km)和缓蚀剂 N_2、C_2H_2、HCN、CH_3NC、环丙烯和CO的(KI)性质 将对它们进行研究，以及它们对温度和pH的依赖。 该酶不同多肽的氨基酸序列 将被确定并与分离的抗体一起用于 合成相应NIF基因的寡核苷酸探针 另一种酶。ESR、Endor、MCD、 穆斯堡尔和x射线吸收光谱将用于 阐明不同成分的物理和化学性质 替代酶中的金属簇。在这些实验中， 该酶将在不同的氧化状态下和在 不同底物和抑制剂的存在和不存在。 此外，使用既定程序，VFe辅因数 集群将根据以下方面进行隔离和表征 元素分析以及电子和磁学性质。这 辅因子将用于重建载脂蛋白组分1的活性 传统的和替代的酶。最后， 将进行常规和常规的提纯 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.