NITROGEN FIXATION IN W CONTAINING MEDIUM

固氮，即将氮素转化为氨，是直接 与世界范围的农作物生产有关，因此是世界上最 自然界中重要的酶反应。然而，尽管受到了极大的鼓舞 已经对这个系统进行了大量的研究，其机制 固氮作用仍不清楚。这项研究的目的是 要更好地理解这一机制，请研究 固氮酶，固氮酶，在突变的系统中。 由于固氮酶是一种含钼酶，而且由于它通常 感觉钼是在酶的活性位置上为两者服务的 氮素的还原和结合，需要研究的突变体有 在没有钼但有钨(W)存在的情况下固定氮素， 一种已被证实的野生型固氮抑制剂。 对这些突变体中的酶的初步研究表明，它包含 在这个方案中，这种酶将被分离出来 并提纯到同质。它将以金属为特征 (即铁、钼和钨)含量、相对分子质量、多肽组成和 相对于野生型酶的底物专一性。光谱学 表征将通过记录酶的ESR来完成， 光学和低温MCD光谱。活动位置辅因数将 也从该突变体中分离出来，并测定其金属组成。 提出的研究的第二阶段是寻找可能的 固氮菌的交替固氮系统。它是由以下人员提出的 其他人则认为，这种备用系统在工作状态下工作 剥夺。C_2H_2和N_2相对还原速率的差异 将对突变体进行测量，以确定是否存在两种不同的 固氮系统。抗正常人组分I的抗体 固氮酶将被用来抑制普通的体外固定 系统，从而允许检测可能的替代系统。 最后，使用野生型固氮菌来耗尽含W的培养基 允许研究突变体在W-培养基中的生长 任何钼的存在，看看在没有钼的情况下是否可以进行固氮。

Nitrogen fixation, the conversion of dinitrogen to ammonia, is directly related to world-wide crop production and, as such, is one of the most important enzymatic reactions in nature. However, inspite of the large volume of research that has been done on this system, the mechanism of nitrogen fixation is still unclear. The purpose of this research is to gain a better understanding of this mechanism by studying the nitrogen-fixing enzyme, nitrogenase, in a mutationally altered system. Since nitrogenase is a molybdenum-containing enzyme and since it generally is felt that Mo is at the active site of the enzyme serving both for reduction and binding of dinitrogen, the mutants to be studied are those that fix dinitrogen in the absence of Mo but the presence of tungsten (W), a proven inhibitor of wild-type nitrogen fixation. A preliminary study of the enzyme in these mutants suggest that it contains equal amounts of Mo and W. In this proposal, this enzyme will be isolated and purified to homogeneity. It will be characterized in terms of metal (i.e. Fe, Mo and W) content, molecular weight, polypeptide composition and substrate specificity relative to the wild-type enzyme. Spectroscopic characterization will be accomplished by recording the enzyme's esr, optical and low-temperature MCD spectra. The active-site cofactor will also be isolated from this mutant and its metal composition determined. The second stage of the research proposed is the search for a possible alternate nitrogen-fixing system in azotobacter. It has been proposed by others that this alternate system functions during conditions of Mo deprivation. Differences in the relative rates of C2H2 and N2 reduction in the mutant will be measured to determine whether there are two different nitrogen-fixing systems. Antibodies against component I of normal nitrogenase will be used to inhibit in vitro fixation by the ordinary system thus allowing the detection of the possible alternate system. Finally, wild-type azotobacter will be used to deplete W-containing medium of Mo to allow the study of the growth of the mutant in W-medium without any Mo present to see if nitrogen fixation can occur in the absence of Mo.