NITROGEN FIXATION IN W CONTAINING MEDIUM

固氮，即二氮转化为氨，是直接 与世界范围内的作物生产有关，因此是最重要的作物之一。 自然界中重要的酶反应。 然而，尽管大 大量的研究已经完成了这个系统，机制， 固氮作用仍不清楚。 本研究的目的是 为了更好地理解这一机制， 固氮酶，固氮酶，在一个突变改变的系统。 由于固氮酶是一种含氘的酶，并且由于它通常 认为Mo位于酶的活性位点， 还原和结合双氮，要研究的突变体是那些 其在不存在Mo但存在钨（W）的情况下固定二氮， 一种被证实的野生型固氮抑制剂。 对这些突变体中酶的初步研究表明，它含有 等量的Mo和W。 在这项提案中，这种酶将被分离出来， 并纯化至均质。 它将以金属为特征 (i.e. Fe、Mo和W）含量、分子量、多肽组成和 相对于野生型酶的底物特异性。 光谱 通过记录酶的ESR来完成表征， 光学和低温MCD光谱。 活性位点辅因子将 也从该突变体中分离并测定其金属组成。 第二阶段的研究建议是寻找一个可能的 固氮菌交替固氮系统。 它是由 其他人认为，这种替代系统的功能，在条件钼 剥夺 C2 H2和N2还原相对速率的差异， 将测量突变体以确定是否存在两种不同的 固氮系统 抗正常人免疫球蛋白I组分抗体 固氮酶将用于抑制体外固定的普通 系统，从而允许检测可能的替代系统。 最后，野生型固氮菌将用于耗尽含W培养基 允许研究突变体在W-培养基中的生长，而不 任何钼存在，看看固氮是否可以发生在钼的情况下。

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.