Enzymology of the Hydroxylamine Oxidizing System of Nitrosomonas

在土壤中，化肥氨被化学自养硝化细菌NitroSomonas uropaea氧化成亚硝酸盐(NH3+1.502-HNO2+H2O)。亚硝酸盐被硝酸杆菌氧化成硝酸盐，这是非豆类作物中N的主要来源。因此，大多数食物氮的初始转化都是由亚硝化单胞菌进行的。在土壤中，亚硝化单胞菌在NH3氧化过程中或反硝化细菌还原硝酸盐过程中产生的氮气损失具有重要的经济意义。通过使用亚硝化单胞菌的氨氧化抑制剂来尝试通过反硝化来控制农业损失。这对更好地认识氨氧化为亚硝酸盐的机理具有重要意义。NH3被O2氧化成NH_2OH，然后被羟胺氧化还原酶“Hao”氧化(NH_2OH+H_2O)-HNO_2+4E-+4H+。HOO是一种大而异常复杂的血红素蛋白(每60,000 mW蛋白有7个c-血红素和1个底物结合“p460”基团)。四个电子从HAO传递到四氢血红素细胞色素c-554。然后，两个电子传递到膜上的电子转移链，最后到达末端的氧化酶，产生水。另外两个电子从细胞色素c-554以单独的电子转移链传递到氨氧化反应。该项目的目标包括分析Hao酶、细胞色素c-554和到氧气的电子传输链。他们打算确定这两种蛋白质的结构。将特别关注郝建国的活跃地点。探讨细胞色素c-554在活性中心的转换和电子从活性中心转移到好的血红素，进而转移到血红素的机制。该方法包括蛋白质纯化、光学光谱、多肽分析、DNA测序、动力学、自杀底物、电子顺磁共振光谱、穆斯堡尔光谱和X射线结晶学分析。

In soils, fertilizer ammonia is oxidized to nitrite (NH3 +1.502 - HNO2 +H2O) by the chemoautotrophic nitrifying bacterium Nitrosomonas europaea. Nitrite is oxidized by Nitrobacter to nitrate, a primary source of N for food protein in non-leguminous crops. Thus the initial transformation of most food nitrogen is carried out by Nitrosomonas. Of great economic importance is loss of N in soils as N-gases produced by Nitrosomonas during oxidation of NH3 or by reduction of nitrate by denitrifying bacteria. Control of agricultural loss by denitrification is attempted by the use of inhibitors of ammonia oxidation by Nitrosomonas. This lends significance to improved knowledge of the mechanism of oxidation of ammonia to nitrite. NH3 is oxygenated by O2 to NH2OH which is then oxidized (NH2OH +H2O)- HNO2 + 4e- +4H+) by the enzyme hydroxylamine oxidoreductase "HAO". HAO is a large and unusually complex hemoprotein (7 c-hemes and 1 substrate binding "P460" group per 60,000 MW protein). Four electron pass from HAO to a tetraheme cytochrome c-554. Two electrons then pass to a membrane electron transfer chain and finally to a terminal oxidase, producing water. Two additional electrons pass from cytochrome c-554 in a separate electron transfer chain to the ammonia oxidizing reaction. Goals of the project include an analysis of the enzyme HAO, the cytochrome c-554 and the electron transport chain to dioxygen. They intend to determine the structure of the two proteins. Particular attention will be paid to the active site of HAO. The mechanism of turnover in the active site and electron transfer from active site to HAO hemes and then hemes of cytochrome c-554 will be pursued. The approach involves protein purification, optical spectroscopy, peptide analysis, DNA sequencing, kinetics, suicide substrates, electron paramagnetic resonance spectroscopy, Mossbauer spectroscopy and X-ray crystallographic analysis.