The Hydroxylamine Oxidizing System of Nitrosomonas Europaea

Hooper NH2OH被羟胺氧化还原酶（HAO）氧化（NH2OH + H2O - HNO2 + 4e- + 4H+），这是已知最复杂的血红蛋白，每63 kDa亚基含有7个c-血红素和1个底物结合血红素P460。后者是一种新的酪氨酸衍生的c-血红素。至少两对血红素以电子方式相互作用，其中一对形成活性位点。另一种羟胺氧化酶“细胞色素P460”有一个金属中心，是赖氨酸衍生的c-血红素，它将与荚膜霉的细胞色素P-460一起进行研究。最近出现了一种HAO的晶体结构，它允许人们提出和测试涉及Fe-N或C-N中间体的不同催化机制，识别和探测活性位点中的重要假基和氨基酸，并研究负责先前观察到的金属中心之间磁相互作用的结构元素。本项目重点研究HAO和细胞色素P460。方法包括肽分析，DNA和蛋白质测序，动力学，自杀底物，电位测定法，光学，EPR-，穆斯堡尔-，共振拉曼-和ENDOR-光谱和x射线晶体学分析。在土壤中，亚硝化单胞菌有助于将氨氧化为作物所需的氮。在淹水土壤中，氮肥也会通过亚硝基单胞菌转化为一氧化二氮（N20）而流失，这是一种温室气体，也是破坏臭氧的催化剂。本文研究的工艺也是废水处理中除氨的限制步骤。最后，亚硝基单胞菌还能催化许多含氯污染物的降解。对所涉及的生物化学的了解可以导致更有效地利用肥料-氮，减少氧化亚氮的产生，改善废水处理，并改善污染土壤的现场生物修复。

9723608 Hooper NH2OH is oxidized (NH2OH + H2O - HNO2 + 4e- + 4H+) by the enzyme hydroxylamine oxidoreductase, "HAO", the most complex hemoprotein known, which contains 7 c-hemes and 1 substrate-binding heme P460 per 63 kDa subunit. The latter is a novel tyrosine-derivatized c-heme. At least two pairs of the hemes interact electronically and one of the pairs forms the active site. A second hydroxylamine-oxidizing enzyme, "cytochrome P460", has one metal center which is a lysine-derivatized c-heme and it, together with a cytochrome P-460 from M.capsulatus, will be studied. A crystal structure for HAO has recently appeared which allows one to propose and test distinct catalytic mechanisms involving Fe-N or C-N intermediates, identify and probe important prosthetic groups and amino acids in the active site, and investigate structural elements responsible for the magnetic interactions previously observed between metal centers. The focus of this project is on HAO and cytochromes P460. Approaches include peptide analysis, DNA and protein sequencing, kinetics, suicide substrates, potentiometry, Optical-, EPR-, Mossbauer-, resonance Raman-, and ENDOR- spectroscopies and X-ray crystallographic analysis. In soils, Nitrosomonas helps oxidize ammonia to the form of nitrogen needed by crops. In flooded soils fertilizer nitrogen can also be lost by conversion by Nitrosomonas to the gas, nitrous oxide (N20), a greenhouse gas and a catalyst of ozone destruction. The process studied here is also the limiting step in the removal of ammonia in waste water treatment. Finally, Nitrosomonas will also catalyze degradation of many chlorinated pollutants. An understanding of the biochemistry of the organism involved could lead to more efficient use of fertilizer-nitrogen, decreased production of nitrous oxide, improvements in the waste water treatment, and improved on-site bioremediation of contaminated soils.