The Hydroxylamine Oxidizing System of Nitrosomonas europaea

Hooper 0093447本研究的目的包括理解（a）单血红素细胞色素P460，（B）多血红素c-细胞色素羟胺氧化还原酶（HAO）的催化机制。(c)四血红素细胞色素C554，（d）单血红素细胞色素C552和（e）二-c-血红素细胞色素过氧化物酶。 这些是参与自养细菌N.欧洲。 细菌催化全球氮循环的硝化途径中的一个步骤。 它的活动在农业、废水处理、环境污染物的现场生物修复、温室气体形成和臭氧消耗等方面至关重要。 羟胺氧化酶的活性位点含有一种新的血红素样大环化合物，血红素P460，一种与蛋白酪氨酸具有独特共价交联的c-血红素衍生物。 在另一种来自N.称为细胞色素P460。两种血红素P460具有相似的独特光学性质。. 催化血红素作为还原酶或加氧酶在许多细胞过程中起着关键作用。 另一方面，HAO催化生物相关的脱氢反应，这是催化细胞色素中前所未有的功能，并且在一般金属蛋白中很少观察到。 待测试的机制包括：底物的去质子化;参与催化的交联酪氨酸或赖氨酸;和促进电子去除的c-血红素。 具体目标如下。 (a)细胞色素P460。 纯化现有的定点突变蛋白K70 A、K70 R和K70 Y，并表征其催化、光学、EPR和拉曼光谱以及氧化还原电位。 研究X射线晶体结构的工作将继续下去。(b)浩 建立hao基因的表达系统并进行定点突变。通过蛋白水解和MALDI鉴定三硝基甲烷衍生的或天然蛋白中修饰的残基。 完成X光结构。(c)细胞色素c554。 鉴定现有的定点突变体F156 A。 (d)细胞色素c552。 纯化和鉴定现有的v63缺失位点定向突变体，并建立HALS信号的结构基础。 (e)细胞色素c过氧化物酶。完成晶体结构分析，制备定点突变体。

Hooper 0093447The objectives of this research include understanding the catalytic mechanism of (a) The monoheme cytochrome P460, (b) the multiheme c-cytochrome, hydroxylamine oxidoreductase (HAO). (c) the tetraheme cytochrome c554, (d) the monoheme cytochrome c552 and (e) the di-c-heme cytcperoxidase. These are reactive cyochromes which participate in the energy-generating reactions of the autotrophic bacterium, N. europaea. The bacterium catalyzes a step in the nitrification pathway of the global nitrogen cycle. Its activity is key in agriculture, waste water treatment, on-site bioremediation of environmental pollutants, greenhouse gas formation and ozone depletion. The active site of the hydroxylamine oxidizing enzyme contains a novel heme-like macrocycle, heme P460, a derivative of a c-heme which has a unique, covalent crosslink to a protein tyrosine. A similar crosslink, but to a lysine, is observed in anotherhydroxylamine oxidizing enzyme from N. europaea called cytochrome P460. Both heme P460s have similar unique optical properties. . Catalytic hemes play critical roles in many cellular processes functioning as either reductases or oxygenases. HAO, on the other hand, catalyzes a biologically relevant dehydrogenation reaction, a function unprecedented among catalytic cytochromes, and rarely observed in metalloproteins in general. The mechanism to be tested includes: deprotonation of substrate; involvement of the cross-linked tyrosine or lysine in catalysis; and facilitation of electron removal by c-hemes. Specific goals are as follows. (a) Cytochrome P460. To purify the existing site directed mutant proteins K70A, K70R and K70Y and characterize their catalytic, optical-, EPR- and Raman-spectra and their redox potentials. To work on the X-ray crystallographic structure will continue. (b) HAO. To develop an expression system for the hao gene and make site-directed mutants. To identify by proteolysis and MALDI residue(s) which are derivatized by trinitromethane or which are modified in the native protein. To complete the X-ray structure. (c) Cytochrome c554. To characterize the existing site directed mutant F156A. (d) Cytochrome c552. To purify and characterize the existing v63 deletion site directed mutant and establish the structural basis for the HALS signal. (e) Cytochrome c Peroxidase. To complete analysis of the crystal structure and prepare site-directed mutants.