ELECTRON TRANSFER PATHWAYS AND BLUE COPPER PROTEINS

NO的化学性质在高等生物的细胞过程中很重要， 以及在环境过程中。 NO、NO2和NO3最近被 显示是巨噬细胞和嗜中性粒细胞的产物，具有细胞毒性或 细胞抑制作用 在环境过程中，分解细菌 是处理废物的重要媒介，也是硝酸盐的来源。 大气中的一氧化物（NO）。 细胞系统在很大程度上是氧化的， 而在细菌途径中，硝酸盐被还原转化， 最终通过NO和N2 O转化为N2。 来自细菌的含铜蛋白质提供了一种极好的 系统来研究NxOy化合物的化学性质，并探索影响NxOy化合物形成的因素。 影响氧化还原电位、电子转移途径和特异性。 在分解细菌粪产碱杆菌中， 蛋白质（pseudoazurin，一种铜氧还蛋白）将电子提供给铜 含有亚硝酸盐还原酶，如果NO2是底物，该亚硝酸盐还原酶产生NO，和 如果O2为底物，则为过氧化物。 无色杆菌（Achromobacter cycloclastes） 在反硝化途径中含有一对类似的分子， 此外还含有一个含铜的N2 O还原酶。 我们测定了A.粪 和来自A.碎裂环已结晶 每一对的候补成员，现在有机会获得现场指导 pseudoazurin和A.粪便 我们将包括X- 射线结构测定的定点突变的A。粪 pseudoazurin和Achromobacter cycloclastes NIR，并开始对三个新的 蛋白质类：A. faecalis NIR、多铜A.环裂体N2 O还原酶 和D.脱硫菌 在 为了深入了解影响氧化还原电位的因素，我们将 计算静电对所观察到的 假天青蛋白突变体P80 A的氧化还原电位增加139 mV 与野生型蛋白质相比，使用我们已知的氧化和 每个人的缩小形式。 我们将对天然蛋白质进行核磁共振研究 为了研究晶体学检测到的结构 解决方案的差异，以及测量电子转移自我， 汇率与其他cupredoxins比较。 对处理NxOy化合物的蛋白质结构的了解将 有助于理解重要生命过程的化学， 以及电子传递和蛋白质-蛋白质相互作用的基本过程 互动

The chemistry of NO is important in cellular processes of higher organisms, as well as in environmental processes. NO, NO2 AND NO3 have recently been shown to be a product of macrophages and neutrophils with cytotoxic or cytostatic effects. In environmental processes, denitrifying bacteria are important agents in managing waste, as well as being a source of nitric oxide (NO) in the atmosphere. The cellular system is largely oxidative, while in the bacterial pathway, nitrates are reductively converted eventually to N2 via NO and N2O. Copper-containing proteins from denitrifying bacteria provide an excellent system to study the chemistry of NxOy compounds and to explore factors affecting redox potentials, electron transfer pathways, and specificity. In the denitrifying bacteria Alcaligenes faecalis, a small blue copper protein (pseudoazurin, a cupredoxin) donates electrons to a copper containing nitrite reductase which produces NO if NO2 is the substrate, and peroxide if O2 is the substrate. The organism Achromobacter cycloclastes contains a similar pair of molecules in its denitrification pathway, and in addition has a copper-containing N2O reductase. We have determined the X-ray structures of pseudoaxurin from A. faecalis and nitrite reductase (NIR) from A. cycloclastes, have crystallized alternate members of each pair, and now have access to site-directed mutants of pseudoazurin and NIR from A. faecalis. We will include the X- ray structure determination of site-directed mutants of A. faecalis pseudoazurin, and Achromobacter cycloclastes NIR, and start on three new proteins: A. faecalis NIR, the multicopper A. cycloclastes N2O reductase and the hexaheme-containing nitrite reductase from D. desulfuricans. In order to gain insight into the factors affecting redox potentials we will perform calculations of electrostatic contributions to the observed increase of 139 mV in the redox potential of the pseudoazurin mutant P80A over the wild type protein, using our known structures of the oxidized and reduced forms of each. We will initiate NMR studies of the native protein in order to investigate crystallographically detected structural differences in solution, as well as to measure electron transfer self- exchange rates for comparison with other cupredoxins. Knowledge of the structures of proteins which process NxOy compounds will contribute to understanding the chemistry of important life processes, as well as fundamental processes of electron transfer and protein-protein interaction.