A CYTOCHROME CD(1)-TYPE NITRITE REDUCTASE ISOLATED FROM THE MARINE DENITRIFIER PSEUDOMONAS-NAUTICA-617 - PURIFICATION AND CHARACTERIZATION
A CYTOCHROME CD(1)-TYPE NITRITE REDUCTASE ISOLATED FROM THE MARINE DENITRIFIER PSEUDOMONAS-NAUTICA-617 - PURIFICATION AND CHARACTERIZATION
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DOI：
10.1006/anae.1995.1021
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发表时间：
1995-08-01
期刊：
影响因子：
2.3
通讯作者：
FAUQUE, G
中科院分区：
文献类型：
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作者：
BESSON, S;CARNEIRO, C;FAUQUE, G
从海洋反硝化细菌假单胞菌617的可溶性提取物中纯化出亚硝酸盐还原酶（细胞色素cd(1)），达到电泳均匀性。细胞以10mm硝酸盐作为最终电子受体厌氧培养。通过连续4个色谱步骤纯化可溶部分，纯度最高的细胞色素cd(1)的A（280nm（氧化））/A（410nm（氧化））系数为0.90。纯化过程中，细胞色素cd(1)比活性最大值为0.048单位/mg蛋白。这种周质酶是一种同型二聚体，每个60 kDa的亚基含有一个血红素c和一个血红素d(1)作为假体，都处于低自旋状态。血红素c和d(1)的氧化还原电位在三种不同的pH值（6.6,7.6和8.6）下测定，没有任何pH依赖性。该蛋白nh2末端区域的前20个氨基酸序列与铜绿假单胞菌亚硝酸盐还原酶对应区域的同源性为45%，而与假单胞菌stutzeri和反硝化副球菌酶无同源性。描述了海洋假单胞菌617细胞色素cd(1)的紫外可见光谱和电子顺磁共振光谱特性。电子顺磁共振实验证实了血红素d(1)-一氧化氮配合物作为亚硝酸盐还原中间体的形成。(C) 1995年学术出版社
Nitrite reductase (cytochrome cd(1)) was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica strain 617. Cells were anaerobically grown with 10 mM nitrate as final electron acceptor. The soluble fraction was purified by four successive chromatographic steps and the purest cytochrome cd(1) exhibited an A(280nm(oxidized))/A(410nm(oxidized)) coefficient of 0.90. In the course of purification, cytochrome cd(1) specific activity presented a maximum value of 0.048 units/mg of protein. This periplasmic enzyme is a homodimer and each 60 kDa subunit contains one heme c and one heme d(1) as prosthetic moieties, both in a low spin state. Redox potentials of hemes c and d(1) were determined at three different pH values (6.6, 7.6 and 8.6) and did not show any pH dependence. The first 20 amino acids of the NH2-terminal region of the protein were identified and the sequence showed 45% identity with the corresponding region of Pseudomonas aeruginosa nitrite reductase but no homology to Pseudomonas stutzeri and Paracoccus denitrificans enzymes. Spectroscopic properties of Pseudomonas nautica 617 cytochrome cd(1) in the ultraviolet-visible range and in electron paramagnetic resonance are described. The formation of a heme d(1)-nitric-oxide complex as an intermediate of nitrite reduction was demonstrated by electron paramagnetic resonance experiments. (C) 1995 Academic Press