Brito F, DeMoss J A, Dubourdieu M
Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston 77030, USA.
J Bacteriol. 1995 Jul;177(13):3728-35. doi: 10.1128/jb.177.13.3728-3735.1995.
On the basis of the observation that nitrate reductase from Escherichia coli is sensitive to UV irradiation with an action spectrum indicative of a naphthoquinone (F. Brito and M. Dubourdieu, Biochem. Int. 15:1079-1088, 1987), we extracted and characterized quinone components from two different preparations of purified nitrate reductase. A soluble form of nitrate reductase, composed of alpha and beta subunits, was purified after release from the membrane fraction by heat treatment, and a detergent-solubilized form, containing alpha, beta, and gamma (cytochrome bNR) subunits, was purified in the presence of Triton X-100. Extraction of soluble alpha beta form with chloroform-methanol yielded several UV-absorbing components, which were characterized as menaquinone-9 with an oxidized side chain and further photodestruction products of the menaquinone. The total amount of menaquinone extracted into the organic phase was estimated to be 0.97 mol/mol of alpha beta dimer. Extraction of the detergent-solubilized alpha beta gamma form by a similar procedure yielded two naphthoquinone-like components which were characterized by mass spectrometry as the oxidized forms of menaquinone-9 and demethylmenaquinone-9. In this case, the molar ratio of total naphthoquinone to the alpha beta dimer was estimated to be greater than 6:1. When cytochrome bNR and detergent were eliminated from the detergent-solubilized enzyme by heat treatment and ion-exchange chromatography, only menaquinone-9 could be identified in the organic extract of the active alpha beta product. These results suggest that menaquinone-9 is specifically bound to the alpha beta dimer and may be the UV-sensitive component in the pathway of electron transfer catalyzed by nitrate reductase.
基于大肠杆菌硝酸还原酶对紫外线辐射敏感且其作用光谱表明存在萘醌这一观察结果（F. 布里托和M. 迪布尔迪厄，《生物化学国际》15:1079 - 1088，1987年），我们从两种不同的纯化硝酸还原酶制剂中提取并鉴定了醌类成分。一种由α和β亚基组成的可溶性硝酸还原酶形式，经热处理从膜组分中释放后进行纯化；一种含α、β和γ（细胞色素bNR）亚基的去污剂增溶形式，在Triton X - 100存在下进行纯化。用氯仿 - 甲醇提取可溶性αβ形式产生了几种紫外线吸收成分，其被鉴定为具有氧化侧链的甲基萘醌 - 9以及甲基萘醌的进一步光解产物。提取到有机相中的甲基萘醌总量估计为每摩尔αβ二聚体0.97摩尔。通过类似方法提取去污剂增溶的αβγ形式产生了两种萘醌样成分，通过质谱鉴定为甲基萘醌 - 9和去甲基甲基萘醌 - 9的氧化形式。在这种情况下，总萘醌与αβ二聚体的摩尔比估计大于6:1。当通过热处理和离子交换色谱从去污剂增溶的酶中去除细胞色素bNR和去污剂时，在活性αβ产物的有机提取物中仅能鉴定出甲基萘醌 - 9。这些结果表明甲基萘醌 - 9特异性结合到αβ二聚体上，并且可能是硝酸还原酶催化的电子传递途径中的紫外线敏感成分。
