Yano T, Yagi T, Sled V D, Ohnishi T
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 1995 Aug 4;270(31):18264-70. doi: 10.1074/jbc.270.31.18264.
The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 dissimilar subunits which are designated NQO1-14 and contains one noncovalently bound FMN and at least five EPR-visible iron-sulfur clusters (N1a, N1b, N2, N3, and N4) as prosthetic groups. Comparison of the deduced primary structures of the subunits with consensus sequences for the cofactor binding sites has predicted that NQO1, NQO2, NQO3, NQO9, and probably NQO6 subunits are cofactor binding subunits. Previously, we have reported that the NQO2 (25 kDa) subunit was overexpressed as a water-soluble protein in Escherichia coli and was found to ligate a single [2Fe-2S] cluster with rhombic symmetry (gx,y,z = 1.92, 1.95, and 2.00) (Yano, T., Sled', V.D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). In the present study, the NQO3 (66 kDa) subunit, which is equivalent to the 75-kDa subunit of bovine heart Complex I, was overexpressed in E. coli. The expressed NQO3 subunit was found predominantly in the cytoplasmic phase and was purified by ammonium sulfate fractionation and anion-exchange chromatography. The chemical analyses and UV-visible and EPR spectroscopic studies showed that the expressed NQO3 subunit contains at least two distinct iron-sulfur clusters: a [2Fe-2S] cluster with axial EPR signals (g perpendicular, parallel = 1.934 and 2.026, and L perpendicular parallel = 1.8 and 3.0 millitesla) and a [4Fe-4S] cluster with rhombic symmetry (gx,y,z = 1.892, 1.928, and 2.063, and Lx,y,z = 2.40, 1.55, and 1.75 millitesla). The midpoint redox potentials of [2Fe-2S] and [4Fe-4S] clusters at pH 8.6 are -472 and -391 mV, respectively. The tetranuclear cluster in the isolated NQO3 subunit is sensitive toward oxidants and converts into [3Fe-4S] form. The assignment of these iron-sulfur clusters to those identified in the P. denitrificans NDH-1 enzyme complex and the possible functional role of the NQO3 subunit is discussed.
反硝化副球菌的质子转运型NADH-醌氧化还原酶（NDH-1）由至少14个不同的亚基组成，这些亚基被命名为NQO1-14，并且含有一个非共价结合的FMN和至少五个EPR可见的铁硫簇（N1a、N1b、N2、N3和N4）作为辅基。将这些亚基的推导一级结构与辅因子结合位点的共有序列进行比较，预测NQO1、NQO2、NQO3、NQO9以及可能的NQO6亚基是辅因子结合亚基。此前，我们报道过NQO2（25 kDa）亚基在大肠杆菌中作为水溶性蛋白过表达，并且发现它连接了一个具有菱形对称性的单[2Fe-2S]簇（gx,y,z = 1.92、1.95和2.00）（矢野，T.，斯莱德，V.D.，大西，T.，和矢木，T.（1994年）《生物化学》33卷，494 - 499页）。在本研究中，与牛心复合体I的75 kDa亚基相当的NQO3（66 kDa）亚基在大肠杆菌中过表达。所表达的NQO3亚基主要存在于细胞质相中，通过硫酸铵分级分离和阴离子交换色谱法进行纯化。化学分析以及紫外可见和EPR光谱研究表明，所表达的NQO3亚基含有至少两个不同的铁硫簇：一个具有轴向EPR信号的[2Fe-2S]簇（g垂直，平行 = 1.934和2.026，以及L垂直平行 = 1.8和3.0毫特斯拉）和一个具有菱形对称性的[4Fe-4S]簇（gx,y,z = 1.892、1.928和2.063，以及Lx,y,z = 2.40、1.55和1.75毫特斯拉）。在pH 8.6时，[2Fe-2S]和[4Fe-4S]簇的中点氧化还原电位分别为 - 472和 - 391 mV。分离出的NQO3亚基中的四核簇对氧化剂敏感，并转化为[3Fe-4S]形式。讨论了这些铁硫簇与在反硝化副球菌NDH-1酶复合体中鉴定出的铁硫簇的对应关系以及NQO3亚基可能的功能作用。
