Expression of the 25-kilodalton iron-sulfur subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans.
Expression of the 25-kilodalton iron-sulfur subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans.
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脱氮副球菌能量转导 NADH-泛醌氧化还原酶 25 千道尔顿铁硫亚基的表达。
DOI：
10.1021/bi00168a014
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Yagi,T
中科院分区：
文献类型：
--
作者：
Yano,T;Sled,VD;Ohnishi,T;Yagi,T
摘要：副球菌的能量转导NADH-泛醌（Q）氧化还原酶由14个不同的亚基组成，并含有至少4个铁-硫簇[Yagi，T.（1993）Biochim. Biophys. Acta 1141，1-11]，已经确定了编码嗜热假单胞菌的能量转导NADH-Q氧化还原酶的基因簇的完整DNA序列。本文报道了在大肠杆菌中表达的P. acetificans酶复合物的25千道尔顿（kDa）（NQ 02）亚基和表征的铁-硫簇结合到表达的亚基。25-kDa亚基在E. coli细胞，然后使用亲和镍螯合柱纯化。纯化的亚基含有1.44摩尔的非血红素铁和1.33摩尔的酸不稳定的硫化物/摩尔亚基。EPR分析表明，该亚基的还原形式，表达的亚基包含一个单一的双核[2Fe-2S]簇。该簇表现出菱形对称的光谱，g值为gxya=1.913、1.942和1.996，这与牛心脏复合物I的解析黄素蛋白II亚组分（亚基24+ 9 kDa）中[2Fe-2S]簇的光谱非常相似[Ragan，C.一、加兰特岛M.，Hatefi，Y.，& Ohnishi，T.（1982）Biochemistry 21，590-594; Ohnishi，T.，拉根角一、& Hatefi，Y.（1985）J.Biol.Chem.260，2782-2788]。Paracoccus nadh-Q氧化还原酶的25 kDa亚基的双核铁-硫簇原位EPR可见的铁-硫簇的分配进行了讨论。Paracoccus nadh-Q氧化还原酶是一种革兰氏阴性的土壤细菌，被称为“一个自由生活的细菌”（Steinriicke & Ludwig，1993; Kurowski & Ludwig，1987; Ludwig，1987; Stouthamer，1980）。需氧生长的嗜热假单胞菌表达哺乳动物呼吸型呼吸链（Stouthamer，1992），其含有能量转导的NADH-泛醌氧化还原酶（NDH-1）1（Yagi，1991，1993）。副球菌NDH-1含有非共价结合的FMN和4-5个EPR可见的铁-硫簇，并且似乎由至少14个不同的亚基组成（Yagi，1986; Meinhardt等人，1987; Yagi等人，1992年）。最近，由徐埃塔尔。（1991 a，B，1992 a，B，1993），对编码副球菌NDH-1的基因簇进行了克隆和DNA测序。副球菌NDH-1的基因簇包含14个结构基因和6个URFs，命名为
Revised Manuscript Received November 4, 1993® abstract: The energy-transducing NADH-ubiquinone (Q) oxidoreductase of Paracoccus denitrificans is composed of 14 dissimilar subunitsand contains at least four iron-sulfur clusters [Yagi, T.(1993) Biochim. Biophys. Acta 1141, 1-11], The complete DNA sequence of the gene cluster encoding the energy-transducing NADH-Q oxidoreductase of P. denitrificans has been determined. This paper reports the expression of the 25-kilodalton (kDa)(NQ02) subunit of the P. denitrificans enzyme complex in Escherichia coli and the characterization of the iron-sulfur cluster bound to the expressed subunit. The 25-kDa subunit was expressed in the cytoplasmic phase but not in the membrane fraction of E. coli cells and then purified using an affinity nickel chelation column. The purified subunit contains 1.44 mol of non-heme iron and 1.33 mol of acid-labile sulfide/molof subunit. EPR analysis of the reduced form of this subunit indicates that the expressed subunit contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with g values of gxya=1.913, 1.942, and 1.996, which is very similar to the spectrum of the [2Fe-2S] clusterin the resolved flavoprotein II subfraction (subunit 24+ 9 kDa) of bovine heart complex I [Ragan, C. I., Galante, Y. M., Hatefi, Y., & Ohnishi, T.(1982) Biochemistry 21, 590-594; Ohnishi, T., Ragan, C. I., & Hatefi, Y.(1985) J. Biol. Chem. 260, 2782-2788]. The assignment of the binuclear iron-sulfur cluster of the 25-kDa subunit to an EPR-visible iron-sulfur cluster in the Paracoccus NADH-Q oxidoreductase in situ is discussed.Paracoccus denitrificans is a Gram-negative soil bacterium and has been called “a free-living mitochondrion”(Steinriicke & Ludwig, 1993; Kurowski & Ludwig, 1987; Ludwig, 1987; Stouthamer, 1980). Aerobically grown P. denitrificans expresses a mammalian mitochondrial-type respiratory chain (Stouthamer, 1992) which contains the energy-transducing NADH-ubiquinone oxidoreductase (NDH-1) 1 (Yagi, 1991, 1993). The Paracoccus NDH-1 contains noncovalently bound FMN and 4-5 EPR-visible iron-sulfur clusters and appears to be composed of at least 14 unlike subunits (Yagi, 1986; Meinhardt et al., 1987; Yagi et al., 1992). Recently, by Xu etal.(1991a, b, 1992a, b, 1993), cloning and DNA sequencing of a gene cluster encoding the Paracoccus NDH-1 have been carried out. The gene cluster of the Paracoccus NDH-1 was found to contain 14 structural genes and 6 URFs, designated
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线粒体 NADH 脱氢酶的解析和两种铁硫蛋白的分离。
影响因子：
2.9
作者：
Ragan,CI;Galante,YM;Hatefi,Y;Ohnishi,T
通讯作者：
Ohnishi,T
DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
P. Matsudaira
通讯作者：
P. Matsudaira
脱氮副球菌 bc1 复合体的基因。
影响因子：
4.8
作者：
B. Kurowski;B. Ludwig
通讯作者：
B. Ludwig
DOI：
10.1016/s0021-9258(18)92736-5
发表时间：
1991-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
C. Hekman;J. Tomich;Y. Hatefi
通讯作者：
C. Hekman;J. Tomich;Y. Hatefi
脱氮副球菌的能量转导 NADH-醌氧化还原酶 (NDH-1)。
DOI：
--
发表时间：
1992
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Yagi,T;Xu,X;Matsuno-Yagi,A
通讯作者：
Matsuno-Yagi,A