Yano T, Sled V D, Ohnishi T, Yagi T
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
FEBS Lett. 1994 Nov 7;354(2):160-4. doi: 10.1016/0014-5793(94)01107-9.
In order to identify the ligand residues of the [2Fe-2S] cluster of the 25 kDa (NQO2) subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans, we mutated individually all seven cysteine residues (C61, C96, C101, C104, C113, C137, and C141) and one conserved histidine residue (H92) to Ser or Ala and expressed them in E. coli. After purification of the mutated 25 kDa subunits, the effect of mutations on the iron-sulfur cluster were characterized by chemical analyses and UV-visible and EPR spectroscopy. All mutated subunits, especially mutants of conserved cysteines, contained lower amounts of non-heme iron than wild-type. The subunits of three non-conserved cysteine residues (C61, C104, and C113) mutated to Ser and a histidine residue (H92) mutated to Ala exhibited essentially the same spectroscopic properties as those of the wild-type subunit. In contrast, mutation of the four conserved cysteine residues (C96, C101, C137, and C141) to Ser or Ala considerably altered the UV-visible and EPR spectra from the wild-type subunit. These results indicate that the four conserved cysteine residues coordinate the [2Fe-2S] cluster in the P. denitrificans 25 kDa subunit.
为了鉴定反硝化副球菌质子转运型NADH-醌氧化还原酶25 kDa（NQO2）亚基的[2Fe-2S]簇的配体残基，我们将所有七个半胱氨酸残基（C61、C96、C101、C104、C113、C137和C141）以及一个保守的组氨酸残基（H92）分别突变为丝氨酸或丙氨酸，并在大肠杆菌中表达。纯化突变的25 kDa亚基后，通过化学分析以及紫外可见光谱和电子顺磁共振光谱对突变对铁硫簇的影响进行了表征。所有突变的亚基，尤其是保守半胱氨酸的突变体，所含的非血红素铁量均低于野生型。三个非保守半胱氨酸残基（C61、C104和C113）突变为丝氨酸以及一个组氨酸残基（H92）突变为丙氨酸的亚基表现出与野生型亚基基本相同的光谱特性。相反，四个保守半胱氨酸残基（C96、C101、C137和C141）突变为丝氨酸或丙氨酸，使紫外可见光谱和电子顺磁共振光谱与野生型亚基相比发生了很大变化。这些结果表明，四个保守半胱氨酸残基在反硝化副球菌25 kDa亚基中与[2Fe-2S]簇配位。
