Identification of amino acid residues associated with the [2Fe‐2S] cluster of the 25 kDa (NQO2) subunit of the proton‐translocating NADH‐quinone oxidoreductase of Paracoccus denitrificans

FEBS Letters

Yano, Takahiro¹  Yagi, Takao¹  Ohnishi, Tomoko²  Sled, Vladimir D.² 

[1] Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10666 North Torrey Pines Rd., La Jolla, CA 92037, USA;Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

关键词: Iron-sulfur cluster;    NADH-quinone oxidoreductase;    Expression;    Site-directed mutation;    Paracoccus denitrificans;    NDH-1;    bacterial proton-translocating NADH-quinone oxidoreductase;    [2Fe-2S];    binuclear iron-sulfur cluster;    Complex I;    mitochondrial proton-translocating NADH-quinone oxidoreductase;    EPR;    electron paramagnetic resonance;    MCD;    magnetic circular dichroism;   

DOI  :  10.1016/0014-5793(94)01107-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown