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.
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.
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
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D000596:Amino Acids / Q000737:chemistry*; D001483:Base Sequence; D001692:Biological Transport; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D019159:Nonheme Iron Proteins; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011522:Protons; D011808:Quinone Reductases / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D000596:Amino Acids; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D019159:Nonheme Iron Proteins; D011522:Protons; D006639:Histidine; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D003545:Cysteine