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 th e 25 kDa (NQO2) subunit of the proton-translocating NADH-quinone oxido reductase of Paracoccus denitrificans, we mutated individually all sev en 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 ef fect of mutations on the iron-sulfur cluster were characterized by che mical analyses and W-visible and EPR spectroscopy. All mutated subunit s, especially mutants of conserved cysteines, contained lower amounts of non-heme iron than wild-type. The subunits of three non-conserved c ysteine residues (C61, C104, and C113) mutated to Ser and a histidine residue (H92) mutated to Ala exhibited essentially the same spectrosco pic properties as those of the wild-type subunit. Tn contrast, mutatio n 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. denitrifi cans 25 kDa subunit.