The essential role of the Cu(II) state of Sco in the maturation of the CuA center of cytochrome oxidase: Evidence from H135Met and H135SeM variants of the Bacillus subtilis Sco

Gnana S. Siluvai, Michiko Nakano, Mary Mayfield, Ninian J. Blackburn

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Sco is a red copper protein that plays an essential yet poorly understood role in the metalation of the CuA center of cytochrome oxidase, and is stable in both the Cu(I) and Cu(II) forms. To determine which oxidation state is important for function, we constructed His135 to Met or selenomethionine (SeM) variants that were designed to stabilize the Cu(I) over the Cu(II) state. H135M was unable to complement a scoΔ strain of Bacillus subtilis, indicating that the His to Met substitution abrogated cytochrome oxidase maturation. The Cu(I) binding affinities of H135M and H135SeM were comparable to that of the WT and 100-fold tighter than that of the H135A variant. The coordination chemistry of the H135M and H135SeM variants was studied by UV/vis, EPR, and XAS spectroscopy in both the Cu(I) and the Cu(II) forms. Both oxidation states bound copper via the S atoms of C45, C49 and M135. In particular, EXAFS data collected at both the Cu and the Se edges of the H135SeM derivative provided unambiguous evidence for selenomethionine coordination. Whereas the coordination chemistry and copper binding affinity of the Cu(I) state closely resembled that of the WT protein, the Cu(II) state was unstable, undergoing autoreduction to Cu(I). H135M also reacted faster with H2O 2 than WT Sco. These data, when coupled with the complete elimination of function in the H135M variant, imply that the Cu(I) state cannot be the sole determinant of function; the Cu(II) state must be involved in function at some stage of the reaction cycle.

Original languageEnglish (US)
Pages (from-to)285-297
Number of pages13
JournalJournal of Biological Inorganic Chemistry
Issue number2
StatePublished - Feb 1 2011



  • Copper
  • Cytochrome oxidase
  • Sco
  • Selenomethionine
  • X-ray absorption

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

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