Spectroscopic studies of metal binding and metal selectivity in Bacillus subtilis BSco, a homologue of the yeast mitochondrial protein Sco1p

Luisa Andruzzi, Michiko Nakano, Mark J. Nilges, Ninian J. Blackburn

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Sco1 is a mitochondrial membrane protein involved in the assembly of the CuA site of cytochrome c oxidase. The Bacillus subtilis genome contains a homologue of yeast Sco1, YpmQ (hereafter termed BSco), deletion of which leads to a phenotype lacking in caa3 (CuA- containing) oxidase activity but expressing normal levels of aa3 (quinol) oxidase activity. Here, we report the characterization of the metal binding site of BSco in its Cu(I)-, Cu(II)-, Zn(II)-, and Ni(II)-bound forms. Apo BSco was found to bind Cu(II), Zn(II), and Ni(II) at a 1:1 protein/metal ratio. The Cu(I) protein could be prepared by either dithionite reduction of the Cu(II) derivative or by reconstitution of the apo protein with Cu(I). X-ray absorption (XAS) spectroscopy showed that Cu(I) was coordinated by two cysteines at 2.22 ± 0.01 Å and by a weakly bound low-Z scatterer at 1.95 ± 0.03 Å. The Cu(II) derivative was reddish-orange and exhibited a strong type-2 thiolate to Cu(II) transition around 350 nm. Multifrequency electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM) studies on the Cu(II) derivative provided evidence of one strongly coupled histidine residue, at least one strongly coupled cysteine, and coupling to an exchangeable proton. XAS spectroscopy indicated two cysteine ligands at 2.21 Å and two O/N donor ligands at 1.95 Å, at least one of which is derived from a coordinated histidine. The Zn(II) and Ni(II) derivatives were 4-coordinate with MS2N(HiS)X coordination. These results provide evidence that a copper chaperone can engage in redox chemistry at the metal center and may suggest interesting redox-based mechanisms for metalation of the mixed-valence Cu A center of cytochrome c oxidase.

Original languageEnglish (US)
Pages (from-to)16548-16558
Number of pages11
JournalJournal of the American Chemical Society
Issue number47
StatePublished - Nov 30 2005

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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