Anatomy of a red copper center

Spectroscopic identification and reactivity of the copper centers of bacillus subtilis Sco and its Cys-to-ala variants

Gnana S. Siluvai, Mary Mayfield, Mark J. Nilges, Serena Debeer George, Ninian Blackburn

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Sco is a mononuclear red copper protein involved in the assembly of cytochrome c oxidase. It is spectroscopically similar to red copper nitrosocyanin, but unlike the latter, which has one copper cysteine thiolate, the former has two. In addition to the two cysteine ligands (C45 and C49), the wild-type (WT) protein from Bacillus subtilis (hereafter named BSco) has a histidine (H135) and an unknown endogenous protein oxygen ligand in a distorted tetragonal array. We have compared the properties of the WT protein to variants in which each of the two coordinating Cys residues has been individually mutated to Ala, using UV/visible, Cu and S K-edge X-ray absorption, electron paramagnetic resonance, and resonance Raman spectroscopies. Unlike the Cu(II) form of native Sco, the Cu(II) complexes of the Cys variants are unstable. The copper center of C49A undergoes autoreduction to the Cu(I) form, which is shown by extended X-ray absorption fine structure to be composed of a novel two-coordinate center with one Cys and one His ligand. C45A rearranges to a new stable Cu(II) species coordinated by C49, H135 and a second His ligand recruited from a previously uncoordinated protein side chain. The different chemistry exhibited by the Cys variants can be rationalized by whether a stable Cu(I) species can be formed by autoredox chemistry. For C49A, the remaining Cys and His residues are trans, which facilitates the formation of the highly stable two-coordinate Cu(I) species, while for C45A such a configuration cannot be attained. Resonance Raman spectroscopy of the WT protein indicates a net weak Cu-S bond strength at ∼2.24 Å corresponding to the two thiolate-copper bonds, whereas the single variant C45A shows a moderately strong Cu-S bond at ∼2.16 Å. S K-edge data give a total covalency of 28% for both Cu-S bonds in the WT protein. These data suggest an average covalency per Cu-S bond lower than that observed for nitrosocyanin and close to that expected for type-2 Cu(II)-thiolate systems. The data are discussed relative to the unique Cu-S characteristics of cupredoxins, from which it is concluded that Sco does not contain highly covalent Cu-S bonds of the type expected for long-range electron-transfer reactivity.

Original languageEnglish (US)
Pages (from-to)5215-5226
Number of pages12
JournalJournal of the American Chemical Society
Volume132
Issue number14
DOIs
StatePublished - Apr 14 2010

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Bacilli
Bacillus subtilis
Copper
Anatomy
Proteins
Ligands
Raman Spectrum Analysis
X ray absorption
Raman spectroscopy
X-Rays
Electron Spin Resonance Spectroscopy
Electron Transport Complex IV
Histidine
Cysteine
Paramagnetic resonance
Electrons
Oxygen

ASJC Scopus subject areas

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

Cite this

Anatomy of a red copper center : Spectroscopic identification and reactivity of the copper centers of bacillus subtilis Sco and its Cys-to-ala variants. / Siluvai, Gnana S.; Mayfield, Mary; Nilges, Mark J.; Debeer George, Serena; Blackburn, Ninian.

In: Journal of the American Chemical Society, Vol. 132, No. 14, 14.04.2010, p. 5215-5226.

Research output: Contribution to journalArticle

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abstract = "Sco is a mononuclear red copper protein involved in the assembly of cytochrome c oxidase. It is spectroscopically similar to red copper nitrosocyanin, but unlike the latter, which has one copper cysteine thiolate, the former has two. In addition to the two cysteine ligands (C45 and C49), the wild-type (WT) protein from Bacillus subtilis (hereafter named BSco) has a histidine (H135) and an unknown endogenous protein oxygen ligand in a distorted tetragonal array. We have compared the properties of the WT protein to variants in which each of the two coordinating Cys residues has been individually mutated to Ala, using UV/visible, Cu and S K-edge X-ray absorption, electron paramagnetic resonance, and resonance Raman spectroscopies. Unlike the Cu(II) form of native Sco, the Cu(II) complexes of the Cys variants are unstable. The copper center of C49A undergoes autoreduction to the Cu(I) form, which is shown by extended X-ray absorption fine structure to be composed of a novel two-coordinate center with one Cys and one His ligand. C45A rearranges to a new stable Cu(II) species coordinated by C49, H135 and a second His ligand recruited from a previously uncoordinated protein side chain. The different chemistry exhibited by the Cys variants can be rationalized by whether a stable Cu(I) species can be formed by autoredox chemistry. For C49A, the remaining Cys and His residues are trans, which facilitates the formation of the highly stable two-coordinate Cu(I) species, while for C45A such a configuration cannot be attained. Resonance Raman spectroscopy of the WT protein indicates a net weak Cu-S bond strength at ∼2.24 {\AA} corresponding to the two thiolate-copper bonds, whereas the single variant C45A shows a moderately strong Cu-S bond at ∼2.16 {\AA}. S K-edge data give a total covalency of 28{\%} for both Cu-S bonds in the WT protein. These data suggest an average covalency per Cu-S bond lower than that observed for nitrosocyanin and close to that expected for type-2 Cu(II)-thiolate systems. The data are discussed relative to the unique Cu-S characteristics of cupredoxins, from which it is concluded that Sco does not contain highly covalent Cu-S bonds of the type expected for long-range electron-transfer reactivity.",
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T2 - Spectroscopic identification and reactivity of the copper centers of bacillus subtilis Sco and its Cys-to-ala variants

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