Copper–sulfenate complex from oxidation of a cavity mutant of Pseudomonas aeruginosa azurin

Nathan A. Sieracki; Shiliang Tian; Ryan G. Hadt; Jun-Long Zhang; Julia S. Woertink; Mark J. Nilges; Furong Sun; Edward I. Solomon; Yi Lu

doi:10.1073/pnas.1316483111

New Research In

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved November 27, 2013 (received for review September 3, 2013)

Significance

Posttranslational modification of cysteinyl thiolate to sulfenate has been found to play important roles in biology, such as redox signaling, and enzyme and gene regulation. Nitrile hydratase and thiocyanate hydrolase with cobalt and iron cofactors are the few known metalloenzymes requiring sulfenate coordination for reactivity. No other metal ions have been found to stably bind sulfenate in a biological context. Here we report a copper–sulfenate complex characterized in a protein environment, formed at the active site of a cavity mutant of Pseudomonas aeruginosa azurin. Computational studies strongly suggest that noncovalent interactions in the secondary coordination sphere are critical in stabilizing this species.

Abstract

Metal–sulfenate centers are known to play important roles in biology and yet only limited examples are known due to their instability and high reactivity. Herein we report a copper–sulfenate complex characterized in a protein environment, formed at the active site of a cavity mutant of an electron transfer protein, type 1 blue copper azurin. Reaction of hydrogen peroxide with Cu(I)–M121G azurin resulted in a species with strong visible absorptions at 350 and 452 nm and a relatively low electron paramagnetic resonance g_z value of 2.169 in comparison with other normal type 2 copper centers. The presence of a side-on copper–sulfenate species is supported by resonance Raman spectroscopy, electrospray mass spectrometry using isotopically enriched hydrogen peroxide, and density functional theory calculations correlated to the experimental data. In contrast, the reaction with Cu(II)–M121G or Zn(II)–M121G azurin under the same conditions did not result in Cys oxidation or copper–sulfenate formation. Structural and computational studies strongly suggest that the secondary coordination sphere noncovalent interactions are critical in stabilizing this highly reactive species, which can further react with oxygen to form a sulfinate and then a sulfonate species, as demonstrated by mass spectrometry. Engineering the electron transfer protein azurin into an active copper enzyme that forms a copper–sulfenate center and demonstrating the importance of noncovalent secondary sphere interactions in stabilizing it constitute important contributions toward the understanding of metal–sulfenate species in biological systems.

Footnotes

↵¹N.A.S., S.T., and R.G.H. contributed equally to this work.
↵²To whom correspondence may be addressed. E-mail: edward.solomon{at}stanford.edu or yi-lu{at}illinois.edu.

Author contributions: N.A.S., S.T., J.-L.Z., and Y.L. designed research; N.A.S., S.T., R.G.H., J.-L.Z., and J.S.W. performed research; N.A.S., S.T., R.G.H., J.-L.Z., M.J.N., F.S., E.I.S., and Y.L. analyzed data; and N.A.S., S.T., R.G.H., E.I.S., and Y.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structures factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4MFH and 4AZU).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1316483111/-/DCSupplemental.

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