Resonance Raman characterization of a stable tryptophan radical in an azurin mutant

J Phys Chem B. 2009 Jan 8;113(1):382-8. doi: 10.1021/jp809329a.


Tryptophan radicals play a significant role in mediating biological electron transfer and catalytic processes. Here, we employ visible and UV resonance Raman, EPR, and absorption spectroscopy along with pH/isotope studies and calculations to probe a neutral closed-shell tryptophan and its oxidized radical counterpart in a modified azurin protein. Comparison of the resonance Raman spectra of the radical and closed-shell species combined with vibrational analysis reveals important structural differences between these two tryptophan species. We experimentally observe a significant reduction in bond order of the pyrrole ring of the radical, as evidenced by a 208 cm(-1) downshift of the W3 mode (predominantly C(2)-C(3) stretch). Analysis of the spectra acquired at acidic pH and in deuterated buffer highlights those vibrational modes of the radical that are sensitive to the hydrogen-bonding environment. The most significant change caused by the deuterated buffer is a 45 cm(-1) downshift of an indole nitrogen displacement mode (W17). Our spectra provide evidence that the radical species is a strong hydrogen bond acceptor, particularly in an acidic environment. Furthermore, the pK(a) for this tryptophan radical must be less than 4.0, which falls below previously reported values for l-tryptophan in aqueous solution. The normal mode assignments of the tryptophan radical help characterize its local environment, conformation, hydrogen bonding, and protonation state within a protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azurin / chemistry*
  • Azurin / genetics
  • Crystallography, X-Ray
  • Deuterium Oxide / chemistry
  • Free Radicals / chemistry
  • Hydrogen Bonding
  • Hydrogen-Ion Concentration
  • Mutant Proteins / chemistry
  • Spectrum Analysis, Raman
  • Tryptophan / chemistry*
  • Vibration


  • Free Radicals
  • Mutant Proteins
  • Azurin
  • Tryptophan
  • Deuterium Oxide