Resonance Raman characterization of a stable tryptophan radical in an azurin mutant.
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.
Shafaat
Hannah S
HS
Department of Chemistry & Biochemistry, University of California at San Diego, La Jolla, California 92093, USA.
Leigh
Brian S
BS
Tauber
Michael J
MJ
Kim
Judy E
JE
D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D017666:Deuterium Oxide / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D050505:Mutant Proteins / Q000737:chemistry; D013059:Spectrum Analysis, Raman; D014364:Tryptophan / Q000737:chemistry*; D014732:Vibration
D005609:Free Radicals; D050505:Mutant Proteins; D001400:Azurin; D014364:Tryptophan; D017666:Deuterium Oxide