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Resonance Raman Characterization of a Stable Tryptophan Radical in an Azurin Mutant

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journal contribution
posted on 08.01.2009 by Hannah S. Shafaat, Brian S. Leigh, Michael J. Tauber, Judy E. Kim
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 C2−C3 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 pKa 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.