Spectroscopic Comparison of Photogenerated Tryptophan Radicals in Azurin: Effects of Local Environment and Structure

Tryptophan radicals play a significant role in mediating biological electron transfer. We report the photogeneration of a long-lived, neutral tryptophan radical (Az48W center dot) from the native residue tryptophan48 in the hydrophobic core of azurin The optical absorption, electron paramagnetic resonance, and resonance Raman spectra strongly support the formation of a neutral radical, and the data are consistent with direct electron transfer between tryptophan and the copper(II) center Spectra of the long-lived Az48W center dot species are compared to those of a previously studied, solvent-exposed radical at position 108 to identify signatures of tryptophan radicals that are sensitive to the local environment. The absorption maxima of Az48W center dot display an similar to 23 nm hypsochromic shift in the nonpolar environment The majority of the resonance Raman frequencies are downshifted by similar to 7 cm(-1) relative to the solvent-exposed radical, and large changes in intensity are observed for some modes. The resonance Raman excitation profiles for Az48W center dot exhibit distinct maxima within the absorption envelope. Electron paramagnetic resonance spectroscopy yields spectra with partially resolved lines caused by hyperfine couplings, the differences between the coupling constants for the buried and solvent-exposed radical are primarily caused by variations in structure The insights gained by electronic, vibrational, and magnetic resonance spectroscopy enhance our fundamental understanding of the effects of protein environment on radical properties Hypotheses for the proton transfer pathway within azurin and a deprotonation rate of similar to 5 x 10(6) s(-1) are proposed.