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

Abstract

Tryptophan radicals play a significant role in mediating biological electron transfer. We report the photogeneration of a long-lived, neutral tryptophan radical (Az48W*) from the native residue tryptophan-48 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* 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* display an approximately 23 nm hypsochromic shift in the nonpolar environment. The majority of the resonance Raman frequencies are downshifted by approximately 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* 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 approximately 5 x 10(6) s(-1) are proposed.

Effects of the Protein Environment on the Spectral Properties of Tryptophan Radicals in Pseudomonas aeruginosa Azurin

“…Both dihedral angles are in accord with those experimentally derived for ReAz108W • (−76°) and Az48W • (88°) 21 computed ϕ values of the optimized Trp • are different from those derived from the X-ray structures (−81.87°and 89.83°f or ReAz108W and Az48W, respectively). Thus, as reported also in a previous work, 28 the orientation of the oxidized residue diverges from the orientation in its reduced state.…”

“…Despite this, optical and vibrational spectroscopies, especially resonance Raman (RR) spectroscopies, have been rarely employed to study these species. 30,31 The recent photogeneration of a long-lived Trp • in two Pseudomonas aeruginosa azurin mutants, [Y72F/Y108F]AzCu-(II) (Az48W) and the rhenium-labeled Az108W mutant [Re(I)(CO) 3 (4,7-dimethyl-1,10-phenanthroline)(Q107H)]-(W48F/Y72F/H83Q/Y108W)AzM(II) [M = Cu, Zn] (ReAz108W), provided the unique opportunity to obtain the electronic, magnetic, and vibrational spectra of these radical species 21,22,32 and identify the spectral signatures of Trp radicals that are sensitive to the local environment. Indeed, in Az48W the radical (W48 • ) is buried in the hydrophobic core (see Figure 1, left), while it is solvent-exposed (W108 • ) in ReAz108W (see Figure 1, right).…”

Role of zero-point vibrational corrections to carbon hyperfine coupling constants in organic π radicals

“…Despite the fact that electron paramagnetic resonance (EPR) spectroscopy for a long time has been instrumental in analyzing spin density distributions in organic π radicals, [1][2][3][4][5][6] it is not until quite recently that first principles theories have been developed, having an accuracy that allow them to be useful for interpreting experimental spectra of such radicals. [7][8][9][10][11][12][13][14][15][16][17][18] The lack of such analysis tools forced early investigations of EPR data to rely on rules-of-thumb or simple principles that relate the spin density to the measured hyperfine coupling constants (HFCCs).…”

“…One such relation is the McConnell relation that states that the spin density ρ C on the carbon of a C-H fragment in an organic π radical is linearly dependent on the isotropic hyperfine constant of hydrogen. [1][2][3] Although such relations or structure-property tools have frequently been used in the field of EPR, [4][5][6] recent advances in the quantum chemical modeling of HFCCs in organic radicals [7][8][9][10][11][12][13][14][15][16][17][18] offer an alternative way for exploring the physical origin of these constants.…”

The Effects of Biological Environments on the Electron‐Relay Functionality of Tryptophan Residues in Proteins

“…[1][2][3][4][5][6][7][8][9][10][11][12][13] In most cases, Trp residues act as middle relay stations to facilitate ET by frequent interconversion between their reduced and oxidized states. The relay function of Trp residue comes from Trp having the lowest ionization potential among the 20 natural amino acids, and ET through the indole side chain is a rapid reversible process that supports the hopping mechanism of long-range ET in proteins.…”