Electronic structure of a transient histidine radical in liquid aqueous solution: EPR continuous-flow studies and density functional calculations

Transient histidine radicals formed in aqueous solutions by oxidation of hi stidine with a Ti3+/H2O2 Fenton system at pH 2.0 have been studied by EPR u sing a fast continuous-flow setup and a dielectric ring resonator equipped with a mixing chamber. A histidine peroxy radical with a single EPR line at g = 2.0151 and a histidine cation radical with a complex hyperfine structu re and g = 2.0023 have been detected. The hyperfine structure of the latter radical was analyzed by investigating two selectively deuterated histidine s and using an EPR simulation and fit program for analysis of the spectra. Isotropic hyperfine coupling constants of two beta-protons, three ring prot ons, and two nitrogen nuclei have been determined in this way and assigned to a histidine-OH adduct cation radical. Density functional theory (DFT) ca lculations at the B3LYP and PWP86 levels have been performed on protonated cation radicals of 4-ethylimidazole (as histidine models), yielding isotrop ic hyperfine coupling constants for three different positions of OH additio n. The C5 position for OH addition (a 5-oxohistidine cation radical) is cle arly supported by the calculated hyperfine coupling constants. The agreemen t between DFT and EPR is further improved when hydrogen-bonding interaction s to the N1 and C2 protons are introduced in the calculations.