Structure of a Transient Neutral Histidine Radical in Solution: EPR Continuous-Flow Studies in a Ti3+/EDTA−Fenton System and Density Functional Calculations

Abstract

Oxidation of histidine by OH* radicals has been studied at room temperature over a large range of pH values in a Ti3+ /EDTA/H2O2−Fenton system using a special EPR continuous-flow setup. At pH 7 to 8, during fast flow, an EPR spectrum from a new transient histidine radical with a well-resolved hyperfine structure (hfs) has been observed at giso = 2.0026. The hfs changed significantly in the cases of methylene-deuterated histidine in H2O and of histidine in D2O. EPR spectral simulations resulted in an assignment of two slightly different splittings of the two methylene protons (1.27 and 1.59 mT); one large splitting from an out-of-plane proton of the imidazole ring (2.47 mT); and splittings of a ring proton (0.99 mT), a NH (or OH) proton (0.14 mT), and two ring nitrogens (0.27 and 0.11 mT). The large proton-splitting indicates an addition of an OH* radical to the neutral imidazole ring. Such an addition of an OH* radical was already observed for the histidine cation radical in a Ti3+/H2O2 Fenton system at pH 2.1 Density functional theory (DFT) calculations of hyperfine coupling constants were performed at the PCM/B3LYP/6-311G(2df,p) level for three isomeric forms of a histidine model (4-ethyl imidazole) in which an OH* radical is added to different positions (C2, C4, and C5) of the neutral imidazole ring. Theoretical hyperfine data are in excellent agreement with the experiment and clearly support an assignment to a neutral histidine radical formed by addition of an OH* radical at the C5 position of the imidazole ring. Extensive studies of histidine oxidation in a Ti3+/EDTA−Fenton system, at acidic pH as well as at neutral and basic pH values, have advanced our understanding of the Fenton chemistry of histidine and the electronic structure of involved paramagnetic species. The pH profile of the formation of histidine OH-addition radicals shows that the cation radical dominates at pH 2 to 4, and the neutral radical at pH 5 to 9. At pH ≥7, in the presence of histidine, a titanium complex with giso = 1.9632 and a significant hfs from naturally occurring 47Ti and 49Ti nuclei with aiso(Ti) = 1.54 mT has been observed. This complex, which dominates at pH 9 to 10, is also visible in the absence of H2O2 and without flow, and is thus assigned to a stable mixed-ligand Ti3+ complex containing EDTA and histidine. This is the first communication on a neutral histidine radical with a complete set of hyperfine coupling constants derived by EPR as well as by DFT calculations.