Structure of a transient neutral histidine radical in solution: EPR continuous-flow studies in a Ti3+/EDTA-Fenton system and density functional calculations

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 s pectrum from a new transient histidine radical with a well-resolved hyperfi ne structure (hfs) has been observed at g(iso) = 2.0026. The hfs changed si gnificantly in the cases of methylene-deuterated histidine in H2O and of hi stidine in D2O. EPR spectral simulations resulted in an assignment of two s lightly 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-splitti ng indicates an addition of an OH* radical to the neutral imidazole ring. S uch an addition of an OH* radical was already observed for the histidine ca tion radical in a Ti3+/H2O2 Fenton system at pH 2.(1) Density functional th eory (DFT) calculations of hyperfine coupling constants were performed at t he PCM/B3LYP/6-311G(2df,p) level for three isomeric forms of a histidine mo del (4-ethyl imidazole) in which an OH* radical is added to different posit ions (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* radi cal at the C5 position of the imidazole ring. Extensive studies of histidin e oxidation in a Ti3+/EDTA-Fenton system, at acidic pH as well as at neutra l and basic pH values, have advanced our understanding of the Fenton chemis try of histidine and the electronic structure of involved paramagnetic spec ies. The pH profile of the formation of histidine OH-addition radicals show s that the cation radical dominates at pH 2 to 4, and the neutral radical a t pH 5 to 9. At pH greater than or equal to 7, in the presence of histidine , a titanium complex with g(iso) = 1.9632 and a significant hfs from natura lly occurring Ti-47 and Ti-49 nuclei with a(iso)(Ti) = 1.54 mT has been obs erved. 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-li gand Ti3+ complex containing EDTA and histidine. This is the first communic ation on a neutral histidine radical with a complete set of hyperfine coupl ing constants derived by EPR as well as by DFT calculations.