Methionine Radical Cation:  Structural Studies as a Function of pH Using X- and Q-Band Time-Resolved Electron Paramagnetic Resonance Spectroscopy

A comprehensive high resolution electron paramagnetic resonance (EPR) characterization of the l-methionine radical cation and its N-acetyl derivative in liquid solution at room temperature is presented. The cations were generated photochemically in high yield by excimer laser excitation of a water soluble dye, anthraquinone sulfonate sodium salt, the excited triplet state of which is quenched by electron transfer from the side chain sulfur atom of methionine or N-acetylmethionine. The radicals were detected by continuous wave (CW) time-resolved electron paramagnetic resonance (TREPR) spectroscopy at X-band (9.5 GHz) and Q-band (35 GHz) microwave frequencies. At pH values well below the pK a of the protonated amine nitrogen, the cation forms a dimer with another ground-state methionine molecule through a S−S three-electron bond. In basic solution, the lone pair on the nitrogen of the amino acid is available to make an intramolecular S−N three-electron bond with the side chain sulfur atom, leading to a five-membered ring structure for the cation. When the amino acid nitrogen is unsubstituted (methionine itself), rapid deprotonation to an aminyl radical takes place at high pH values. If the nitrogen is substituted (N-acetylmethionine), the cyclic structure is observed within its electron spin relaxation time at about 1 μs. Spectral simulation provides chemical shifts (g-factors) and hyperfine coupling constants for all structures, and isotopic labeling experiments strongly support the assignments.