X-ray absorption spectroscopy of aqueous amino acids
The carbon, nitrogen, and oxygen K-edge spectra were measured for aqueous solutions of glycine, proline, and diglycine by total electron yield near-edge X-ray absorption fine structure (TEY NEXAFS) spectroscopy, enabled by using liquid microjets to introduce the samples into a high vacuum apparatus. The bulk solution pH was systematically varied while maintaining a constant amino acid concentration. Spectra were assigned through comparisons with both previous studies and ab initio computed spectra of isolated molecules and hydrated clusters. The carbon and oxygen spectra of glycine show little sensitivity to pH, although at low pH the oxygen 1s → π*C=O transition exhibits a 0.25-eV red shift due to the protonation of the carboxylic acid terminus. The nitrogen K-edge solution spectra of aqueous glycine recorded at low and moderate pH are nearly identical to those of solid glycine, whereas the anionic glycine solution spectrum strongly resembles that of the gas phase. The sharp preedge resonances at 401.3 and 402.6 eV observed in the spectrum of anionic glycine indicate that the nitrogen terminus is in an "acceptor-only" configuration at high pH, wherein neither amine proton is involved in, hydrogen bonding to the solvent. These "acceptor-only" transitions are absent in the NEXAFS spectrum of anionic proline, implying that the acceptor-only conformation observed in anionic glycine arises from steric shielding induced by free rotation of the amine terminus about the glycine CN bond. Anionic diglycine solutions exhibit a broadened 1s → π*CN resonance at 401.2 eV and a broad shoulder resonance at 403 eV, also suggesting the presence of an acceptor-only species. Although this assignment is not as unambiguous as for glycine, it implies that the nitrogen terminus of most proteins is capable of existing in an acceptor-only conformation at high pH. These spectral differences indicate that the variations in electronic structure observed in the NEXAFS spectra are determined by the internal charge state and hydration environment of the molecule in solution.