Vibrational Spectra and Ab Initio DFT Calculations of 4-Methylimidazole and Its Different Protonation Forms:  Infrared and Raman Markers of the Protonation State of a Histidine Side Chain

Abstract

The imidazole group of a histidine side chain has four different protonation forms, i.e., the two neutral tautomers (N1-and N3-protonated forms), imidazolium cation, and imidazolate anion. Owing to the presence of these convertible protonation forms, histidine plays important roles in proton-transfer reactions in various enzymes. Vibrational spectroscopy is one of the most powerful methods to study the protonation state of histidine in proteins. For systematic investigation of IR and Raman markers of the protonation state of histidine, we have performed ab initio normal-mode calculations using the density function theory (DFT) method for all of the four protonation forms of 4-methylimidazole (a simple model compound of a histidine side chain) and their N-deuterated analogues. FTIR and Raman spectra of all of these compounds were measured, and the observed bands were assigned according to the calculated frequencies and intensities. Differences in the optimized geometries and changes in the vibrational couplings explained the differences in band frequencies and N-deuteration shifts among the protonation forms. These analyses provided theoretical bases for the IR and Raman markers of the protonation state, including known markers, such as the C4C5 stretching and the C5N1 stretching bands, as well as some new potential markers.

Infrared and Raman spectroscopic study of pH-induced structural changes of l-histidine in aqueous environment

“…Furthermore, it can be concluded that the sensitivity of the C=C vibrational frequency for (de)protonation of N p or N t points to either an inductive effect of N-protonation or to coupling of the C=C vibration with =C-N, =C-C or =C-N-H modes. According to DFT calculations on 4-methylimidazole and 4-ethyl-imidazole [15,16,18] coupling of dN-H and nC=C indeed occurs, but only in the N t -protonated tautomer. This explains the difference in band position compared to the N p -protonated form.…”

“…The band has been reported as an N-H in-plane bending [21] and as a ring stretching combined with =C-H bending [23,29] for histidine, and to a coupled ring dN-H/nC=N mode for 4-methyl-imidazole and 4-ethyl-imidazole [15,16,18]. Coupling of vibrations undoubtedly plays a role, and in agreement with [15,16,18,21] we believe the band to be predominantly an in-plane N-H bending/C=N stretching vibration for two reasons. Firstly, the band disappears upon deuteration, as shown by Toyoma et al [16], which implies that the ring nitrogens are largely involved.…”

“…The fact that the Raman intensity drops at pH >6 upon partial deprotonation of the ring and completely disappears at pH 14, where both N p and N t are deprotonated, is in line with this assignment. The same holds for the weak IR activity as a band at the same position is only observed at pH 8-12. Assignment of the Raman band at 1414 cm À1 to a coupled ring N-H bending/C-N stretching mode as indicated by the results of DFT calculations on 4-methylimidazole and 4-ethyl-imidazole [15,18], is rejected as the band is still present when the ring is completely deprotonated, i.e. at pH 14.…”

“…Literature assignments of the very weak IR bands that we observe in the same region are absent, probably due to the lack of intensity, but as the IR bands around 1284 and 1265 cm À1 largely overlap with the positions found in Raman it seems obvious to follow the same assignment. Indeed, it probably concerns coupled =C-N stretching modes [15][16][17][18], and/or =C-H deformation modes [6,12,14], but in view of the weak intensities, the usefulness as tautomer markers is poor.…”

“…For that reason, only results of theoretical calculations on the model compounds imidazole [12,13], 4-methyl-imidazole [12,[14][15][16][17] and 4-ethyl-imidazole [18] have been used to support the conclusions from empirical IR and Raman studies of histidine [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] and closely related molecules [37][38][39][40][41]. The majority of this research has been focussed on IR or Raman bands that can be used as a marker to determine the state of protonation of the imidazole nitrogen atoms [15,16,[20][21][22][23][24][25][26][27] or the type of metal ion binding [24,29,32,34,37,39,…”