Infrared multiple photon dissociation spectroscopy of protonated histidine and 4-phenyl imidazole
Graphical abstract
Highlights
► Protonated histidine (His) and 4-phenyl imidazole (PhIm) are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy. ► Structures are determined by comparing the spectra with B3LYP/6–311+G(d,p) predictions. ► The dominant structure of H+(His) has the protonated nitrogen atom of imidazole hydrogen bonding to the backbone amino nitrogen. ► Results are compared to IRMPD studies of protonated histamine, radical His+, H+(HisArg), H22+(HisArg), and M+(His), where M+ = alkali cations.
Introduction
Histidine (His) is chemically one of the most flexible protein residues because the imidazole side chain can function as both an acid and base near neutral pH [1]. The histidine molecule presents three potential coordination sites in aqueous solution. The carboxyl group (pKa = 1.9), the imidazole nitrogen (pKa = 6.1), and the amino nitrogen (pKa = 9.1) become available for complexation as pH increases. The acid–base properties of a biomolecule affect physicochemical activities such as solubility, hydrophobicity, and electrostatic interactions that directly impact the biological activity of the molecule in a living system. Because the pKa of the imidazolium form is around 6, histidine can undergo protonation and deprotonation reactions at physiological pH. Therefore, it functions as a competent proton-transfer mediator in various proteins [2], [3], [4], [5]. Histidine also serves as both a hydrogen-bond donor and an acceptor, and this hydrogen-bonding property is of importance in proton-transfer reactions [5] and in organizing the active centers of enzymes. As a consequence, histidine is often found at the catalytic sites of protein enzymes.
In recent years, the structures of many proteins have been studied by X-ray crystallography; however, because of the poor sensitivity of X-ray crystallography in detecting hydrogen atoms, the protonation structures and hydrogen-bonding interactions of amino acid side chains are not well resolved in many cases. To obtain such information, vibrational spectroscopy is a powerful method as it is more sensitive to chemical bonds and molecular interactions. In the case of histidine, its protonation state, metal binding, and hydrogen bonding interactions have been investigated using both Raman [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18] and Fourier transform infrared (FTIR) [19] spectroscopy. Huang et al. [20] investigated the neutral, protonated, and deprotonated histidine conformers in the gas phase using time-dependent density functional theory (TDDFT) to calculate the electronic spectra and charge-transfer processes. Also, Hasegawa et al. [21] studied 4-methylimidazole (a simple model compound of the histidine side chain) and its different protonation forms using FTIR and Raman spectra for systematic investigation of vibrational markers of the protonation state of histidine.
Infrared multiple photon dissociation (IRMPD) spectroscopy has been used to probe the structures of ionized complexes in the gas phase and can be a powerful tool for understanding ion–protein interactions. An important advantage of this technique is the ability to investigate the structures of biomolecules in isolation, where complicating structural effects of solvent and counter-ions are absent. Recently, gas-phase structures of protonated histamine [22], histidine radical cation (His+) [23], H+(HisArg) and H22+(HisArg) [24], and M+(His) [25], where M+ = Li+, Na+, K+, Rb+, and Cs+, have all been investigated by IRMPD spectroscopy utilizing light generated by a free electron laser. In the present study, we measure the IRMPD action spectra for photodissociation of protonated His and 4-phenyl imidazole (PhIm), where the latter provides a model for assessing the vibrations of the imidazole side-chain ring. Conformations of these molecules are identified by comparing the experimental spectra to IR spectra of the low-lying structures of the cationized His and PhIm complexes predicted by quantum chemical calculations at the B3LYP/6–311+G(d,p) level of theory. IRMPD action spectra for H+(His) and H+(PhIm) are also compared to the previous results for H+(histamine), His+, H+(HisArg), H22+(HisArg), and M+(His), where M+ = Li+, Na+, K+, Rb+, and Cs+.
Section snippets
Mass spectrometry and photodissociation
Experiments were performed using the Free Electron Laser for Infrared eXperiments (FELIX) [26] in combination with a home-built Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, which has been described in detail elsewhere [27], [28], [29]. Protonated histidine and protonated phenyl imidazole complexes were generated using a Z-spray (Micromass UK Ltd.) electrospray ionization source. Solutions used were 1.0–3.0 mM His with 1 mM acetic acid in 50% MeOH and 50% H2O for H+(His)
IRMPD action spectroscopy
The photodissociation spectra of protonated (m/z 156) His both as depletion of the parent ion and the yield for loss of H2O + CO (m/z 110) (corrected for laser power and uncorrected) are shown in Fig. S1 of the Supporting Information. Very small amounts (>10 times smaller) of just H2O loss were also detected. These decomposition pathways match the lowest energy products observed in the collision-induced dissociation (CID) spectrum of H+(His), as observed previously [50], [51]. Parent and product
Conclusions
The gas-phase structures of protonated histidine (His) and the side-chain model, protonated 4-phenyl imidazole (PhIm), are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy utilizing light generated by the free electron laser FELIX. Comparison of the measured IRMPD spectra to single photon absorption results calculated at a B3LYP/6–311+G(d,p) level of theory show that H+(His) is characterized by a [Nπ,Nα] conformer along with some [Nπ,CO]. These conformers have the
Acknowledgments
Financial support was provided by the National Science Foundation, Grants PIRE-0730072 and CHE-1049580. This work is also part of the research program of FOM, which is financially supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). The skillful assistance of the FELIX staff, in particular Lex van der Meer, Britta Redlich, Giel Berden, and Josipa Grzetic, is gratefully acknowledged. In addition, we thank the Center for High Performance Computing at the University of
References (54)
- et al.
Proceedings of the National Academy of Sciences of the United States of America
(1998) - et al.
Biochemistry
(1998) - et al.
Journal of the American Chemical Society
(1986) - et al.
Biochemistry
(1989) - et al.
Science
(1994) - et al.
Inorganic Chemistry
(1983) - et al.
Journal of the American Chemical Society
(1986) - et al.
Biochemistry
(1989) - et al.
Journal of the American Chemical Society
(1997) - et al.
Inorganic Chemistry
(1998)