Infrared multiple photon dissociation spectroscopy of protonated histidine and 4-phenyl imidazole

Abstract

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. To identify the structures present in the experimental studies, the measured IRMPD spectra are compared to spectra calculated at a B3LYP/6–311+G(d,p) level of theory. Relative energies of various conformers are provided by single point energy calculations carried out at the B3LYP, B3P86, and MP2(full) levels using the 6–311+G(2d,2p) basis set. On the basis of these experiments and calculations, the IRMPD action spectrum for H⁺(His) is characterized by a mixture of [N_π,N_α] and [N_π,CO] conformers, with the former dominating. These conformers have the protonated nitrogen atom of imidazole adjacent to the side-chain (N_π) hydrogen bonding to the backbone amino nitrogen (N_α) and to the backbone carbonyl oxygen, respectively. Comparison of the present results to recent IRMPD studies of protonated histamine, the radical His⁺ cation, H⁺(HisArg), H₂²⁺(HisArg), and M⁺(His), where M⁺ = Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺, allows evaluation of the vibrational motions associated with the observed bands.

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 (pK_a = 1.9), the imidazole nitrogen (pK_a = 6.1), and the amino nitrogen (pK_a = 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 pK_a 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⁺.