Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin

Abstract

The positions of protons are not available in most high-resolution structural data of biomolecules, thus the identity of proton storage sites in biomolecules that transport proton is generally difficult to determine unambiguously. Using combined quantum mechanical/ molecular mechanical computations, we demonstrate that a pair of conserved glutamate residues (Glu 194/204) bonded by a delocalized proton is the proton release group that has been long sought in the proton pump, bacteriorhodopsin. This model is consistent with all available experimental structural and infrared data for both the wild-type bacteriorhodopsin and several mutants. In particular, the continuum infrared band in the 1,800-to 2,000-cm ؊1 region is shown to arise due to the partially delocalized nature of the proton between the glutamates in the wild-type bacteriorhodopsin; alternations in the flexibility of the glutamates and electrostatic nature of nearby residues in various mutants modulate the degree of proton delocalization and therefore intensity of the continuum band. The strong hydrogen bond between Glu 194/204 also significantly shifts the carboxylate stretches of these residues well <1,700 cm ؊1 , which explains why carboxylate spectral shift was not observed experimentally in the typical >1,700-cm ؊1 region upon proton release. By contrast, simulations with the proton restrained on the nearby water cluster, as proposed by several recent studies [see, for example, Garezarek K, Gerwert K (2006) Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy. Nature 439:109], led to significant structural deviations from available X-ray structures. This study establishes a biological function for strong, low-barrier hydrogen bonds.infrared spectroscopy ͉ proton pumping ͉ water cluster ͉ quantum mechanical/molecular mechanical simulations

Mechanism of a proton pump analyzed with computer simulations

“…1). In test QM/MM computations with E204 protonated, the relative orientation and the distance between E194 and E204 was in good agreement with the crystal structure [41,86]. In contrast, when the proton release group was modeled as a Zundel ion, the distance between E194 and E204 sampled values of 3-6.5 Å , significantly higher than the hydrogen-bonding distance in the crystal structure [41].…”

“…The MP2/ 6-311?G** relative deprotonation energy was best reproduced with B3LYP/6-31G** (3.3 kcal/mol error relative to MP2/6-311?G**), and with Self-Consistent Charge Density Functional Tight Binding SCC-DFTB [51] with a specific parametrization of the Schiff base nitrogen atom (3.6 kcal/mol error) [22]. That SCC-DFTB is reliable for describing the structure and energetics of hydrogen-bonded systems, the ground-state properties of the retinal molecule, and the energetics of proton transfer, has been documented based on a large set of benchmark computations (see, e.g., [22,30,41,50,52,53].…”

“…For computations of the first protontransfer step we included in the QM region the retinal, the side-chains of K216, D85, D212, and T89, and water molecule w402 [22]. To investigate the identity of the proton release group (proton transfer step two) we treated with QM the side-chains of E194 and E204, and three water molecules close to E194/E204 [41]. The empty valence of the QM host atoms was satisfied with hydrogen link atoms [28] placed on the C b atom in the case of the lysine and glutamate side-chains, and on the C a atom in the case of the aspartate and threonine residues.…”

“…[52]. B3LYP/MM computations [22,30,41] were performed using TURBOMOLE [55] or Q-Chem [56] with CHARMM. Vibrational spectra were computed from the Fourier Transform of the auto-correlation function of the dipole moment extracted from SCC-DFTB/MM MD trajectories [41].…”

“…B3LYP/MM computations [22,30,41] were performed using TURBOMOLE [55] or Q-Chem [56] with CHARMM. Vibrational spectra were computed from the Fourier Transform of the auto-correlation function of the dipole moment extracted from SCC-DFTB/MM MD trajectories [41]. The QM computations on gas phase models of the retinal [22], and of the retinal binding pocket [32] were performed using GAUSSIAN [57] or SCC-DFTB [51].…”