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

Identifying the group that acts as the proton storage/loading site is a challenging but important problem for understanding the mechanism of proton pumping in biomolecular proton pumps, such as bacteriorhodopsin (bR) and cytochrome c oxidase. Recent experimental studies of bR propelled the idea that the proton storage/release group (PRG) in bR is not an amino acid but a water cluster embedded in the protein. We argue that this idea is at odds with our knowledge of protein electrostatics, since invoking the water cluster as PRG would require the protein to raise the pKa of a hydronium by almost 11 pKa units, which is difficult considering known cases of pKa shifts in proteins. Our recent QM/MM simulations suggested an alternative “intermolecular proton bond” model in which the stored proton is shared between two conserved Glu residues (194 and 204). Here we show that this model leads to microscopic pKa values consistent with available experimental data and the functional requirement of a PRG. Extensive QM/MM simulations also show that, independent of a number of technical issues, such as the influence of QM region size, starting x-ray structure and nuclear quantum effects, the “intermolecular proton bond” model is qualitatively consistent with available spectroscopic data. Potential of mean force calculations show explicitly that the stored proton strongly prefers the pair of Glu residues over the water cluster. The results and analyses help highlight the importance of considering protein electrostatics and provide arguments for why the “intermolecular proton bond” model is likely applicable to PRG in biomolecular proton pumps in general.

The structure and spectroscopy of rhodopsin have been intensely studied in the past decade both experimentally and theoretically; however, important issues still remain unresolved. Of central interest is the protonation state of Glu181, where controversial and contradictory experimental evidence has appeared. While FTIR measurements indicate this residue to be unprotonated, preresonance Raman and UV-vis spectra have been interpreted in favor of a protonated Glu181. Previous computational approaches were not able to resolve this issue, providing contradicting data as well. Here, we perform hybrid QM/MM calculations using DFT methods for the electronic ground state, MRCI methods for the electronically excited states, and a polarization model for the MM part in order to investigate this issue systematically. We constructed various active-site models for protonated as well as unprotonated Glu181, which were evaluated by computing NMR, IR, Raman, and UV-vis spectroscopic data. The resulting differences in the UV-vis and Raman spectra between protonated and unprotonated models are very subtle, which has two major consequences. First, the common interpretation of prior Raman and UV-vis experiments in favor of a neutral Glu181 appears questionable, as it is based on the assumption that a charge at the Glu181 location would have a sizable impact. Second, also theoretical results should be interpreted with care. Spectroscopic differences between the structural models must be related to modeling uncertainties and intrinsic methodological errors. Despite a detailed comparison of various rhodopsins and mutants and consistently favorite results with charged Glu181 models, we find merely weak evidence from UV-vis and Raman calculations. On the contrary, difference FTIR and NMR chemical shift measurements on Rh mutants are indicative of the protonation state of Glu181. Supported by our results, they provide strong and independent evidence for a charged Glu181.

The effect of water on the stability and vibrational states of a hydroxy-isoprene adduct is probed through the introduction of 1-15 water molecules. It is found that when a static nuclear harmonic approximation is invoked there is a substantial red-shift of the alcohol O-H stretch (of the order of 800 cm(-1)) as a result of introduction of water. When potential energy surface sampling and associated anharmonicities are introduced through finite temperature ab initio dynamics, this hydroxy-isoprene OH stretch strongly couples with all the water vibrational modes as well as the hydroxy-isoprene OH bend modes. A new computational technique is introduced to probe the coupling between these modes. The method involves a two-dimensional, time-frequency analysis of the finite temperature vibrational properties. Such an analysis not only provides information about the modes that are coupled as a result of finite-temperature analysis, but also the temporal evolution of such coupling.

We present a computational methodology to sample rare events in large biological enzymes that may involve electronically polarizing, reactive processes. The approach includes simultaneous dynamical treatment of electronic and nuclear degrees of freedom, where contributions from the electronic portion are computed using hybrid density functional theory and the computational costs are reduced through a hybrid quantum mechanics/molecular mechanics (QM/MM) treatment. Thus, the paper involves a QM/MM dynamical treatment of rare events. The method is applied to probe the effect of the active site elements on the critical hydrogen transfer step in the soybean lipoxygenase-1 (SLO-1) catalyzed oxidation of linoleic acid. It is found that the dynamical fluctuations and associated flexibility of the active site are critical towards maintaining the electrostatics in the regime where the reactive process can occur smoothly. Physical constraints enforced to limit the active site flexibility are akin to mutations and, in the cases studied, have a detrimental effect on the electrostatic fluctuations, thus adversely affecting the hydrogen transfer process.