Catalysis of Ground State cis[Formula: see text] trans Isomerization of Bacteriorhodopsin's Retinal Chromophore by a Hydrogen-Bond Network.

For the photocycle of the membrane protein bacteriorhodopsin to proceed efficiently, the thermal 13-cis to all-trans back-isomerization of the retinal chromophore must return the protein to its resting state on a time-scale of milliseconds. Here, we report on quantum mechanical/molecular mechanical energy calculations examining the structural and energetic determinants of the retinal cis-trans isomerization in the protein environment. The results suggest that a hydrogen-bonded network consisting of the retinal Schiff base, active site amino acid residues, and water molecules can stabilize the twisted retinal, thus reducing the intrinsic energy cost of the cis-trans thermal isomerization barrier.

Active Site Dynamical Effects in the Hydrogen Transfer Rate-limiting Step in the Catalysis of Linoleic Acid by Soybean Lipoxygenase-1 (SLO-1): Primary and Secondary Isotope Contributions.

Using ab initio molecular dynamics (AIMD) simulations that facilitate the treatment of rare events, we probe the active site participation in the rate-determining hydrogen transfer step in the catalytic oxidation of linoleic acid by soybean lipoxygenase-1 (SLO-1). The role of two different active site components is probed. (a) On the hydrogen atom acceptor side of the active site, the hydrogen bonding propensity between the acceptor side hydroxyl group, which is bound to the iron cofactor, and the backbone carboxyl group of isoleucine (residue number 839) is studied toward its role in promoting the hydrogen transfer event. Primary and secondary (H/D) isotope effects are also probed and a definite correlation with subtle secondary H/D isotope effects is found. With increasing average nuclear kinetic energy, the increase in transfer probability is enhanced due to the presence of the hydrogen bond between the backbone carbonyl of I839 and the acceptor oxygen. Further increase in average nuclear kinetic energy reduces the strength of this secondary hydrogen bond which leads to a deterioration in hydrogen transfer rates and finally embrances an Arrhenius-like behavior. (b) On the hydrogen atom donor side, the coupling between vibrational modes predominantly localized on the donor-side linoleic acid group and the reactive mode is probed. There appears to be a qualitative difference in the coupling between modes that belong to linoleic acid and the hydrogen transfer mode, for hydrogen and deuterium transfer. For example, the donor side secondary hydrogen atom is much more labile (by nearly a factor of 5) during deuterium transfer as compared to the case for hydrogen transfer. This appears to indicate a greater coupling between the modes belonging to the linoleic acid scaffold and the deuterium transfer mode and also provides a new rationalization for the abnormal (nonclassical) secondary isotope effect results obtained by Knapp, Rickert, and Klinman in J. Am. Chem. Soc. , 2002 , 124 , 3865 . To substantiate our findings noted in point a above, we have suggested an I839 → A839 or I839 → V839 mutation. This will modify the bulkiness of hydrogen the bonding residue, allowing greater flexibility in the secondary hydrogen bond formation highlighted above and adversely affecting the reaction rate.

Excitation energies of a water-bridged twisted retinal structure in the bacteriorhodopsin proton pump: a theoretical investigation.

The first proton transfer in the bacteriorhodopsin photocycle takes place during the L → M transition. Structural details of the pre proton transfer L intermediate have been investigated using experiments and computations. Here, we assess L-state structural models by performing hybrid quantum mechanical/molecular mechanical molecular dynamics and excitation energy calculations. The computations suggest that a water-bridged twisted retinal structure gives the closest agreement with the experimental L/bR shift in the excitation energy.

Gauging the flexibility of the active site in soybean lipoxygenase-1 (SLO-1) through an atom-centered density matrix propagation (ADMP) treatment that facilitates the sampling of rare events.

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 toward 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.

Influence of water on anharmonicity, stability, and vibrational energy distribution of hydrogen-bonded adducts in atmospheric reactions: case study of the OH + isoprene reaction intermediate using ab initio molecular dynamics.

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.

Proton storage site in bacteriorhodopsin: new insights from quantum mechanics/molecular mechanics simulations of microscopic pK(a) and infrared spectra.

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 the PRG would require the protein to raise the pK(a) of a hydronium by almost 11 pK(a) units, which is difficult considering known cases of pK(a) shifts in proteins. Our recent quantum mechanics/molecular mechanics (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 pK(a) 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 the PRG in biomolecular proton pumps in general.

Role of Arg82 in the early steps of the bacteriorhodopsin proton-pumping cycle.

Proton-transfer reactions in the bacteriorhodopsin light-driven proton pump are coupled with structural rearrangements of protein amino acids and internal water molecules. It is generally thought that the first proton-transfer step from retinal Schiff base to the nearby Asp85 is coupled with movement of the Arg82 side chain away from Asp85 and toward the extracellular proton release group. This movement of Arg82 likely triggers the release of the proton from the proton release group to the extracellular bulk. The exact timing of the movement of Arg82 and how this movement is coupled with proton transfer are still not understood in molecular detail. Here, we address these questions by computing the free energy for the movement of the Arg82 side chain. The calculations indicate that protonation of Asp85 leads to a fast reorientation of the Arg82 side chain toward the extracellular proton release group.

The protonation state of Glu181 in rhodopsin revisited: interpretation of experimental data on the basis of QM/MM calculations.

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.

Long-distance proton transfer with a break in the bacteriorhodopsin active site.

Bacteriorhodopsin is a proton-pumping membrane protein found in the plasma membrane of the archaeon Halobacterium salinarium. Light-induced isomerization of the retinal chromophore from all-trans to 13-cis leads to a sequence of five conformation-coupled proton transfer steps and the net transport of one proton from the cytoplasmic to the extracellular side of the membrane. The mechanism of the long-distance proton transfer from the primary acceptor Asp85 to the extracellular proton release group during the O --> bR is poorly understood. Experiments suggest that this long-distance transfer could involve a transient state [O] in which the proton resides on the intermediate carrier Asp212. To assess whether the transient protonation of Asp212 participates in the deprotonation of Asp85, we performed hybrid Quantum Mechanics/Molecular Mechanics proton transfer calculations using different protein structures and with different retinal geometries and active site water molecules. The structural models were assessed by computing UV-vis excitation energies and C=O vibrational frequencies. The results indicate that a transient [O] conformer with protonated Asp212 could indeed be sampled during the long-distance proton transfer to the proton release group. Our calculations suggest that, in the starting proton transfer state O, the retinal is strongly twisted and at least three water molecules are present in the active site.

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

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.