Reversible proton transfer dynamics in bacteriorhodopsin

In a previous study of ab initio dynamics, the proton transfer in bacteriorhodopsin from protonated asp96 in the cytoplasmic region toward the deprotonated Schiff base was investigated. A quantum mechanics/molecular mechanics model was constructed from the X-ray structure of bacteriorhodopsin E204Q mutant. In this model, asp96, asp85, and thr89 as well as most of the retinal chromophore and the Schiff base link of lys216 were treated quantum mechanically while the rest of the atoms were treated molecular mechanically. A channel was found in the X-ray structure allowing a water chain to form between the asp96 and Schiff base. In the present study, a chain of four waters from asp96 to the Schiff base N coupled with one branching water supports proton transfer as a concerted event in about 3.5 ps. With both a neutral asp85 and a branched water, the dynamics is now found to be more complicated than observed in the initial study for the transition from the photocycle late M state to the N state. Proton transfer is also observed from the Schiff base back to asp96 demonstrating that there is no effective barrier to proton transfer larger than kT in a strong H-bonded network. The binding of the branched water to the four water chains can dynamically hinder the proton transfer.     

Key role of electrostatic interactions in bacteriorhodopsin proton transfer

The first proton transport step following photon absorption in bacteriorhodopsin is from the 13-cis retinal Schiff base to Asp85. Configurational and energetic determinants of this step are investigated here by performing quantum mechanical/molecular mechanical minimum-energy reaction-path calculations. The results suggest that retinal can pump protons when in the 13-cis, 15-anti conformation but not when 13-cis, 15-syn. Decomposition of the proton transfer energy profiles for various possible pathways reveals a conflict between the effect of the intrinsic proton affinities of the Schiff base and Asp85, which favors the neutral, product state (i.e., with Asp85 protonated), with the mainly electrostatic interaction between the protein environment with the reacting partners, which favors the ion pair reactant state (i.e., with retinal protonated). The rate-limiting proton-transfer barrier depends both on the relative orientations of the proton donor and acceptor groups and on the pathway followed by the proton; depending on these factors, the barrier may arise from breaking and forming of hydrogen bonds involving the Schiff base, Asp85, Asp212, and water w402, and from nonbonded interactions involving protein groups that respond to the charge rearrangements in the Schiff base region.     

细菌紫红质中氢键相互作用和质子转移机理的分子模拟

用CHARMM程序以细菌紫红质1R84晶体为模型, 模拟了在等温定容条件下细菌紫红质在1 ps过程中的变化, 分析了与质子传递相关的ASP85, ASP212和水分子与视黄醛间氢键的结构变化情况. 考虑到氨基酸残基和席夫碱质子的不同距离, 考察了EC和PC两种结构的变化情况, 探讨了紫红质中质子传递的可能途径. 模拟结果表明1R84中可能的质子连续传递的机理是质子由席夫碱向水传递, 再由水向ASP85传递. 发现Asp212在模拟过程中保持EC结构, 这样可能更有利于顺序质子传递.     

Water as a cofactor in the unidirectional light-driven proton transfer steps in bacteriorhodopsin

Recent evidence for involvement of internal water molecules in the mechanism of bacteriorhodopsin is reviewed. Water O-H stretching vibration bands in the Fourier transform IR difference spectra of the L, M and N intermediates of bacteriorhodopsin were analyzed by photoreactions at cryogenic temperatures. A broad vibrational band in L was shown to be due to formation of a structure of water molecules connecting the Schiff base to the Thr46-Asp96 region. This structure disappears in the M intermediate, suggesting that it is involved in transient stabilization of the L intermediate prior to proton transfer from the Schiff base to Asp85. The interaction of the Schiff base with a water molecule is restored in the N intermediate. We propose that water is a critical mobile component of bacteriorhodopsin, forming organized structures in the transient intermediates during the photocycle and, to a large extent, determining the chemical behavior of these transient states.     

Dynamics of proton transfer and vibrational relaxation in dilute hydrofluoric acid

The molecular mechanisms in both vibrational relaxation and proton transfer (PT) associated with infrared (IR)-induced PT in a dilute hydrofluoric acid solution at ambient temperature are studied by molecular dynamics (MD) simulations with the multistate empirical valence bond model. To investigate the solvation dynamics, a collective solvent coordinate and its perpendicular bath modes are defined from the diabatic energy gap and their motions are examined by the generalized Langevin equation (GLE) formalism. The GLE analysis using the equilibrium MD simulation shows that the major solvent reorganizations in the PT are represented by the libration and hindered translation. In particular, the libration gives the stronger coupling to the solvent reorganization and the faster relaxation. The nonequilibrium MD simulation demonstrated that both the HF stretching vibration and the solvent reorganization relax on a similar time scale and thus compete in the PT. It also supported the "presolvation mechanism" for the PT in this system.     

A role for internal water molecules in proton affinity changes in the Schiff base and Asp85 for one-way proton transfer in bacteriorhodopsin

Light-induced proton pumping in bacteriorhodospin is carried out through five proton transfer steps. We propose that the proton transfer to Asp85 from the Schiff base in the L-to-M transition is accompanied by the relocation of a water cluster on the cytoplasmic side of the Schiff base from a site close to the Schiff base in L to the Phe219-Thr46 region in M. The water cluster present in L, formed at 170 K, is more rigid than that at room temperature. This may be responsible for blocking the conversion of L to M at 170 K. In the photocycle at room temperature, this water cluster returns to the site close to the Schiff base in N, with a rigid structure similar to that of L at 170 K. The increase in the proton affinity of Asp85, which is a prerequisite for the one-way proton transfer in the M-to-N transition, is suggested to be facilitated by a structural change which disrupts interactions between Asp212 and the Schiff base, and between Asp212 and Arg82. We propose that this liberation of Asp212 is accompanied by a rearrangement of the structure of water molecules between Asp85 and Asp212, stabilizing the protonated Asp85 in M.     

Dynamics of competitive reactions: endothermic proton transfer and exothermic substitution

Dynamics of an endothermic proton-transfer reaction, F(-) with dimethyl sulfoxide, and an endothermic proton-transfer reaction with a competing exothermic substitution (S(N)2) channel, F(-) with borane-methyl sulfide complex, were investigated using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR) and kinetic modeling. The two proton-transfer reactions have slightly positive and a small negative overall free energy changes, respectively. Energy-dependent rate constants were measured as a function of F(-) ion translational energy, and the resulting kinetics were modeled with the RRKM (Rice-Ramsperger-Kassel-Marcus) theory. The observed rate constants for the proton-transfer reactions of F(-) with dimethyl sulfoxide and with borane-methyl sulfide complex are identical, with a value of 0.17 x 10(-9) cm(3) molecule(-1) s(-1); for the S(N)2 reaction, k = 0.90 x 10(-9) cm(3) molecule(-1) s(-1) at 350 K. Both proton-transfer reactions have positive entropy changes in the forward direction and show positive energy dependences. The competing S(N)2 reaction exhibits negative energy dependence and becomes less important at higher energies. The changes of the observed rate constants agree with RRKM theory predictions for a few kcal/mol of additional kinetic energy. The dynamic change of the branching ratio for the competing proton transfer and the substitution reactions results from the competition between the microscopic rate constants associated with each channel.     

Dynamics of proton transfer at nonactivated carbons from laser flash electron photoinjection experiments

The investigation of proton exchange dynamics at carbon atoms has been so far limited to molecules activated by an electron-withdrawing substituent or by the removal of one electron yielding the corresponding cation radical. A method is proposed to overcome this limitation and extend the gathering of data to nonactivated carbon acids, RH. It consists of using photoinjected electrons to generate the radical R. from a rapidly or concertedly cleaving substrate, RX. The variations of the radical "polarogram" (in which R. is converted into R-) upon addition of an acid are then exploited to derive the protonation rate constant of R-. The method is demonstrated with the example of the diphenylmethyl carbanion. The Br?nsted plot thus obtained indicates that proton transfer to this carbanion is intrinsically slow, with a barrier on the order of 1 eV. An inverted region behavior seems to appear at large driving forces.     

Measurement of proton release and uptake by analogs of bacteriorhodopsin

Proton release and subsequent uptake by several forms of bacteriorhodopsin (bR), including 4-keto analogs of wild-type (WT) and D96N and D85N mutants as well as the 9-demethylretinal analog of WT and D96N mutants, have been measured using a highly sensitive electrochemical technique. Release and uptake of protons by bR in membrane patches on a tin oxide electrode produce a current transient whose amplitude is proportional to the rate of pH change at the electrode surface. Profiles of proton release by the analogs vs. pH are substantially different from the profiles of the native proteins.     

Intramolecular proton transfer

[structure: see text] Reversal of the normal kinetic protonation stereochemistry results as a consequence of intramolecular delivery.     

Tunnel effect in proton transfer

Semiempirical computations were carried out to determine the tunneling rates in the case of coupled motion of two protons along the reaction coordinate. The following molecular systems were studied for medium intermolecular distances (A—B = 2.72 or 2.75 Å); ⁺AH—BH—A, where A was NH₃ or H₂O and BH was HF or H₂O. In the cases where the bridge was HF, solvation was modeled with just one water molecule attached to each side of the perpendicular axis through HF at 2.75 Å. Coupled motion of three protons was also included in the case of H₃O—H₂O—H₂O—H₂O.     

Model for proton transport coupled to protein conformational change: application to proton pumping in the bacteriorhodopsin photocycle

A modeling method is presented for protein systems in which proton transport is coupled to conformational change, as in proton pumps and in motors driven by the proton-motive force. Previously developed methods for calculating pKa values in proteins using a macroscopic dielectric model are extended beyond the equilibrium case to a master-equation model for the time evolution of the system through states defined by ionization microstate and a discrete set of conformers. The macroscopic dielectric model supplies free energy changes for changes of protonation microstate, while the method for obtaining the energetics of conformational change and the relaxation rates, the other ingredients needed for the master equation, are system dependent. The method is applied to the photoactivated proton pump, bacteriorhodopsin, using conformational free energy differences from experiment and treating relaxation rates through three adjustable parameters. The model is found to pump protons with an efficiency relatively insensitive to parameter choice over a wide range of parameter values, and most of the main features of the known photocycle from very early M to the return to the resting state are reproduced. The boundaries of these parameter ranges are such that short-range proton transfers are faster than longer-range ones, which in turn are faster than conformational changes. No relaxation rates depend on conformation. The results suggest that an "accessibility switch", while not ruled out, is not required and that vectorial proton transport can be achieved through the coupling of the energetics of ionization and conformational states.     

Dynamics of the concerted triple proton transfer in cyclic water trimer using the multiconfiguration molecular mechanics algorithm

Cyclic water clusters are important molecular species to understand the nature of hydrogen bonded networks. Theoretical studies for the dynamics of triple proton transfer in the cyclic water trimer were performed. The potential energy surface (PES) of triple proton transfer is generated by the multiconfiguration molecular mechanics (MCMM) algorithm. We have used the MP2/6-31G(d,p) level for high-level ab initio data (energies, gradients, and Hessians), which are used in the Shepard interpolation. Eight high-level reference points were added step by step, including two points for the critical configurations of the large curvature tunneling paths. The more high-level points are used, the better the potential energy surfaces become. The rate constant and kinetic isotope effect (KIE) for the triple proton transfer at 300 K, which have been calculated by the canonical variational transition-state theory with microcanonical optimized multidimensional semiclassical tunneling approximation, are 1.6 x 10(-3) s(-1) and 230, respectively. Tunneling is very important not only for the triple proton transfer but also for the triple deuterium transfer. The MCMM results show good agreement with those from the direct ab initio dynamics calculations.     

The proton uptake channel of bacteriorhodopsin as studied by a photoelectrochemical method

A series of the mutant proteins (D96N, D96N/D85N, D115N, L93T, T46V, V49A) where the residues are located at the cytoplasmic domain of bacteriorhodopsin (bR) were studied photoelectrochemically and their photocurrent response characteristics at the electrode/electrolyte interface were compared with those of the wild-type bR. While the wild-type bR of normal proton pumping activity yields symmetrical cathodic (positive) and anodic (negative) responses, corresponding to proton release and proton uptake, respectively, these mutants, with the exception of D115N, showed diminished amplitudes in the negative response. This indicates retardation of proton translocation from the cytoplasmic surface to the retinal Schiff base. The mutation that gave the strongest influence on the negative response was D96N while moderate influence was obtained with L93T, T46V, and V49A. These results suggest that residues other than D96 also participate in the cytoplasmic proton uptake channel, either by interacting with D96 directly or by forming a hydrogen-bonded network with water molecules. The D96N/D85N double mutant yielded little response at neutral pH, but the response was partially recovered by addition of azide, while it was fully recovered in the single mutant D96N. The D115N mutant showed the response profile that closely resembles the wild-type, indicating that D115 is not crucially involved in the event of proton transfer relay at the cytoplasmic region. It was also found that every mutant in this study releases protons prior to uptake at the other membrane surface, as does the wild-type.     

Elementary steps in excited-state proton transfer

The absorption of a photon by a hydroxy-aromatic photoacid triggers a cascade of events contributing to the overall phenomenon of intermolecular excited-state proton transfer. The fundamental steps involved were studied over the last 20 years using a combination of theoretical and experimental techniques. They are surveyed in this sequel in sequential order, from fast to slow. The excitation triggers an intramolecular charge transfer to the ring system, which is more prominent for the anionic base than the acid. The charge redistribution, in turn, triggers changes in hydrogen-bond strengths that set the stage for the proton-transfer step itself. This step is strongly influenced by the solvent, resulting in unusual dependence of the dissociation rate coefficient on water content, temperature, and isotopic substitution. The photolyzed proton can diffuse in the aqueous solution in a mechanism that involves collective changes in hydrogen-bonding. On longer times, it may recombine adiabatically with the excited base or quench it. The theory for these diffusion-influenced geminate reactions has been developed, showing nice agreement with experiment. Finally, the effect of inert salts, bases, and acids on these reactions is analyzed.     

Computational analysis of the proton translocation from Asp96 to schiff base in bacteriorhodopsin

The potential energy change during the M --> N process in bacteriorhodopsin has been evaluated by ab initio quantum chemical and advanced quantum chemical calculations following molecular dynamics (MD) simulations. Many previous experimental studies have suggested that the proton transfer from Asp96 to the Schiff base occurs under the following two conditions: (1) the hydrogen bond between Thr46 and Asp96 breaks and Thr46 is detached from Asp96 and (2) a stable chain of four water molecules spans an area from Asp96 --> Schiff base. In this work, we successfully reproduced the proton-transfer process occurring under these two conditions by molecular dynamics and quantum chemical calculations. The quantum chemical computation revealed that the proton transfer from Asp96 to Shiff base occurs in two-step reactions via an intermediate in which an H(3)O(+) appears around Ala215. The activation energy for the proton transfer in the first reaction was calculated to be 9.7 kcal/mol, which enables fast and efficient proton pump action. Further QM/MM (quantum mechanical/molecular mechanical) and FMO (fragment molecular orbital) calculations revealed that the potential energy change during the proton transfer is tightly regulated by the composition and the geometry of the surrounding amino acid residues of bacteriorhodopsin. Here, we report in detail the Asp96 --> Schiff base proton translocation mechanism of bacteriorhodopsin. Additionally, we discuss the effectiveness of combining quantum chemical calculations with truncated cluster models followed by advanced quantum chemical calculations applied to a whole protein to elucidate its reaction mechanism.     

Pathways of proton transfer in acetal hydrolysis

The process of acetal hydrolysis is analyzed in terms of the competing steps of proton transfer and heavy atom reorganization. The results of this analysis are portrayed in a series of three-dimensional reaction coordinate diagrams. The observed pathway of hydrolysis (A-1, general acid catalyzed, or spontaneous) is shown to depend on the energy of the various possible intermediates in these reactions.     

The mechanism of bacteriorhodopsin functioning. II. Ejection and injection of proton

Transmission of vibrational excitation energy conserved in cis-conformation of retinal to the outlet proton channel is considered from the perspective of quantum theory. A distribution of vibrational excitations in the channel is found; it allowed to calculate the magnitude of the directed drift proton current. The differences between velocities of proton movement in active and passive channels are considered. A transition of retinal from cis- to all -trans- conformation with the subsequent capture of proton by Schiff base out of the inlet channel is described. The lack of proton in this channel, i.e., in the H-bonded chain, is eliminated at the expense of the capture of a proton out of the cytoplasmic water enviroment. The correspondence between theoretically established states and spectroscopically identified forms of bacteriorhodopsin (inter-mediates L, M, N, and O) is proposed.     

Electron and proton transfer in acid-base catalysis

The role of electron and proton transfer in acid-base catalysis is discussed, with two reactions as examples, in one of which (polymerization of cyclobutenes) an acid, and in another (nitramide decomposition), a base acts as the catalyst.