Proton Transfer In Bacteriorhodopsin Research Articles

Mechanism of proton transfer in bacteriorhodopsin

Photoinduced proton pumping in bateriorhodopsin (bR) was extensively studied using multiple experimental methods and utilizing various theoretical modelling approaches. These studies usually refer to the well-resolved structural data of bacteriorhodopsin. However, despite the obtained results, the origin of the proton pumping force initiated by the electronic excitation of retinal remains questionable. Using quantum chemical calculations, we have revealed that the retinal molecule after its excitation is fixed in the ground state of 13-cis,15-syn configuration, as a result of interaction with specific protein residuals. Reaching this fixed configuration, the proton is first transferred to the aspartic acid No. 85 (Asp-85) residue from the water molecule, which is subsequently restored by the proton initially located in the Schiff base. We discuss the challenges and approaches to modelling the proton transfer in bR and demonstrate that the process, which starts from the electronic excitation of the retinal molecule, is mainly due to the detailed arrangement of the protein environment.

Hydroxide Ion Carrier for Proton Pumps in Bacteriorhodopsin: Primary Proton Transfer.

Bacteriorhodopsin (BR) is a model protein for light-driven proton pumps, where the vectorial active proton transport results in light-energy conversion. To clarify the microscopic mechanism of primary proton transfer from retinal Schiff base (SB) to Asp85 in BR, herein, we performed quantum-mechanical metadynamics simulations with the isolated BR model (∼3750 atoms). The simulations showed a novel proton transfer mechanism, viz. the hydroxide ion mechanism, in which the deprotonation of specific internal water (Wat452) yields the protonation of Asp85 via Thr89, after which the resulting hydroxide ion accepts the remaining proton from retinal SB. Systematic investigations adopting four sequential snapshots obtained by the time-resolved serial femtosecond crystallography revealed that proton transfer took 2-5.25 μs on the photocycle. The presence of Wat401, which is the main difference between snapshots at 2 and 5.25 μs, is found to be essential in assisting the primary proton transfer. Furthermore, the hydroxide ion mechanism was confirmed by the minimum energy path for the primary proton transfer in BR obtained by the nudged elastic band calculations with the embedded BR model (10,119 atoms), in which BR was embedded within lipid membranes in between water solvents.

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.

The energetics of the primary proton transfer in bacteriorhodopsin revisited: It is a sequential light-induced charge separation after all

The light-induced proton transport in bacteriorhodopsin has been considered as a model for other light-induced proton pumps. However, the exact nature of this process is still unclear. For example, it is not entirely clear what the driving force of the initial proton transfer is and, in particular, whether it reflects electrostatic forces or other effects. The present work simulates the primary proton transfer (PT) by a specialized combination of the EVB and the QCFF/PI methods. This combination allows us to obtain sufficient sampling and a quantitative free energy profile for the PT at different protein configurations. The calculated profiles provide new insight about energetics of the primary PT and its coupling to the protein conformational changes. Our finding confirms the tentative analysis of an earlier work (A. Warshel, Conversion of light energy to electrostatic energy in the proton pump of Halobacterium halobium, Photochem. Photobiol. 30 (1979) 285–290) and determines that the overall PT process is driven by the energetics of the charge separation between the Schiff base and its counterion Asp85. Apparently, the light-induced relaxation of the steric energy of the chromophore leads to an increase in the ion-pair distance, and this drives the PT process. Our use of the linear response approximation allows us to estimate the change in the protein conformational energy and provides the first computational description of the coupling between the protein structural changes and the PT process. It is also found that the PT is not driven by twist-modulated changes of the Schiff base's p K a, changes in the hydrogen bond directionality, or other non-electrostatic effects. Overall, based on a consistent use of structural information as the starting point for converging free energy calculations, we conclude that the primary event should be described as a light-induced formation of an unstable ground state, whose relaxation leads to charge separation and to the destabilization of the ion-pair state. This provides the driving force for the subsequent PT steps.

Structural and energetic determinants of primary proton transfer in bacteriorhodopsin

In the light-driven bacteriorhodopsin proton pump, the first proton transfer step is from the retinal Schiff base to a nearby carboxylate group. The mechanism of this transfer step is highly controversial, in particular whether a direct proton jump is allowed. Here, we review the structural and energetic determinants of the direct proton transfer path computed by using a combined quantum mechanical/molecular mechanical approach. Both protein flexibility and electrostatic interactions play an important role in shaping the proton transfer energy profile. Detailed analysis of the energetics of putative transitions in the first half of the photocycle focuses on two elements that determine the likelihood that a given configuration of the active site is populated during the proton-pumping cycle. First, the rate-limiting barrier for proton transfer must be consistent with the kinetics of the photocycle. Second, the active-site configuration must be compatible with a productive overall pumping cycle.

Mechanism of Primary Proton Transfer in Bacteriorhodopsin

Recent structures of putative intermediates in the bacteriorhodopsin photocycle have provided valuable snapshots of the mechanism by which protons are pumped across the membrane. However, key steps remain highly controversial, particularly the proton transfer occurring immediately after retinal trans-->cis photoisomerization. The gradual release of stored energy is inherently nonequilibrium: which photocycle intermediates are populated depends not only on their energy but also on their interconversion rates. To understand why the photocycle follows a productive (i.e., pumping), rather than some unproductive, relaxation pathway, it is necessary to know the relative energy barriers of individual steps. To discriminate between the many proposed scenarios of this process, we computed all its possible minimum-energy paths. This reveals that not one, but three very different pathways have energy barriers consistent with experiment. This result reconciles the conflicting views held on the mechanism and suggests a strategy by which the protein renders this essential step resilient.

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.

Dynamics of proton transfer in bacteriorhodopsin.

Proton transfer in bacteriorhodopsin from the cytoplasm to the extracellular side is initiated from protonated asp96 in the cytoplasmic region toward the deprotonated Schiff base. This occurs in the transition from the photocycle late M state to the N state. To investigate this proton-transfer process, a quantum mechanics/molecular mechanics (QM/MM) model is constructed from the bacteriorhodopsin E204Q mutant crystal structure. Three residues, asp96, asp85, and thr89, as well as most of the retinal chromophore and the Schiff base link of lys216 are treated quantum mechanically and connected to the remaining classical protein through linker atom hydrogens. Structural transformation in the M state results in the formation of a water channel between the Schiff base and asp96. Since a part of this channel is lined with hydrophobic residues, there has been a question on the mechanism of proton transfer in a hydrophobic channel. Ab initio dynamics using the CHARMM/GAMESS methodology is used to simulate the transfer of the proton through a partially hydrophobic channel. Once sufficient water molecules are added to the channel to allow the formation of a single chain of waters from asp96 to the Schiff base, the transfer occurs as a fast (less than a picosecond) concerted event irrespective of the protonation state of asp85. Dynamic transfer of the proton from asp96 to the nearest water initiates the organization of a strongly bonded water chain conducive to the transfer of the proton to the Schiff base nitrogen.

Kinetics of picosecond laser pulse induced charge separation and proton transfer in bacteriorhodopsin.

Bacteriorhodopsin (BR) films oriented by an electrophoretic method are deposited on a transparent conductive ITO glass. A counterelectrode of copper and gelose gel is used to compose a sandwich-type photodetector with the structure of ITO/BR film/gelose gel/Cu. A single 30-ps laser pulse and a mode-locked pulse train are respectively used to excite the BR photodetector. The ultrafast falling edge and the bipolar response signal are measured by the digital oscilloscope under seven different time ranges. Marquardt nonlinear least squares fitting is used to fit all the experimental data and a good fitting equation is found to describe the kinetic process of the photoelectric signal. Data fitting resolves six exponential components that can be assigned to a seven-step BR photocycle model: BR-->K-->KL-->L-->M-->N-->O-->BR. Comparing tests of the BR photodetector with a 100-ps Si PIN photodiode demonstrates that this type of BR photodetector has at least 100-ps response time and can also serve as a fast photoelectric switch.

A factor to determine the direction of the proton transfer in bacteriorhodopsin

Ab initio quantum chemical calculations have been performed to figure out a hypothetical structure that allows for reversible proton transport among Asp85, Schiff base, and Asp96 in bacteriorhodopsin. As an example of the hypothetical structure, we constructed a model system in which Asp85 and Asp96 are located in the Cs symmetry with respect to a vertical plane containing the N atom of the Schiff base and the O atom of the water molecule bound to the Schiff base. The proton transport reaction usually proceeds with the L→M→N in bacteriorhodopsin. In the present hypothetical structure, both of the proton transports (L→M→N and N→M→L) were revealed to occur reversibly.

Proton Transfer in Bacteriorhodopsin: Structure, Excitation, IR Spectra, and Potential Energy Surface Analyses by an ab Initio QM/MM Method

The structures, the optical properties, and the proton-transfer mechanism of bacteriorhodopsin (bR) are investigated by using an ab initio QM/MM calculation. The nature of the hydrogen bond network (HBN) formed in the retinal pocket is examined. The analysis is made for the “environmental” effect from the molecules involved in this HBN on the geometry and the excitation spectrum of retinal. It is found that IR spectra of the O−D stretching of the bound water molecules and the N−D stretching of the Schiff base retinal are very sensitive to the vibration−electron coupling among these molecules in the pocket. The hypothetical proton-transfer reaction path in the bR ground state is investigated, and the “environmental effect” on it is analyzed. The present calculation reveals that electronic interactions such as charge transfer and polarization, not included in most empirical methods, are very important to describe these properties of bR, the structures of HBN, the IR activities, and the proton-transfer mecha...

Proton Polarizability of Hydrogen-Bonded Network and its Role in Proton Transfer in Bacteriorhodopsin

Room-temperature time-resolved step-scan Fourier Transform Infrared (FTIR) spectroscopy has been used to study the photocycle of native bacteriorhodopsin (bR) suspension in both H2O and D2O. The kinetics of the retinal isomerization, and that of the protonation/deprotonation of the proton acceptor, Asp85, are compared in the μs to ms time domain. It is found that hydrogen/deuterium (H/D) isotope exchange does not significantly affect the kinetics of the retinal isomerization and relaxation processes. However, the protonation/deprotonation processes of Asp85 COO- become slower in D2O. We also studied the kinetics of the continuum absorbance change in the 1850−1800 cm-1 frequency region, which has previously been proposed to correspond to the absorption of the delocalized proton that is involved in the proton transport to the surface during the photocycle. An H/D isotope shift of the frequency range of this continuum absorbance has been confirmed by the observation that the band in the 1850−1800 cm-1 disappears in the photocycle of bR in D2O. These results could support the previous proposal that the intramolecular proton release pathway consists of an H-bonded network. Our results also suggest that the two independent processes, the transfer of a proton from the Schiff base to Asp85 and the release of a different proton to the extracellular surface, are closely coupled events.

Kinetic isotope effects reveal an ice-like and a liquid-phase-type intramolecular proton transfer in bacteriorhodopsin

The mechanism of the intramolecular proton transfer in the membrane protein bacteriorhodopsin (bR) is studied. The kinetic isotope effects after H/D exchange were determined for the individual photocycle reactions and used as an indicator. Significant differences in the kinetic isotope effects are observed between the intramolecular proton transfer on the release and the uptake pathways. The results suggest a fast intramolecular proton transfer mechanism in the proton release pathway, which is similar to the one proposed for ice, where the rate limiting step is the proton movement within the H bond. However, the reactions in the intramolecular proton uptake pathway occur in a mechanism similar to the one suggested for liquid water, where the rate limiting step is given by a rotational rearrangement of H bonded network groups. We propose that the experimental evidence for a proton wire mechanism given here for bacteriorhodopsin is of general relevance also for other proton transporting proteins.

The two pKa's of aspartate-85 and control of thermal isomerization and proton release in the arginine-82 to lysine mutant of bacteriorhodopsin.

To explore the role of Arg82 in the catalysis of proton transfer in bacteriorhodopsin, we replaced Arg82 with Lys, which is also positively charged at neutral pH but has an intrinsic pKa of about 1.7 pH units lower than that of Arg. In the R82K mutant expressed in Halobacterium salinarium, we found the following: (1) The pKa of the purple-to-blue transition at low pH (which reflects the pKa of Asp85) is 3.6 +/- 0.1. At high pH a second inflection in the blue-to-purple transition with pKa = 8.0 is found. The complex titration behavior of Asp85 indicates that the pKa of Asp85 depends on the protonation state of another amino acid residue, X', which has a pKa = 8.0 in R82K. The fit of the experimental data to a model of two interacting residues shows that deprotonation of X' at high pH causes a shift in the pKa of Asp85 from 3.7 to 6.0. In turn, protonation of Asp85 decreases the pKa of X' by 2.3 pH units. This suggests that X' can release a proton upon formation of the M intermediate and the concomitant protonation of Asp85 in the photocycle. (2) The rate constant of dark adaptation, kda, is proportional to the fraction of blue membrane between pH 2 and 10, indicating that thermal isomerization proceeds through the transient protonation of Asp85. The pH dependence of kda shows that two groups with pKal = 3.9 and pKa2 = 8.0 control the rate of dark adaptation in R82K. The 1.7 pH unit shift in pKa2 in R82K compared to the wild type (WT) (pKa2 = 9.7) supports the hypothesis that X' is Arg82 in WT and Lys82 in R82K (or at least that these groups are the principal part of a cluster of residues that constitute X'). (3) Under steady state illumination, the efficiency of proton transport in R82K incorporated in phosphatidylcholine vesicles is at least 40% of that in the WT. A flash-induced transient signal of the pH-sensitive dye pyranine is similar to that in the WT (proton release precedes uptake), but the amplitude is small in R82K (about 15% of that found in the WT), indicating that only a small fraction of protons is released fast in R82K. This supports the suggestions that Arg82 is associated with the proton release pathway (acts as a proton release group or part of a proton release complex) and that Lys cannot efficiently substitute for Arg in this process.(ABSTRACT TRUNCATED AT 400 WORDS)