Understanding structure and function in the light-driven proton pump bacteriorhodopsin

The atomic structure of bacteriorhodopsin and the outlines of its proton transport mechanism are now available. Photoisomerization of the retinal in the chromophore creates a steric and electrostatic conflict at the retinal binding site. The free energy gain sets off a sequence of reactions in which directed proton transfers take place between the protonated retinal Schiff base, Asp-85, and Asp-96. These internal steps, and other proton transfers at and near the two aqueous interfaces, add up to the translocation of a proton from the cytoplasmic to the extracellular side of the membrane. Bound water plays a crucial role in proton conduction in both extracellular and cytoplasmic regions, but the means by which the protons move from site to site differ. Proton release to the extracellular surface is through interaction of a hydrogen-bonded chain of identified aspartic acid, arginine, water, and glutamic acid residues with Asp-85, while proton uptake from the cytoplasmic surface utilizes a single aspartic acid, Asp-96, whose protonation state appears to be regulated by the protein conformation dependent hydration of this region. The directionality of the translocation is ensured by the accessibility of the Schiff base to the extracellular and cytoplasmic directions after the retinal is photoisomerized, as well as the changing proton affinities of the acceptor Asp-85 and donor Asp-96.     

Structural changes during the formation of early intermediates in the bacteriorhodopsin photocycle

Early intermediates of bacteriorhodopsin's photocycle were modeled by means of ab initio quantum mechanical/molecular mechanical and molecular dynamics simulations. The photoisomerization of the retinal chromophore and the formation of photoproducts corresponding to the early intermediates were simulated by molecular dynamics simulations. By means of the quantum mechanical/molecular mechanical method, the resulting structures were refined and the respective excitation energies were calculated. Two sequential intermediates were found with absorption maxima that exhibit red shifts from the resting state. The intermediates were therefore assigned to the K and KL states. In K, the conformation of the retinal chromophore is strongly deformed, and the N--H bond of the Schiff base points almost perpendicular to the membrane normal toward Asp-212. The strongly deformed conformation of the chromophore and weakened interaction of the Schiff base with the surrounding polar groups are the means by which the absorbed energy is stored. During the K-to-KL transition, the chromophore undergoes further conformational changes that result in the formation of a hydrogen bond between the N--H group of the Schiff base and Thr-89 as well as other rearrangements of the hydrogen-bond network in the vicinity of the Schiff base, which are suggested to play a key role in the proton transfer process in the later phase of the photocycle.     

Subpicosecond resonance Raman spectra of the early intermediates in the photocycle of bacteriorhodopsin

The resonance Raman spectra are presented for the species formed during the photocycle of bacteriorhodopsin (bR) on a timescale of 800-900 fs. In the ethylenic stretch region two intermediates were found with frequencies of 1,510 and 1,518 cm^-1, corresponding to species with optical absorption maxima at 660 and 625 nm, respectively. This leads to the assignment of the 1,518 cm^-1 band to the J₆₂₅ intermediate. In the fingerprint region, the appearance of a vibration at 1,195 cm^-1 strongly suggests that the isomerization indeed has taken place in a time less than the pulsewidth of our laser. This supports the previous proposals made on the basis of the optical spectra. The spectra are compared with those observed in tens of picoseconds up to nanoseconds.     

pH and salt effects on the slow intermediates of the bacteriorhodopsin photocycle

Purple membrane fragments of Halobacterium halobium were used to investigate pH and salt effects on the kinetics of M ₄₁₂, O ₆₆₀ and BR ₅₆₈. The flash-induced absorbance changes were measured in the 5–9 pH range, at low ionic strength and at 4 M NaCl. The results are consistent with a model which implies a branching in the last part of the bacteriorhodopsin photocycle.     

Interaction of internal water molecules with the schiff base in the L intermediate of the bacteriorhodopsin photocycle

In the photocycle of bacteriorhodopsin (BR), the first proton movement, from the Schiff base to Asp85, occurs after the formation of the L intermediate. In L, the C [double bond] N bond of the Schiff base is strained, and the nitrogen interacts strongly with its counterion. The present study seeks to detect the interaction of internal water molecules with the Schiff base in L using difference FTIR spectroscopy at 170 K. The coupled modes of the hydrogen-out-of plane bending vibrations (HOOPs) of the N-H and C(15)-H of the protonated Schiff base are detected as a broad band centered at 911 cm(-1) for BR. A set of bands at 1073, 1064, and 1056 cm(-1) for L is shown to arise from the coupling of the HOOP with the overtones of interacting water O-H vibrations. Interaction with water was shown by the decreased intensity of the HOOPs of L in H(2)(18)O and by the influence of mutants that have been shown to perturb specific internal water molecules in BR. In contrast, the HOOP band of initial BR was not affected by these mutations. In D85N, the coupled HOOP of BR is depleted, while the coupled HOOPs of L are shifted. The results indicate that the Schiff base interacts with water in the L state but in a different manner than in the BR state. Moreover, the effects of mutations suggest that cytoplasmic water close to Thr46 (Wat46) either interacts stronger with the Schiff base in L or that it is important in stabilizing another water that does.     

Thermal equilibration between the M and N intermediates in the photocycle of bacteriorhodopsin.

The stages in the photocycle of bacteriorhodopsin (BR) involving the M and N intermediates are investigated using a double pulse excitation method. A first (cycling) pulse at 532 nm is followed, with an appropriate time delay, by a second pulse (337, 406, 446, or 470 nm) which induces the M-->BR back-photoreaction. After depletion by the second pulse a repopulation of M in the millisecond range is observed which is interpreted in terms of a thermal N-->M relaxation. It is thus concluded that a (thermal) M<-->N equilibrium accounts for the biphasic decay of M in the BR photocycle. Other models for this stage of the light-driven proton-pump are therefore unnecessary.     

High-resolution crystal structures of transient intermediates in the phytochrome photocycle

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The photocycle of the chloride pump halorhodopsin. I: Azide-catalyzed deprotonation of the chromophore is a side reaction of photocycle intermediates inactivating the pump

Halorhodopsin, the light-driven chloride pump of halobacteria, undergoes a photochemical cycle in the 10 ms range. Two intermediates, HR640 and HR520, accumulate in the photosteady state after short times (within 100 ms) of illumination. Upon prolonged illumination a third species, HRL410 accumulates, which is formed from HR520/HR640 by deprotonation of the chromophore in a side reaction of the photocycle. In the dark, HRL410 requires several minutes to reconvert thermally to HR478. Thus, molecules in the HRL410 state must be inactive pumps since their maximal turnover number could only be a few per hour. Inorganic bases, such as azide, catalyze the deprotonation of HR520/HR640 as well as the reprotonation of HRL410. Both reactions are accelerated several hundred times by azide but the photosteady-state concentration of HRL410 remains unchanged.     

Lipidic cubic phase crystallization of bacteriorhodopsin and cryotrapping of intermediates: towards resolving a revolving photocycle

Bacteriorhodopsin is a small retinal protein found in the membrane of the halophilic bacterium Halobacterium salinarum, whose function is to pump protons across the cell membrane against an electrostatic potential, thus converting light into a proton-motive potential needed for the synthesis of ATP. Because of its relative simplicity, exceptional stability and the fundamental importance of vectorial proton pumping, bacteriorhodopsin has become one of the most important model systems in the field of bioenergetics. Recently, a novel methodology to obtain well-diffracting crystals of membrane proteins, utilizing membrane-like bicontinuous lipidic cubic phases, has been introduced, providing X-ray structures of bacteriorhodopsin and its photocycle intermediates at ever higher resolution. We describe this methodology, the new insights provided by the higher resolution ground state structures, and review the mechanistic implications of the structural intermediates reported to date. A detailed understanding of the mechanism of vectorial proton transport across the membrane is thus emerging, helping to elucidate a number of fundamental issues in bioenergetics.     

Protonation and deprotonation of the M, N, and O intermediates during the bacteriorhodopsin photocycle

Transient pH changes were measured with phenol red and chlorophenol red in the 30-microseconds-50-ms time range during the photocycle of bacteriorhodopsin (BR), the light-driven proton pump. At pH greater than or equal to 7, the results confirmed earlier data and suggestions that one proton is released during the L----M reaction, and taken up again during the decay of N. These are likely to be steps in the proton transport process. At pH less than 7, however, the time-resolved pH traces were complex and indicated additional protonation reactions. The data were explained by a model which assumed pH-dependent protonation states for M and N which varied from -1 to 0, and for O which varied from 0 to + 2, relative to BR. If the kinetics of the vectorial proton translocation process were taken as pH independent, this treatment of the data suggested that a residue with a pKa of 5.9 was made protonable in M and N and two residues with pKa's of 6.5 were made cooperatively protonable in O. The additional protons detected are not necessarily in the vectorial proton transfer pathway (i.e., they are probably "Bohr protons"), and while they must reflect conformational and/or neighboring ionization changes in the BR as it passes through the M, N, and O states, their role, if any, in the transport is uncertain.     

Role of internal water molecules in bacteriorhodopsin

Internal water molecules are considered to play a crucial role in the functional processes of proton pump proteins. They may participate in hydrogen-bonding networks inside proteins that constitute proton pathways. In addition, they could participate in the switch reaction by mediating an essential proton transfer at the active site. Nevertheless, little has been known about the structure and function of internal water molecules in such proteins. Recent progress in infrared spectroscopy and X-ray crystallography provided new information on water molecules inside bacteriorhodopsin, the light-driven proton pump. The accumulated knowledge on bacteriorhodopsin in the last decade of the 20th century will lead to a realistic picture of internal water molecules at work in the 21st century. In this review, I describe how the role of water molecules has been studied in bacteriorhodopsin, and what should be known about the role of water molecules in the future.     

Crystallographic structures of the M and N intermediates of bacteriorhodopsin: assembly of a hydrogen-bonded chain of water molecules between Asp-96 and the retinal Schiff base

An M intermediate of wild-type bacteriorhodopsin and an N intermediate of the V49A mutant were accumulated in photostationary states at pH 5.6 and 295 K, and their crystal structures determined to 1.52A and 1.62A resolution, respectively. They appear to be M(1) and N' in the sequence, M(1)<-->M(2)<-->M'(2)<-->N<-->N'-->O-->BR, where M(1), M(2), and M'(2) contain an unprotonated retinal Schiff base before and after a reorientation switch and after proton release to the extracellular surface, while N and N' contain a reprotonated Schiff base, before and after reprotonation of Asp96 from the cytoplasmic surface. In M(1), we detect a cluster of three hydrogen-bonded water molecules at Asp96, not present in the BR state. In M(2), whose structure we reported earlier, one of these water molecules intercalates between Asp96 and Thr46. In N', the cluster is transformed into a single-file hydrogen-bonded chain of four water molecules that connects Asp96 to the Schiff base. We find a network of three water molecules near residue 219 in the crystal structure of the non-illuminated F219L mutant, where the residue replacement creates a cavity. This suggests that the hydration of the cytoplasmic region we observe in N' might have occurred spontaneously, beginning at an existing water molecule as nucleus, in the cavities from residue rearrangements in the photocycle.     

M-decay in the bacteriorhodopsin photocycle: effect of cooperativity and pH

The dependence of the bacteriorhodopsin (bR) photocycle on the intensity of the exciting flash was investigated in purple membranes. The dependence was most pronounced at slightly alkaline pH values. A comparison study of the kinetics of the photocycle and proton uptake at different intensities of the flash suggested that there exist two parallel photocycles in purple membranes at a high intensity of the flash. The photocycle of excited bR in a trimer with the two other bR molecules nonexcited is characterized by an almost irreversible M --> N transition. Excitation of two or three bR in a trimer induces the N --> M back reaction and accelerates the N --> bR transition. Based on the qualitative similarity of the pH dependencies of the photocycles of solubilized bR and excited dimers and trimers we proposed that the interaction of nonexcited bR in trimers alters the photocycle of the excited monomer as compared to solubilized bR and the changes in the photocycles in excited dimers and trimers are the result of decoupling of this interaction.     

Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.

The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.     

Microsecond exchange of internal water molecules in bacteriorhodopsin

The proton-conducting pathway of bacteriorhodopsin (BR) contains at least nine internal water molecules that are thought to be key players in the proton translocation mechanism. Here, we report the results of a multinuclear (1H, 2H, 17O) magnetic relaxation dispersion (MRD) study with the primary goal of determining the rate of exchange of these internal water molecules with bulk water. This rate is of interest in current attempts to elucidate the molecular details of the proton translocation mechanism. The relevance of water exchange kinetics is underscored by recent crystallographic findings of substantial variations in the number and locations of internal water molecules during the photocycle. Moreover, internal water exchange is believed to be governed by conformational fluctuations in the protein and can therefore provide information about the thermal accessibility of functionally important conformational substates. The present 2H and 17O MRD data show that at least seven water molecules, or more if they are orientationally disordered, in BR have residence times (inverse exchange rate constant) in the range 0.1-10 micros at 277 K. At least five of these water molecules have residence times in the more restrictive range 0.1-0.5 micros. These results show that most or all of the deeply buried water molecules in BR exchange on a time-scale that is short compared to the rate-limiting step in the photocycle. The MRD measurements were performed on BR solubilized in micelles of octyl glucoside. From the MRD data, the rotational correlation time of detergent-solubilized BR was determined to 35 ns at 300 K, consistent with a monomeric protein in complex with about 150 detergent molecules. The solubilized protein was found to be stable in the dark for at least eight months at 277 K.     

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.     

Pathways of proton release in the bacteriorhodopsin photocycle.

The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.