The effect of pH on proton transport by bacteriorhodopsin

The pH-dependence of proton motion during the photocycle was investigated by measuring the photoelectric signals due to charge displacement inside bacteriorhodopsin molecules. Measurements were performed on purple membranes oriented in suspension and the kinetics of flash excited electric and light absorption signals was compared. It was found that in the pH range 4.5–8 the photocycle and the successive proton movements have identical kinetics, and do not depend on pH. In the pH range 8–10 both kinetics change, though differently; the charge motion decouples from the photocycle and the photocycle seems to split up into two parallel paths, the photoelectric signal becomes faster. However, the net proton transfer remains the same as at lower pH values. Above pH ≈ 10, the photocycle behaves differently and cannot be described by the parallel pathway model and the net proton displacement drops. The results are explained by the successive titration of two groups (probably tyrosine) participating in proton translocation.     

The bacteriorhodopsin proton pump: effect of crosslinkings of lysine residues

All six available lysine residues in bacteriorhodopsin were amidinated with dimethyl-3,3'-dithiobispropionimidate, which is a crosslinking agent. The photocycle was studied by measuring light absorption and electric signals. The data show an essential change in the photocycle: instead of single components, the rise of the signal due to the M intermediate can be decomposed into two components, and the decay into three. The life-times and the intensities of these components and in general the proton pumping activity of bacteriorhodopsin depend only negligibly upon pH. Changes upon removing the crosslinks are not significantly different from those in the crosslinked samples. The lysine residues therefore may not be considered of primary importance in proton translocation.     

Proteorhodopsin is a light-driven proton pump with variable vectoriality

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions.The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.     

Chemical and physical evidence for multiple functional steps comprising the M state of the bacteriorhodopsin photocycle

In the photocycle of bacteriorhodopsin (bR), light-induced transfer of a proton from the Schiff base to an acceptor group located in the extracellular half of the protein, followed by reprotonation from the cytoplasmic side, are key steps in vectorial proton pumping. Between the deprotonation and reprotonation events, bR is in the M state. Diverse experiments undertaken to characterize the M state support a model in which the M state is not a static entity, but rather a progression of two or more functional substates. Structural changes occurring in the M state and in the entire photocycle of wild-type bR can be understood in the context of a model which reconciles the chloride ion-pumping phenotype of mutants D85S and D85T with the fact that bR creates a transmembrane proton-motive force.     

Aspartic acid 85 in bacteriorhodopsin functions both as proton acceptor and negative counterion to the Schiff base.

In bacteriorhodopsin Asp85 has been proposed to function both as a negative counterion to the Schiff base and as proton acceptor in the early stages of the photocycle. To test this proposal further, we have replaced Asp85 by His. The rationale for this replacement is that although His can function as a proton acceptor, it cannot provide a negative charge at residue 85 to serve as a counterion to the protonated Schiff base. We show here that the absorption spectrum of the D85H mutant is highly sensitive to the pH of the external medium. From spectroscopic titrations, we have determined the apparent pK for deprotonation of the Schiff base to be 8.8 +/- 0.1 and the apparent pK for protonation of the His85 side chain to be approximately 3.5. Between pH 3.5 and 8.8, where the Schiff base is protonated, and the His side chain is deprotonated, the D85H mutant is completely inactive in proton transport. Time-resolved studies show that there is no detectable formation of an M-like intermediate in the photocycle of the D85H mutant. These experiments show that the presence of a neutral proton-accepting moiety at residue 85 is not sufficient for carrying out light-driven proton transport. The requirements at residue 85 are therefore for a group that serves both as a negatively charged counterion and as a proton acceptor.     

Photoelectrochemical Cycle of Bacteriorhodopsin

The scheme of the bacteriorhodopsin photocycle associated with a transmembrane proton transfer and electrogenesis is considered. The role of conformational changes in the polypeptide chain during the proton transport is discussed.     

Hydration switch model for the proton transfer in the Schiff base region of bacteriorhodopsin

In a light-driven proton-pump protein, bacteriorhodopsin (BR), protonated Schiff base of the retinal chromophore and Asp85 form ion-pair state, which is stabilized by a bridged water molecule. After light absorption, all-trans to 13-cis photoisomerization takes place, followed by the primary proton transfer from the Schiff base to Asp85 that triggers sequential proton transfer reactions for the pump. Fourier transform infrared (FTIR) spectroscopy first observed O-H stretching vibrations of water during the photocycle of BR, and accurate spectral acquisition has extended the water stretching frequencies into the entire stretching frequency region in D(2)O. This enabled to capture the water molecules hydrating with negative charges, and we have identified the water O-D stretch at 2171 cm(-1) as the bridged water interacting with Asp85. We found that retinal isomerization weakens the hydrogen bond in the K intermediate, but not in the later intermediates such as L, M, and N. On the basis of the observation particularly on the M intermediate, we proposed a model for the mechanism of proton transfer from the Schiff base to Asp85. In the "hydration switch model", hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have raised the pK(a) of the proton acceptor, and the proton transfer is from the Schiff base to Asp85.     

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.     

Thermodynamics and energy coupling in the bacteriorhodopsin photocycle

Time-resolved absorption changes of photoexcited bacteriorhodopsin were measured with a gated multichannel analyzer between 100 ns and 100 ms at six temperatures between 5 and 30 degrees C. The energetics of the chromophore reaction cycle were analyzed on the basis of a model containing a single cycle and reversible reactions. The calculated thermodynamic parameters provide insights to general principles of the active transport. They indicate that in this light-driven proton pump the free energy is retained after absorption of the photon as the enthalpy of the pKa shift in the chromophore which allows deprotonation of the Schiff base. Part of the excess free energy is dissipated at the "switch" step where the reaction and transport cycles are coupled, and the rest at the chromophore recovery step. All other reactions take place near equilibrium. The "switch" step is the M1----M2 transition in the reaction cycle [Váró, G., & Lanyi, J. K. (1991) Biochemistry (preceeding paper in this issue)]. It provides for return of the chromophore pKa to its initial value so the Schiff base will become a proton acceptor, for reordering access of the Schiff base from one side of the membrane to the other, and for unidirectionality of the proton transfer. Conformational energy of the protein, acquired during the "switch" step, drives the completion of the photocycle.     

Substitution of amino acids in helix F of bacteriorhodopsin: effects on the photochemical cycle

The effects of amino acid substitutions in helix F of bacteriorhodopsin on the photocycle of this light-driven proton pump were studied. The photocycles of Ser-183----Ala and Glu-194----Gln mutants were qualitatively similar to that of wild-type bacteriorhodopsin produced in Escherichia coli and bacteriorhodopsin from Halobacterium halobium. The substitution of a Phe for either Trp-182 or Trp-189 significantly reduced the fraction of photocycling bacteriorhodopsin. The amino acid substitutions Tyr-185----Phe and Ser-193----Ala substantially increased the lifetime of the photocycle without substantially increasing the lifetime of the M photocycle intermediate. Similar results were also obtained with the Pro-186----Gly substitution. In contrast, replacing Pro-186 with the larger residue Leu inhibited the formation of the M photocycle intermediate. These results are consistent with a structural model of the retinal-binding pocket suggested by low-temperature UV/visible and Fourier transform infrared difference spectroscopies that has Trp-182, Tyr-185, Pro-186, and Trp-189 forming part of the binding pocket.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Unraveling the mechanism of proton translocation in the extracellular half‐channel of bacteriorhodopsin

Bacteriorhodopsin, a light activated protein that creates a proton gradient in halobacteria, has long served as a simple model of proton pumps. Within bacteriorhodopsin, several key sites undergo protonation changes during the photocycle, moving protons from the higher pH cytoplasm to the lower pH extracellular side. The mechanism underlying the long‐range proton translocation between the central (the retinal Schiff base SB216, D85, and D212) and exit clusters (E194 and E204) remains elusive. To obtain a dynamic view of the key factors controlling proton translocation, a systematic study using molecular dynamics simulation was performed for eight bacteriorhodopsin models varying in retinal isomer and protonation states of the SB216, D85, D212, and E204. The side‐chain orientation of R82 is determined primarily by the protonation states of the residues in the EC. The side‐chain reorientation of R82 modulates the hydrogen‐bond network and consequently possible pathways of proton transfer. Quantum mechanical intrinsic reaction coordinate calculations of proton‐transfer in the methyl guanidinium‐hydronium‐hydroxide model system show that proton transfer via a guanidinium group requires an initial geometry permitting proton donation and acceptance by the same amine. In all the bacteriorhodopsin models, R82 can form proton wires with both the CC and the EC connected by the same amine. Alternatively, rare proton wires for proton transfer from the CC to the EC without involving R82 were found in an O′ state where the proton on D85 is transferred to D212. Proteins 2016; 84:639–654. © 2016 Wiley Periodicals, Inc.     

Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin.

Structural changes are central to the mechanism of light-driven proton transport by bacteriorhodopsin, a seven-helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two-dimensional crystals of wild-type bacteriorhodopsin and the Asp96-->Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.     

The photochemical reaction cycle of proteorhodopsin at low pH

The proton acceptor group in the recently described retinal protein, proteorhodopsin has an unusually high pK(a) of 7.1. It was shown that at pH above this pK(a), illumination initiates a photocycle similar to that of bacteriorhodopsin, and the protein transports proton across the cell membrane. Recently it was reported that proteorhodopsin, unlike bacteriorhodopsin, transports protons at pH below the pK(a) of the proton acceptor, and this transport is in the reverse direction. We have investigated the photocycle of proteorhodopsin at such low pH. At pH 5, three spectrally distinct intermediates K, L, and N, and another spectrally silent one, PR', could be identified, but a deprotonated Schiff base containing M-like intermediate, characteristic for proton pumping activity, does not accumulate. All the reactions between the intermediates are close to equilibrium, except the last transition from PR' to PR, when the protein returns to its initial unexcited state in a quasiunidirectional reaction. The electric signal measurements indicate that although charge motions are detected inside the protein, their net dislocation is zero, indicating that contrary to the earlier reported, at low pH no charged particle is transported across the membrane.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Bacteriorhodopsin. Correspondence of the photocycle and electrogenesis with sites of the molecule

Correspondence of phases of electrogenesis, photocycle transitions, and proton transfer with the proton transporting groups of bacteriorhodopsin was studied. The structure of bacteriorhodopsin was considered by the file 1c3w and projections of sites of the proton movement pathway onto the normal to the purple membrane were measured. The dielectric permeability of the terminal site of the semichannel Schiff base external surface of the purple membrane was noticeably higher than in the center of the membrane.Translated from Biokhimiya, Vol. 69, No. 12, 2004, pp. 1725–1728.Original Russian Text Copyright © 2004 by Khitrina, Ksenofontov.     

Bacteriorhodopsin in ice. Accelerated proton transfer from the purple membrane surface

The photocycle and the proton pumping kinetics of bacteriorhodopsin, as well as the transfer rate of protons from the membrane surface into the aqueous bulk phase were examined for purple membranes in water and ice. In water, the optical pH indicator pyranine residing in the aqueous bulk phase monitors the H(+)-release later than the pH indicator fluorescein covalently linked to the extracellular surface of BR. In the frozen state, however, pyranine responds to the ejected H+ as fast as fluorescein attached to BR, demonstrating that the surface/bulk transfer is in ice no longer rate limiting. The pumped H+ appears at the extracellular surface during the transition of the photocycle intermediate L550 to the intermediate M412. The Arrhenius plot of the M formation rate suggests that the proton is translocated through the protein via an ice-like structure.     

Protonation reactions and their coupling in bacteriorhodopsin

Light-induced changes of the proton affinities of amino acid side groups are the driving force for proton translocation in bacteriorhodopsin. Recent progress in obtaining structures of bacteriorhodopsin and its intermediates with an increasingly higher resolution, together with functional studies utilizing mutant pigments and spectroscopic methods, have provided important information on the molecular architecture of the proton transfer pathways and the key groups involved in proton transport. In the present paper I consider mechanisms of light-induced proton release and uptake and intramolecular proton transport and mechanisms of modulation of proton affinities of key groups in the framework of these data. Special attention is given to some important aspects that have surfaced recently. These are the coupling of protonation states of groups involved in proton transport, the complex titration of the counterion to the Schiff base and its origin, the role of the transient protonation of buried groups in catalysis of the chromophore's thermal isomerization, and the relationship between proton affinities of the groups and the pH dependencies of the rate constants of the photocycle and proton transfer reactions.     

Photoelectric conversion by bacteriorhodopsin in charged synthetic membranes.

Photoelectroactivity of oriented purple membrane layers attached to an ion exchange film has been investigated. The action spectrum of the photocurrent followed the absorption spectrum of bacteriorhodopsin. The intactness of structure and function of bacteriorhodopsin was demonstrated by studies of absorption and photocycle kinetics. The direction of the photocurrent suggests that the extracellular surface of purple membrane is more positive. Photocurrents as high as 20 microA cm-2 were obtained in some preparations. The dependence of steady-state photocurrents on intensity of illumination and temperature was also studied. The initial rate of build-up of photocurrent depends linearly on the intensity of illumination while the off rate does not exhibit any dependence on the intensity of illumination. With rise in temperature an increase in the steady state photocurrent has been observed. This dependence was found to be linear when increase of the photocurrent due to proton translocation alone was considered.     

Suppression of the back proton-transfer from Asp85 to the retinal Schiff base in bacteriorhodopsin: a theoretical analysis of structural elements

The transfer of a proton from the retinal Schiff base to the nearby Asp85 protein group is an essential step in the directional proton-pumping by bacteriorhodopsin. To avoid the wasteful back reprotonation of the Schiff base from Asp85, the protein must ensure that, following Schiff base deprotonation, the energy barrier for back proton-transfer from Asp85 to the Schiff base is larger than that for proton-transfer from the Schiff base to Asp85. Here, three structural elements that may contribute to suppressing the back proton-transfer from Asp85 to the Schiff base are investigated: (i) retinal twisting; (ii) hydrogen-bonding distances in the active site; and (iii) the number and location of internal water molecules. The impact of the pattern of bond twisting on the retinal deprotonation energy is dissected by performing an extensive set of quantum-mechanical calculations. Structural rearrangements in the active site, such as changes of the Thr89:Asp85 distance and relocation of water molecules hydrogen-bonding to the Asp85 acceptor group, may participate in the mechanism which ensures that following the transfer of the Schiff base proton to Asp85 the protein proceeds with the subsequent photocycle steps, and not with back proton transfer from Asp85 to the Schiff base.