Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.

A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 µM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

Breaking the carboxyl rule: lysine 96 facilitates reprotonation of the Schiff base in the photocycle of a retinal protein from Exiguobacterium sibiricum.

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ε-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.

Photocycle of Exiguobacterium sibiricum rhodopsin characterized by low-temperature trapping in the IR and time-resolved studies in the visible.

The photocycle of the retinal protein from Exiguobacterium sibiricum, which differs from bacteriorhodopsin in both its primary donor and acceptor, is characterized by visible and infrared spectroscopy. At pH above pKa ~6.5, we find a bacteriorhodopsin-like photocycle, which originates from excitation of the all-trans retinal chromophore with K-, L-, M-, and N-like intermediates. At pH below pKa ~6.5, the M state, which reflects Schiff base deprotonation during proton pumping, is not accumulated. However, using the infrared band at ~1760 cm(-1) as a marker for transient protonation of the primary acceptor, we find that Schiff base deprotonation must have occurred at pH not only above but also below the pKa ~6.5. Thus, the M state is formed but not accumulated for kinetic reasons. Further, chromophore reisomerization from the 13-cis to the all-trans conformation occurs very late in the photocycle. The strongly red-shifted states that dominate the second half of the cycle are produced before the reisomerization step, and by this criterion, they are not O-like but rather N-like states. The assignment of photocycle intermediates enables reevaluation of the photocycle; its specific features are discussed in relation to the general mechanism of proton transport in retinal proteins.

Low-temperature FTIR study of multiple K intermediates in the photocycles of bacteriorhodopsin and xanthorhodopsin.

Low-temperature FTIR spectroscopy of bacteriorhodopsin and xanthorhodopsin was used to elucidate the number of K-like bathochromic states, their sequence, and their contributions to the photoequilibrium mixtures created by illumination at 80-180 K. We conclude that in bacteriorhodopsin the photocycle includes three distinct K-like states in the sequence bR (hv)--> I* --> J --> K(0) --> K(E) --> L --> ..., and similarly in xanthorhodopsin. K(0) is the main fraction in the mixture at 77 K that is formed from J. K(0) becomes thermally unstable above approximately 50 K in both proteins. At 77 K, both J-to-K(0) and K(0)-to-K(E) transitions occur and, contrarily to long-standing belief, cryogenic trapping at 77 K does not produce a pure K state but a mixture of the two states, K(0) and K(E), with contributions from K(E) of approximately 15 and approximately 10% in the two retinal proteins, respectively. Raising the temperature leads to increasing conversion of K(0) to K(E), and the two states coexist (without contamination from non-K-like states) in the 80-140 K range in bacteriorhodopsin, and in the 80-190 K range in xanthorhodopsin. Temperature perturbation experiments in these regions of coexistence revealed that, in spite of the observation of apparently stable mixtures of K(0) and K(E), the two states are not in thermally controlled equilibrium. The K(0)-to-K(E) transition is unidirectional, and the partial transformation to K(E) is due to distributed kinetics, which governs the photocycle dynamics at temperatures below approximately 245 K (Dioumaev and Lanyi, Biochemistry 2008, 47, 11125-11133). From spectral deconvolution, we conclude that the K(E) state, which is increasingly present at higher temperatures, is the same intermediate that is detected by time-resolved FTIR prior to its decay, on a time scale of hundreds of nanoseconds at ambient temperature (Dioumaev and Braiman, J. Phys. Chem. B 1997, 101, 1655-1662), into the K(L) state. We were unable to trap the latter separately from K(E) at low temperature, due to the slow distributed kinetics and the increasingly faster overlapping formation of the L state. Formation of the two consecutive K-like states in both proteins is accompanied by distortion of two different weakly bound water molecules: one in K(0), the other in K(E). The first, well-documented in bacteriorhodopsin at 77 K where K(0) dominates, was assigned to water 401 in bacteriorhodopsin. The other water molecule, whose participation has not been described previously, is disturbed on the next step of the photocycle, in K(E), in both proteins. In bacteriorhodopsin, the most likely candidate is water 407. However, unlike bacteriorhodopsin, the crystal structure of xanthorhodopsin lacks homologous weakly bound water molecules.

Two bathointermediates of the bacteriorhodopsin photocycle, from time-resolved nanosecond spectra in the visible.

Time-resolved measurements were performed on wild-type bacteriorhodopsin with an optical multichannel analyzer in the spectral range 350-735 nm, from 100 ns to the photocycle completion, at four temperatures in the 5-30 degrees C range. The intent was to examine the possibility of two K-like bathochromic intermediates and to obtain their spectra and kinetics in the visible. The existence of a second K-like intermediate, termed KL, had been postulated (Shichida et al., Biochim. Biophys. Acta 1983, 723, 240-246) to reconcile inconsistencies in data in the pico- and microsecond time domains. However, introduction of KL led to a controversy, since neither its visible spectrum nor its kinetics could be confirmed. Infrared data (Dioumaev and Braiman, J. Phys. Chem. B 1997, 101, 1655-1662) revealed a state which might have been considered a homologue to KL, but it had a kinetic pattern different from that of the earlier proposed KL. Here, we characterize two distinct K-like intermediates, K(E) ("early") and K(L) ("late"), by their spectra and kinetics in the visible as revealed by global kinetic analysis. The K(E)-to-K(L) transition has a time constant of approximately 250 ns at 20 degrees C, and describes a shift from K(E) with lambda(max) at approximately 600 nm and extinction of approximately 56,000 M(-1) x cm(-1) to K(L) with lambda(max) at approximately 590 nm and extinction of approximately 50,000 M(-1) x cm(-1). The temperature dependence of this transition is characterized by an enthalpy of activation of DeltaH(++) approximately 40 kJ/mol and a positive entropy of activation of DeltaS(++)/R approximately 4. The consequences of multiple K-like states for interpreting the spectral evolution in the early stages of the photocycle are discussed.

Infrared monitoring of interlayer water in stacks of purple membranes.

The thermodynamic behavior of films of hydrated purple membranes from Halobacterium salinarum and the water confined in it was studied by Fourier transform infrared spectroscopy in the 180-280 K range. Unlike bulk water, water in the thin layers sandwiched between the biological membranes does not freeze at 273 K but will be supercooled to approximately 256 K. The melting point is unaffected, leading to hysteresis between 250 and 273 K. In its heating branch, a gradually increasing light-scattering by ice is observed with rate-limiting kinetics of tens of minutes. Infrared (IR) spectra decomposition provided extinction coefficients for the confined water vibrational bands and their changes upon freezing. Because of the hysteresis, at any given temperature in the 255-270 K range, the interbilayer water could be either liquid or frozen, depending on thermal history. We find that this difference affects the dynamics of the bacteriorhodopsin photocycle in the hysteresis range: the decay of the M and N states and the redistribution between them are different depending on whether or not the water was initially precooled to below the freezing point. However, freezing of interbilayer water does block the M to N transition. Unlike the water, the purple membrane lipids do not undergo any IR-detectable phase transition in the 180-280 K range.

Switch from conventional to distributed kinetics in the bacteriorhodopsin photocycle.

Below 195 K, the bacteriorhodopsin photocycle could not be adequately described with exponential kinetics [Dioumaev, A. K., and Lanyi, J. K. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 9621-9626] but required distributed kinetics, previously found in hemoglobin and myoglobin at temperatures below the vitrification point of the surrounding solvent. The aim of this study is to determine which factors cause the switch from this low-temperature regime to the conventional kinetics observed at ambient temperature. The photocycle was monitored by time-resolved FTIR between 180 and 280 K, using the D96N mutant. Depending on the temperature, decay and temporal redistribution of two or three intermediates (L, M, and N) were observed. Above approximately 245 K, an abrupt change in the kinetic behavior of the photocycle takes place. It does not affect the intermediates present but greatly accelerates their decay. Below approximately 240 K, a kinetic pattern with partial decay that cannot be explained by conventional kinetics, but suggesting distributed kinetics, was dominant, while above approximately 250 K, there were no significant deviations from exponential behavior. The approximately 245 K critical point is >/=10 K below the freezing point of interbilayer water, and we were unable to correlate it with any FTIR-detectable transition of the lipids. Therefore, we attribute the change from distributed to conventional kinetics to a thermodynamic phase transition in the protein. Most probably, it is related to the freezing and thawing of internal fluctuations of the protein, known as the dynamic phase transition, although in bacteriorhodopsin the latter is usually believed to take place at least 15 K below the observed critical temperature of approximately 245 K.

Withdrawn: Role of Lipids in Dynamic Phase Transition: FTIR Spectroscopy of Retinal-Protein Containing Membranes.

Withdrawn.

Bacteriorhodopsin photocycle at cryogenic temperatures reveals distributed barriers of conformational substates.

The time course of thermal reactions after illumination of 100% humidified bacteriorhodopsin films was followed with FTIR spectroscopy between 125 and 195 K. We monitored the conversion of the initial photoproduct, K, to the next, L intermediate, and a shunt reaction of the L state directly back to the initial BR state. Both reactions can be described by either multiexponential kinetics, which would lead to apparent end-state mixtures that contain increasing amounts of the product, i.e., L or BR, with increasing temperature, or distributed kinetics. Conventional kinetic schemes that could account for the partial conversion require reversible reactions, branching, or parallel cycles. These possibilities were tested by producing K or L and monitoring their interconversion at a single temperature and by shifting the temperature upward or downward after an initial incubation and after their redistribution. The results are inconsistent with any conventional scheme. Instead, we attribute the partial conversions to the other alternative, distributed kinetics, observed previously in myoglobin, which arise from an ensemble of frozen conformational substates at the cryogenic temperatures. In this case, the time course of the reactions reflects the progressive depletion of distinct microscopic substates in the order of their increasing activation barriers, with a distribution width for K to L reaction of approximately 7 kJ/mol.

Selectivity of retinal photoisomerization in proteorhodopsin is controlled by aspartic acid 227.

Similarly to bacteriorhodopsin, proteorhodopsin that normally contains all-trans and 13-cis retinal is transformed at low pH to a species containing 9-cis retinal under continuous illumination at lambda > 530 nm. This species, absorbing around 430 nm, returns thermally in tens of minutes to initial pigment and can be reconverted also with blue-light illumination. The yield of the 9-cis species is negligibly small at neutral pH but increases manyfold (>100) at acid pH with a pK(a) of 2.6. This indicates that protonation of acidic group(s) alters the photoreaction pathway that leads normally to all-trans --> 13-cis isomerization. In the D97N mutant, in which one of the two acidic groups in the vicinity of the retinal Schiff base is not ionizable, the yield of 9-cis species at low pH shows a pH dependence similar to that in the wild-type but with a somewhat increased pK(a) of 3.3. In contrast to this relatively minor effect, replacement of the other acidic group, Asp227, with Asn results in a remarkable, more than 50-fold, increase in the yield of the light-induced formation of 9-cis species in the pH range 4-6. It appears that protonation of Asp227 at low pH is what causes the dramatic increase in the yield of the 9-cis species in wild-type proteorhodopsin. We conclude that the photoisomerization pathways in proteorhodopsin to 13-cis or 9-cis photoproducts are controlled by the charge state of Asp227.

Proton transport by proteorhodopsin requires that the retinal Schiff base counterion Asp-97 be anionic.

At pH >7, proteorhodopsin functions as an outward-directed proton pump in cell membranes, and Asp-97 and Glu-108, the homologues of the Asp-85 and Asp-96 in bacteriorhodopsin, are the proton acceptor and donor to the retinal Schiff base, respectively. It was reported, however [Friedrich, T. et al. (2002) J. Mol. Biol., 321, 821-838], that proteorhodopsin transports protons also at pH <7 where Asp-97 is protonated and in the direction reverse from that at higher pH. To explore the roles of Asp-97 and Glu-108 in the proposed pumping with variable vectoriality, we compared the photocycles of D97N and E108Q mutants, and the effects of azide on the photocycle of the E108Q mutant, at low and high pH. Unlike at high pH, at a pH low enough to protonate Asp-97 neither the mutations nor the effects of azide revealed evidence for the participation of the acidic residues in proton transfer, and as in the photocycle of the wild-type protein, no intermediate with unprotonated Schiff base accumulated. In view of these findings, and the doubts raised by absence of charge transfer after flash excitation at low pH, we revisited the question whether transport occurs at all under these conditions. In both oriented membrane fragments and liposomes reconstituted with proteorhodopsin, we found transport at high pH but not at low pH. Instead, proton transport activity followed the titration curve for Asp-97, with an apparent pK(a) of 7.1, and became zero at the pH where Asp-97 is fully protonated.

Proton transfers in the photochemical reaction cycle of proteorhodopsin.

The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were done in the visible and infrared (FTIR and FT-Raman). Assignment of the observed C=O stretch bands indicated that Asp-97 and Glu-108 serve as the proton acceptor and donor, respectively, to the retinal Schiff base, as do the residues at corresponding positions in BR, but there are numerous spectral and kinetic differences between the two proteins. There is no detectable dark-adaptation in PR, and the chromophore contains nearly entirely all-trans retinal. Because the pK(a) of Asp-97 is relatively high (7.1), the proton-transporting photocycle is produced only at alkaline pH. It contains at least seven transient states with decay times in the range from 10 micros to 200 ms, but the analysis reveals only three distinct spectral forms. The first is a red-shifted K-like state. Proton release does not occur during the very slow (several milliseconds) rise of the second, M-like, intermediate, consistent with lack of the residues facilitating extracellular proton release in BR. Proton uptake from the bulk, presumably on the cytoplasmic side, takes place prior to release (tau approximately 2 ms), and coincident with reprotonation of the retinal Schiff base. The intermediate produced by this process contains 13-cis retinal as does the N state of BR, but its absorption maximum is red-shifted relative to PR (like the O state of BR). The decay of this N-like state is coupled to reisomerization of the retinal to all-trans, and produces a state that is O-like in its C-C stretch bands, but has an absorption maximum apparently close to that of unphotolyzed PR.