Characterization of the proton-transporting photocycle of pharaonis halorhodopsin

The photocycle of pharaonis halorhodopsin was investigated in the presence of 100 mM NaN(3) and 1 M Na(2)SO(4). Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR' state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide.     

Charge motions during the photocycle of pharaonis halorhodopsin

Oriented gel samples were prepared from halorhodopsin-containing membranes from Natronobacterium pharaonis, and their photoelectric responses to laser flash excitation were measured at different chloride concentrations. The fast component of the current signal displayed a characteristic dependency on chloride concentration, and could be interpreted as a sum of two signals that correspond to the responses at high-chloride and no-chloride, but high-sulfate, concentration. The chloride concentration-dependent transition between the two signals followed the titration curve determined earlier from spectroscopic titration. The voltage signal was very similar to that reported by another group (Kalaidzidis, I. V., Y. L. Kalaidzidis, and A. D. Kaulen. 1998. FEBS Lett. 427:59-63). The absorption kinetics, measured at four wavelengths, fit the kinetic model we had proposed earlier. The calculated time-dependent concentrations of the intermediates were used to fit the voltage signal. Although no negative electric signal was observed at high chloride concentration, the calculated electrogenicity of the K intermediate was negative, and very similar to that of bacteriorhodopsin. The late photocycle intermediates (O, HR', and HR) had almost equal electrogenicities, explaining why no chloride-dependent time constant was identified earlier by Kalaidzidis et al. The calculated electrogenicities, and the spectroscopic information for the chloride release and uptake steps of the photocycle, suggest a mechanism for the chloride-translocation process in this pump.     

Effects of arginine modification on the photocycle of halorhodopsin

Exhaustive reaction with phenylglyoxal removed 9 of the 12 arginine and 1 of the 2 lysine residues in detergent-solubilized halorhodopsin, without affecting the chromophore. The consequences of this extensive removal of positive charges on various chloride-binding equilibria and the photochemistry were evaluated. No significant effects were seen on the affinity of Site I to chloride and on the increase in the pKa of Schiff-base deprotonation, which is caused by the chloride binding at this site. No significant effects were seen on the affinity of Site II to chloride, either. However, the photocycle of the pigment was affected. Kinetic modeling of the observed changes in flash-induced absorption changes suggests that the modification increases the affinity of the main halorhodopsin photointermediate to chloride by about fourfold. If chloride translocation involves release of chloride from this intermediate during the transport cycle, the result might explain the observed partial inhibitory effects on chloride transport. Plausible models of chloride translocation include reversible binding of the anion by positively charged groups, strategically arranged in the protein. The results indicate that two of the three spectroscopically observable chloride-dependent equilibria do not depend on a large number of positively charged residues in the protein. To the extent that the unaffected equilibria represent association and dissociation which occur during chloride translocation, at least part of the chloride translocation might be accomplished with the participation of only a few positively charged residues.     

Resonance Raman study of intermediates of the halorhodopsin photocycle

The resonance Raman (RR) study of the retinal protein halorhodopsin (HR₅₇₈) was extended to two of its photoproducts: HR and HR^L₄₁₀ RR spectra of both species were recorded in H₂O and D₂O and compared with the RR spectra of the intermediates L₅₅₀ and M₄₁₂ from the bacteriorhodopsin photocycle. HR₅₂₀ was found to be a protonated Schiff base in the 13-cis configuration and HR^L₄₁₀ a deprotonated Schiff base in the 13-cis configuration.     

Flash spectroscopic studies of the kinetics of the halorhodopsin photocycle

The photoreactions of halorhodopsin are complicated by the fact that the parent pigment and its photoproducts interact with chloride. Thus, in any photoreaction scheme at least four species have to be accounted for: HR565 and HR578 Cl-, as well as HR640 and HR520 Cl-. A photocycle scheme proposed earlier places the two main photointermediates of halorhodopsin, HR520 Cl- and HR640, into a single photocycle, with a chloride-dependent equilibrium between them [Oesterhelt, D., Hegemann, P., & Tittor, J. (1985) EMBO J. 4, 2351-2356]. This scheme, with the additional feature of direct photoproduction of HR640 from HR565, was tested in this work by using numerical solutions of the appropriate differential equations to simulate flash-induced absorption changes at 500 nm (production of HR520 Cl-) and at 660 nm (production of HR640). The time scale of the simulation was ms following the flash. Comparison of the simulated curves with experimental traces yielded a unique set of three rate constants. The proposed photocycle scheme and these rate constants predict well the shapes and amplitudes of flash traces at various chloride concentrations. It appears from the photocycle scheme, and the numerical values of rate constants, that chloride is bound with high affinity to the parent halorhodopsin molecule, but with much lower affinity to its main photointermediate. This may be the consequence of the fact that in the parent halorhodopsin in the retinal configuration is all-trans, but in the two photointermediates it is 13-cis.     

Halide dependence of the halorhodopsin photocycle as measured by time-resolved infrared spectra

Time-resolved Fourier transform infrared (FTIR) difference spectra of the halorhodopsin (hR) photocycle have been collected from 3 micros to 100 ms in saturating concentrations of KCl or KBr. Kinetic analysis of these data revealed two decay processes, with time constants of tau(1) approximately 150 micros and tau(2) approximately 16 ms in the presence of either halide, with tau(2) describing the return to the starting (hR) state. Comparison to previous low-temperature FTIR spectra of hR intermediates confirms that characteristic hK and hL spectral features are both present before the tau(1) decay, in a state previously defined as hK(L) (Dioumaev, A., and M. Braiman. 1997. Photochem. Photobiol. 66:755-763). However, the relative sizes of these features depend on which halide is present. In Br-, the hL features are clearly more dominant than in Cl-. Therefore, the state present before tau(1) is probably best described as an hK(L)/hL(1) equilibrium, instead of a single hK(L) state. Different halides affect the relative amounts of hK(L) and hL(1) present, i.e., Cl- produces a much more significant back-reaction from hL(1) to hK(L) than does Br-. The halide dependence of this back-reaction could therefore explain the halide selectivity of the halorhodopsin anion pump.     

Temperature and halide dependence of the photocycle of halorhodopsin from Natronobacterium pharaonis

The photocycle kinetics of halorhodopsin from Natronobacterium pharaonis (pHR(575)) was analyzed at different temperatures and chloride concentrations as well as various halides. Over the whole range of modified parameters the kinetics can be adequately modeled with six apparent rate constants. Assuming a model in which the observed rates are assigned to irreversible transitions of a single relaxation chain, six kinetically distinguishable states (P(1-6)) are discernible that are formed from four chromophore states (spectral archetypes S(j): K(570), L(N)(520), O(600), pHR'(575)). Whereas P(1) coincides with K(570) (S(1)), both P(2) and P(3) have identical spectra resembling L(520) (S(2)), thus representing a true spectral silent transition between them. P(4) constitutes a fast temperature-dependent equilibrium between the chromophore states S(2) and S(3) (L(520) and O(600), respectively). The subsequent equilibrium (P(5)) of the same spectral archetypes is only moderately temperature dependent but shows sensitivity toward the type of anion and the chloride concentration. Therefore, S(2) and S(3) occurring in P(4) as well as in P(5) have to be distinguished and are assigned to L(520)<--> O(1)(600) and O(2)(600)<--> N(520) equilibrium, respectively. It is proposed that P(4) and P(5) represent the anion release and uptake steps. Based on the experimental data affinities of the halide binding sites are estimated.     

Low-temperature photoreactions of halorhodopsin. 2. Description of the photocycle and its intermediates

Photostationary states of halorhodopsin (HR, a retinal protein in the halobacterial membrane) and their further thermal conversions were investigated at 140-230 K by absorption spectroscopy in the visible. The difference spectra confirm several steps of the all-trans-HR photocycle, in the presence of chloride, proposed earlier on the basis of room temperature flash spectroscopy. Thus, at 140 K, the spectra reveal the HR600----HR520 reaction, and at 170-230 K the HR640----HR578 and the HR520----HR578 reactions can be seen. No evidence for the expected HR520 in equilibrium HR640 process was found, however. From the difference spectra at various temperatures, exact absorption spectra of HR600 and HR520 were calculated, and an estimate of the HR640 spectrum in a mixture also containing HR520 was obtained. The low-temperature absorption maxima of HR578 and its photointermediates relate to the room temperature maxima differently from what is expected from the spectra of the corresponding intermediates in the bacteriorhodopsin photocycle.     

Cl(-) concentration dependence of photovoltage generation by halorhodopsin from Halobacterium salinarum

The photovoltage generation by halorhodopsin from Halobacterium salinarum (shR) was examined by adsorbing shR-containing membranes onto a thin polymer film. The photovoltage consisted of two major components: one with a sub-millisecond range time constant and the other with a millisecond range time constant with different amplitudes, as previously reported. These components exhibited different Cl(-) concentration dependencies (0.1-9 M). We found that the time constant for the fast component was relatively independent of the Cl(-) concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl(-) concentrations. The fast and the slow processes were attributed to charge (Cl(-)) movements within the protein and related to Cl(-) ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that they corresponded to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl(-) concentration dependence, and the Cl(-) concentration dependence of its time constant suggested a weak and highly cooperative Cl(-)-binding site(s) on the cytoplasmic side (apparent K(D) of approximately 5 M and Hill coefficient > or =5). The Cl(-) concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be >100 ms.     

Effect of anions on the photocycle of halorhodopsin. Substitution of chloride with formate anion

Halorhodopsin from Natronomonas pharaonis is a light-driven chloride pump which transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. It was shown that the chloride anion bound in the vicinity of retinal PSB can be replaced by several inorganic anions, including azide which converts the chloride pump into a proton pump and induces formation of an M-like intermediate detected in the bR photocycle but not in native halorhodopsin. Here we have studied the possibility of replacing the chloride anion with organic anions and have followed the photocycle under several conditions. It is revealed that the chloride can be replaced with a formate anion but not with larger organic anions such as acetate. Flash photolysis experiments detected in the formate pigment an M-like intermediate characterized by a lifetime much longer than that of the O intermediate. The lifetime of the M-like intermediate depends on the pH, and its decay is significantly accelerated at low pH. The decay rate exhibited a titration-like curve, suggesting that the protonation of a protein residue controls the rate of M decay. Similar behavior was detected in N. pharaonis pigments in which the chloride anion was replaced with NO(2)(-) and OCN(-) anions. It is suggested that the formation of the M-like intermediate indicates branching pathways from the L intermediate or basic heterogeneity in the original pigment.     

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.     

Roles of Ser130 and Thr126 in chloride binding and photocycle of pharaonis halorhodopsin

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump in Natronobacterium pharaonis. In order to clarify the roles of the Ser130(phR) and Thr126(phR) residues, which correspond to Ser115(shR) and Thr111(shR) of salinarum hR (shR), with regard to their Cl(-)binding affinity and the photocycle, the wild-type phR, and S130 and T126 mutants were expressed in Escherichia coli cells. The photocycles of the wild-type phR, and S130 and T126 mutants were investigated in the presence of 1 M NaCl. Based on results of kinetic analysis involving singular value decomposition and global fitting, typical photointermediates K, L and O were identified, and the kinetic constants of decay or formation varied depending on the mutant. The photocycle scheme was linear for the wild-type phR, and S130C, S130T and T126V mutants. On the other hand, the S130A mutant showed a branched pathway between the L-hR and L-O steps. The present study revealed the following two facts with respect to the Ser130(phR) residue: 1) The OH group of this residue is important for Cl(-) ion binding next to the Schiff base nitrogen, and 2) replacement of an Ala residue, which is unable to form a hydrogen bond, results in a branched photocycle. The implication of this branching was discussed.     

Resonance Raman study of halorhodopsin photocycle kinetics, chromophore structure, and chloride-pumping mechanism.

Kinetic resonance Raman spectra of the HR520, HR640, and HR578 species in the halorhodopsin photocycle are obtained using time delays ranging from 5 microseconds to 10 ms in 0.3 M NO3-, 0.3 M Cl-, and 3 M Cl-. The Raman intensities are converted to absolute concentrations by using a conservation of molecules constraint. The simplest kinetic scheme that satisfactorily models the data is HR578-->HR520 in equilibrium with HR640-->HR578. The rate constant for the HR640-->HR578 transition increases with Cl- concentration, suggesting that Cl- is taken up between HR640 and HR578. The ratio of the forward to the reverse rate constants connecting HR520 and HR640 increases as the inverse of the Cl- concentration, suggesting that Cl- is released during the HR520-->HR640 step. The configuration about the C13 = C14 bond of the retinal chromophore in HR640 is examined by regenerating the protein with [12,14-2H2]retinal. The C12-2H + C14-2H rocking vibration for HR640 is observed at 943 cm-1, demonstrating that the chromophore is 13-cis. The changes in the resonance Raman spectrum of HR640 in response to 2H2O suspension indicates that the Schiff base linkage to the protein is protonated. None of the HR640 fingerprint vibrations shift significantly in 2H2O, suggesting that the Schiff base adopts a C = N anti configuration; this assignment is supported by the frequency of the C15-2H rocking mode (1002 cm-1). The 13-cis structure for the chromophore in HR640 requires that thermal isomerization back to all-trans occurs in the HR640-->HR578 transition. These structural and kinetic results are incorporated into a two-state C-T model for Cl- pumping.     

Expression of salinarum halorhodopsin in Escherichia coli cells: solubilization in the presence of retinal yields the natural state

Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all-trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3-4 mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes.     

Effect of salt on photocycle and ion-pumping of halorhodopsin and third rhodopsinlike pigment of Halobacterium halobium

The cytoplasmic membranes of Halobacterium halobium contain at least three retinal pigments: bacteriorhodopsin (bR), halorhodopsin (hR), and a third rhodopsinlike pigment (tR). The amplitudes of the phototransient in the photolysis of hR and tR were measured in various salt solutions. Halogen ion (except fluoride) was required to retain the photocycle of hR. Parallels between the amplitude of the phototransient of hR and the magnitude of the photo-induced tetraphenylphosphonium (TPP+) uptake suggests that hR is a light-driven halogen pump, which supports the hypothesis by Schobert and Lanyi (J. Biol. Chem., 1982, 257:10306-10313). The order of effectiveness of halogen was Br- greater than Cl- greater than I-. On the other hand, no specific ion was required to retain the photocycle of tR, and tR was concluded to be nonelectrogenic.     

Activation parameters for the halorhodopsin photocycle: a phase lifetime spectroscopic study of the 520- and 640-nanometer intermediates

Phase lifetime spectroscopy is used to investigate the kinetics of the 520- and 640-nm intermediates in the halorhodopsin photocycle. These intermediates decay on the millisecond time scale and are strongly implicated in the chloride transport steps. The temperature dependence of the 520 and 640 relaxations was measured for chloride and nitrate buffers at pH 6, 7, and 8 and for iodide buffer at pH 6. The 640 relaxations have small activation energies but large entropy barriers. The two relaxation times observed for the 640 intermediate were interpreted by using a mechanism in which two 640 species exist in equilibrium. The second 640 species is not along the main decay path for the photocycle. A quantitative analysis of the data allowed rate constants and activation parameters to be calculated for the elementary steps of this isomerization process. These parameters are similar for both chloride and nitrate buffers but differ somewhat in iodide. The derived calculated rate constants were consistent with the relaxation times observed for the 520 intermediate. These results indicate that the 520 and two 640 intermediates have very similar free energies as well as similar free energies of activation for the various interconversion processes.     

Transient spectroscopy of bacterial rhodopsins with an optical multichannel analyzer. 2. Effects of anions on the halorhodopsin photocycle

We find that the photocycle of halorhodopsin (HR) in the presence of nitrate (but not chloride) consists of two parallel series of reactions. The first is essentially the same as that which occurs in the presence of chloride: HRhv----HRK----HRKL----HRL----HRO----HR. The second photocycle, however, which we describe as HRhv----HR'K----HRKO----HRO----HR, seems characteristic of what one would observe in the absence of chloride. Absorption spectra are calculated for all species but HRK and HR'K, which occur at shorter times (less than 60 ns) than we can resolve. At nitrate concentrations between 0.1 and 1 M, the proportion of HR which enters the first kind of photocycle increases in such a way as to suggest that nitrate can substitute for chloride, but much less effectively. At lower anion concentrations, the two photocycles are independent of one another, but at higher concentrations, they interact; i.e., the reaction HRKO----HRO----HRL can be observed. Thus, HRO must be common to the two photocycles. Kinetic fitting of the time dependence of HRL and HRO at different chloride concentrations provides evidence for the participation of chloride in the interconversion of HRL and HRO. The results are consistent with a model in which the photoreaction is influenced by the binding of an anion (either chloride or nitrate) to site II in HR: when an anion is bound, the HRK-initiated HRL-type photocycle is observed, but when the site is not occupied, the HR'K-initiated HRO-type photocycle is seen.     

Stopped-flow analysis on anion binding to blue-form halorhodopsin from Natronobacterium pharaonis: comparison with the anion-uptake process during the photocycle

Pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells. The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column. Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore. Addition of Cl- converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)). Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers. The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N3-, which was the same as in the case of phR purified from N. pharaonis membranes. Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing. Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes. The rate constant of the fast component at 100 mM NaCl at 25 degrees C was 260 s(-1) with an apparent activation energy of 35 kJ/mol. These values are in good agreement with the process of Cl- uptake in the photocycle (O --> hR' reaction) reported previously [Váró et al. (1995) Biochemistry 34, 14500-14507]. In addition, the Cl- concentration dependence on both rates was similar to each other. These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process.     

Spectroscopic Characterization of Two Soluble Transducers from the Archaeon Halobacterium salinarum

In the present study, structural aspects of the two soluble transducers, HtrX and HtrXI, from the archaeon H. salinarum have been examined using UV circular dichroism and steady-state fluorescence spectroscopies. Circular dichroism (CD) data indicate that both HtrX and HtrXI exhibit salt-dependent protein folding. Under low-ionic-strength conditions (0.2 M NaCl or KCl) the CD spectra of HtrXI is similar to that of the Gdn-HCl- or urea-denatured forms and is indicative of random coil structure. In contrast, the CD spectrum of HtrX under low-ionic-strength conditions contains roughly 85% α-helical character, indicating a significant degree of folding. Addition of NaCl or KCl to solutions of HtrX or HtrXI results in CD features consistent with predominately α-helical character (>95%) for both proteins. In addition, the transition points (i.e., ionic strengths at which the protein converts from random coil to α-helical character) are quite distinct and dependent upon the type of salt present (i.e., either NaCl or KCl). Accessibility of tryptophan residues to the solvent was also examined for both HtrX and HtrXI in both folded and unfolded states using Kl quenching. The Stern–Volmer constants obtained suggest that the tryptophans (Trp35 in HtrX and both Trp47 and Trp74 in HtrXI) are partially exposed to the solvent, indicating that they are located near the surface of the protein in all three cases. Furthermore, fluorescence quenching with the single Trp mutants Trp74AIa and Trp47AIa of HtrXI indicates different environments for these two residues.