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Membrane Independence of Ultrafast Photochemistry in Pharaonis Halorhodopsin; Testing the Role of Bacterioruberin Itay Gdor, Maya Mani-Hazan, Noga Friedman, Mordechai Sheves, and Sanford Ruhman J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b12698 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on March 1, 2017

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Membrane Independence of Ultrafast Photochemistry in Pharaonis Halorhodopsin; Testing the Role of Bacterioruberin Itay Gdor1, Maya Mani-Hazan2, Noga Friedman2, Mordechai Sheves2, and Sanford Ruhman1* 1) Institute of Chemistry and the Farkas Center for Light Induced Processes, the Hebrew University, Jerusalem 91904, Israel. 2) Department of Organic Chemistry, the Weizmann Institute of Science, Rehovot 76100, Israel.

Abstract Ultrafast photochemistry of p-HR in the intact Natronomonas pharaonic membrane has been studied by photoselective femtosecond pump-hyperspectral probe spectroscopy with high time resolution. Two variants of this sample, one with wild type retinal prosthetic groups, and another after shifting retinal absorption deep into the blue by reducing the Schiff base linkage were studied, and compared with previous results on detergent solubilized p-HR. This comparison shows that retinal photo-isomerization dynamics are identical in the membrane and in the solubilized sample. Selective photoexcitation of bacterioruberin which associates to the protein in the native membrane, in wild type and reduced samples, demonstrates conclusively that unlike associated carotenoids in some bacterial retinal proteins, the carotenoid in p-HR does not act as a light harvesting antenna.

*

Corresponding author [email protected] Tel: (+972-2) 65-86322

ACS Paragon Plus Environment


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Introduction Halorhodopsin (p-HR) is a membranal protein that acts as a light-driven chloride pump in the halophilic archaeon Natronomonas pharaonis which was first discovered in African salt lakes.1,2 Its function is energized by photoisomerization of a retinal chromophore covalently bound to a lysine residue to form a protonated Schiff base (PSB) linkage. As in other archaeal rhodopsins, this photoisomerization leads from an initial all-trans resting state to a 13-cis configuration.3,4,5 This process, which takes place within a few picoseconds, triggers a millisecond scale cyclic transformation which pumps a single chloride ion from the extracellular medium to the cytoplasm.6,7,8 To characterize this rapid photchemistry, subpicosecond transient absorption measurements were carried out by Arlt et al. revealing an initial relaxation phase of 170 ± 70 fs assigned to retinal motions on the excited-state potential surface, followed by biexponential excited electronic state decay with time constants of 1.5 and 8.5 ps.9 More recently, following research performed by Shibata et al., studying the N-D stretching frequency of the (deuterated) Schiff base containing different halide ions,10 Nakamura et al. employed femtosecond pump-probe experiments to record primary events in p-HR upon light absorption, monitoring the dependence of photo-isomerization dynamics on the halide ion being pumped. Their results reveal that the activation barrier height in the excited state is affected by the interaction between the Schiff base and the halide ion, resulting in different isomerization times and quantum yields for K product generation.11 In addition to, they suggested a model to explain the bi-exponential picosecond internal conversion (IC) kinetics in this protein. They proposed that the two IC decay times represent distinct excited state populations, one of which is a photocycle intermediate, the other leading back to the reactant state. This hypothesis was investigated by Bismuth et al.12 using femtosecond visible pumpnear IR dump-hyperspectral probe spectroscopy. Results showed that dumping produces a


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proportionate reduction in photocycle yields throughout the fluorescent state lifetime, and led to the conclusion that despite its nonexponential IC kinetics, the fluorescent state in HR constitutes a single intermediate in the photocycle see p-HR photoreaction scheme 1.

Scheme 1. Model for potential energy surfaces and mechanisms of the initial photoisomerization process of p-HR.

The cited studies were conducted on free protein solubilized in detergent and not on the native membrane where p-HR assumes a close-packed trimeric structure.13 The importance of such oligomeric packing, common to other retinal ion pumps, has fascinated photo-biologists for decades.14,15,16 Thermal stability of bacteriorhodopsin (BR) has been shown to change markedly going from the native membrane to a detergent stabilized state.17,18 Pronounced circular dichroism of the retinal PSB absorption in native ion pump membranes, the most studied being that of BR, was suggested by some to result from strong interactions between the transition dipoles of the trimer constituent proteins.19,20,21 In turn this has led to investigations into cooperative photochemistry of the associated proteins.22,23,24,25 In the case of BR the impact of this packing on the rate of photo-isomerization was also studied using femtosecond transient absorption spectroscopy, and shown to have negligible influence on isomerization rates.26 Photoselective ultrafast studies have shown that energy transfer between trimerically bound proteins is negligible as well, questioning the assignment of CD signatures in purple membrane absorption to excitonic interactions within BR trimers.27,28



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It was recently demonstrated that the Natronomonas pharaonis halorhodopsinoverproducing mutant strain KM-1 contains, in addition to the retinal chromophore, a lipid soluble chromophore, bacterioruberin (a C40 carotenoid (CAR)) which binds to crevices between adjacent protein subunits.13,29 In principle, embedding in the native membrane could affect p-HR photo-isomerization dynamics significantly since trimers of HR are closely associated with the bacterioruberin molecules. Such CAR molecules have been shown to function as light harvesting antennas in some retinal proton pumps coming from bacteria such as Xanthrhodopsin active in Salinibacter ruber.30,31,32 Due to the CAR position and orientation with respect to the retinal chromophore in pHR it is not clear whether it serves as an antenna in this protein. However, due to the perturbation of the bacterioruberin absorption spectra upon the chloride binding it was suggested that the CAR absorption is affected by protein conformation and/or electrostatic perturbations.33,13 In order to test how the membrane environment influences retinal photochemistry in p-HR, including the participation of bacterioruberin in light harvesting, ultrafast transient absorption measurements were conducted after selective excitation of both chromophores. This was performed on the native protein in the mutant KM-1 membrane, and on a modified sample where the protonated Schiff base C N bond has been reduced to a single bond (R-p-HR), shifting the retinal absorption to the blue and out of range for electronic energy transfer. A comparison of results with data obtained in a similar fashion from p-HR solubalized in detergent indicates that despite the close packed trimers and the existence of additional chromophores, inclusion in the membrane does not alter photochemical kinetics in p-HR, and that bacterioruberin does not act as a light harvesting antenna as salinixanthin does in xanthorhodopsin.

Experimental


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p-HR and R-p-HR membrane preparation The KM-1 strain of N. pharaonis was grown in a culture medium (pH 9) as described by Ihara et al.13 The p-HR membranes were isolated using a reported protocol, washed with 100 mM NaCl, suspended in 100 mM NaCl and subjected to several rounds of centrifugation. The reduced sample was prepared as follows. To a solution of native p-HR membranes (50 mM Tris buffer, pH = 8.4, 300 mM NaCl, 30% sucrose), 0.12 M of NaBH4 were added. The reduction process was carried out using illumination for 1 h. The light was filtered through DDW (to prevent over heating of the sample) and a long pass cut-off filter λmax > 550 nm (Schott, Mainz, Germany). The sample was dialyzed against 0.1 M NaCl, to remove NaBH4. Pump–Probe Experiments 30 fs pulses at 790 nm were derived from a homemade multi-pass amplified Ti:Sapph laser system. A few µJ were used to generate a multi-filament white light continuum probe in sapphire, excess energy power ca. 780 nm were removed by spatial obstruction. The continuum pulses were collimated and re-focused into the sample with reflective optics. The remaining fundamental was used to pump a TOPAS (Light Conversion) and produce ~30 fs pump pulses centered at 505 nm by mixing signal with amplifier fundamental and at 600 nm by doubling the signal in 0.25 mm BBO. The pump chirp was compensated in a slightly misaligned zero dispersion grating pulse shaper. The resulting pulse was focused in a 0.4 mm flow cell equipped with 0.15 mm glass windows. Probe pulses were focused to a spot of 0.1 mm, and the pump beam diameters at least twice that. The sample was continuously pumped through the cell at a rate which ensured replenishment between excitations. Probe and reference pulses were directed through fibers to a double diode array spectrograph setup. Data were collected in vertical-vertical polarization conformation unless specified otherwise. Kerr scans in water provided the probe’s wavelength dependent group delay, and indicated a



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pump–probe cross-correlation of ~70 fs throughout the probed range. After collection, the resulting spectra were corrected for group delay dispersion of the probe continuum.

Results and Discussion The ground-state absorption spectra of KM-1 strain p-HR and R-p-HR, membranes along with the intensity spectra of pump pulses employed in our experiments are shown in Figure 1. The absorption spectrum of p-HR comprises bands from both constituent chromophores, with retinal contributing a broad shoulder at ~570 nm due to the allowed S0 to S1 absorption which both harvests the solar energy and processes it via photoisomerization.7,6 This band partially overlaps the strong and more structured S0 to S2 bacterioruberin spectrum in the 400–550 nm range, with vibronic bands at 470, 505, and 540 nm. The central wavelength of 505 nm was chosen for strong resonance with the bacterioruberin chromophores’ absorption, and minimal simultaneous contributions from retinal. Due to spectral overlap, direct absorption of the retinal is unavoidable, and from the spectrum it is estimated to contribute ca. 15% of the absorbed photons. HR abstorption HR-RED pump 505 nm pump 600 nm

OD(a.u.)

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λ nm Figure 1: Ground-state absorption spectra of wild type p-HR membrane and R-p-HR membrane along with pump intensity spectra of the TOPAS excitation pumps.

600 nm (Retinal excitation)


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λ(nm) Figure 2: Spectral evolution after excitation at 600 nm presented as temporal cuts taken at the designated delays.

The spectral evolution following pumping of the HR sample at 600 nm is presented as a sequence of transient difference absorption spectra in Fig. 2. Upon excitation, the blue side of the spectrum is dominated by the S1 absorption band centered at 475 nm. This band partially overlaps the strong excess transmission induced by S0 bleaching. On the red side of the ground state bleach, a double humped stimulated emission band attributed to overlapping emission and absorption bands of S1 rises concurrently. All of these features decay within 10 ps, leaving the longer lived “K” intermediate's difference spectrum reflecting a mild red shift of absorption relative to the ground state.6,34 To compare the retinal dynamics in the solubilized sample with that of the protein in the intact membrane, Fig. 3 presents spectral cuts at the peak of the S0 bleaching (560 nm) and at the peak of stimulated emission. The results reveal that, as in the case of BR, inclusion in the membrane has virtually no effect on the resulting dynamics, as can be seen also in Figure S1 of the supporting information. The emission in both cases fits adequately to a bi-exponential decay with identical decay times (1.9 and 5.6 ps) and relative amplitudes (0.05 and 0.02 respectively) for both samples.



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560 nm

0

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Time ps Figure 3: Spectral cuts comparing retinal dynamics in the p-HR membranes (red squares) and isolated in detergent (black line) following photoexcitation in the retinal band at 600 nm. The upper panel probes at the peak of excited state emission, while that below pertains to the maximum of the ground state bleach.

Even the vibrational coherences in low frequency retinal torsions, which is the most likely to be affected by protein surroundings, seems to be indifferent to the presence or absence of the native membrane. As seen in Fig. S2 in the supporting information, clear vibrational modulations are apparent superimposed on the emission decay with the same 90 cm-1 frequency measured in the previous study on the isolated protein.12

505 nm (Bacterioruberin Excitation)


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Figure 4: Spectral evolution after excitation at 505 nm presented as temporal cuts at the designated pump-probe delays. Left panels present the evolution of the untreated HR membrane, while the right panels present the reduced form.

Transient difference spectra obtained after 505 nm excitation from p-HR and reduced p-HR membranes are presented in Fig. 4 as sequences of temporal cuts, at delays which are detailed in the legend. Early and late delays are presented in the upper and lower rows of the figure, respectively, with a column for each sample. Immediately following photo absorption, increased transmission is observed in both samples, from the blue edge of the probing range, to ~650 nm. Above this wavelength, weak absorption rises rapidly, extending to the red edge of the observation window. These features are the expected signature of the CAR's S2 excited state that decays rapidly (~150 fs),35 leaving increased transmission in the range where bacterioruberin S0 absorbs to S2, and a strong and sharply peaked excited state absorption at 610 nm. The latter attributed to the S1→Sn transition in the carotenoid evolves spectrally, and decays onward within picoseconds primarily back to the ground state. In the case of the reduced sample, this decay leads to an effective erasure of the difference spectrum, while in the untreated sample a weak residual spectrum is observed (see Fig. S3). This is assigned to residual "K" photoproduct resulting from direct retinal excitation. Most importantly, the



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structure and intensity of transient spectra and decay times indigenous to the bacterioruberin are identical in both samples. 0

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WT Reduced -16

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Figure 5: Carotenoid excited state kinetics measured at 540 nm (ground state bleach) and 610 (S1 absorption) in the native membrane (red line) and in the reduced form (black squares). The gradual rise of the S1 absorption is demonstrated by the inset to the lower panel, which depicts the first 750 fs of delay.

Fig. 5 presents a comparison of the transient absorption kinetics following direct photoexcitation of the CAR in the intact membrane and in the reduced sample in the form of spectral cuts at the peak of the S0 bleach (540 nm) and at that of S1 absorption (610 nm). The results reveal that the presence of the retinal does not affect the kinetics of internal conversion from S2 to S1 of bacterioruberin. Photo-selective pump-probe measurements were performed on the untreated p-HR sample in order to verify that indeed, unlike the XR protein, no energy transfer takes place from S2 to retinal. Assuming such energy transfer does take place two channels would contribute to the retinal activation and isomerization. The first is direct retinal photoexcitation, the other being retinal activation through resonance energy transfer from S2 of bacterioruberin. The contribution of direct retinal excitation to photoproduct absorption anisotropy is 0.4 (the dipole moment for the photoproduct absorption is oriented along the retinal backbone, as is that of the ground state).36,37,38 In contrast, that arising from CAR absorption followed by energy transfer depends on the angle


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between the two chromophores, and is almost zero for nearly orthogonal transition dipoles. As can be seen in Figure S3, in the supporting information, the ratio between the VV and VH "K" state transient absorption is equal to 3 giving an anisotropy of 0.4, providing there is no contribution of energy transfer to the retinal activation. In order to investigate the excited-state dynamics of the CAR, global fitting analysis was performed, including convolution with an experimentally determined Gaussian instrument response function, and five phases of exponential spectral evolution. Both data sets were fit to a kinetic scheme summarized as ܵ଴ ← ܵ ∗ ← ࡿ૛ → ܵଵு → ܵଵ஼ → ܵ଴ , where S1H and S1C refer to vibrationally hot and relaxed S1 intermediates leading to species associated difference spectra (SADS) presented in figure 6. The model used is present on the right (branched reaction mechanism) referring the reader to recent discussions of low-lying dark excited states in large CARs.39,40 In a nutshell, after excitation to the bright S2 energy level, the energy flows toward two possible excited dark states, S1 and S*.41 The identification, while there is no debate regarding the nature of S1, S* is more controversial, as, in the past, it was also associated with hot ground state, due to its location, to the red of the main absorption.42 In a previous publication, regarding XR protein,32 anisotropy measurements revealed an angle between the S* dipole moment and the ground state dipole moment (similar to S1) which we believe confirm our assignment as an excited state. From those two dark states, the energy flows back to the ground state, as seen in the scheme in figure 6. It entails branching through IC of the directly accessed S2 singlet to two lower lying states coined S1 and S* (1/k1=80±14 fs and 1/k*=180±22

fs). The former is assumed to

evolve spectrally in a fraction of a picosecond (1/k2=200±18 fs) assigned to a period of vibrational cooling, and convert to S0 within a few picoseconds (1/k3=1.5±0.1 ps). The latter also repopulates the ground state more slowly (1/k4=4.8±0.3 ps). SADS obtained from R-pHR sample results in essentially identical time constants (1/k1=78±15 1/k*=185±16 fs,



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1/k2=195±18 fs, 1/k3==1.4±0.2 ps, and 1/k4=4.9±0.2 ps) associated with nearly identical spectra. The only spectrum which is significantly different in amplitude, relates to the extremely short lived S2 state of the bacterioruberin, most likely reflecting difficulties in perfect deconvolution from the instrument response function. Notice that for all others the spectra match not only in relaxation times but also in amplitude. This similarity provides independent evidence that the CAR molecules which attach to p-HR trimers in the membrane play no role in light harvesting. Reduced

W.T.

S2 S1H

Kinetic Scheme

S*

S2 k1

k*

S1C

∆OD

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λ nm Figure 6: Species associated difference spectra extracted by target analysis using the kinetic scheme on the right from reduced and wild type p-HR as designated.

The absence of detectable light harvesting by bacterioruberin in the native cell membrane is not surprising, given the reported orientation of the chromophores in the trimer structure.29 Both the distance of centers and angle between the molecular axes which define the interaction energies are unfavorable. Directly demonstrating this is important in view of the growing awareness of prevalent tight co-assembly of carotenoids with microbial RPs in their natural membranes.43,44 The pump-probe data also dismiss the notion of strong membrane immersion effects on the course of photochemistry in this protein. One aspect of this has however not been completely explored here. The experiments on solubilized p-HR were conducted in our labs by Bismuth et.al.12 CD spectroscopy studies have shown that this


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protein has a propensity for self-assembly in homo-trimers even in the solubilized state,45,46 given the right detergent environment. It is thus possible that at least part of the detergent solubilized proteins were so aggregated. Figuring this out will require a separate dedicated study. Despite this missing link, this study contributes to a growing appreciation that the key factors governing photoisomerization kinetics in retinal proteins are to be found within the protein surroundings, and that early suggestions that specific interactions with the membrane constituents are incorrect. This is not to say that later stages of the photocycle are not significantly affected, but that the initial stage of ultrafast photochemistry is not significantly influenced. This is logical since on the timescale of retinal isomerization even parts of the opsin itself beyond the retinal binding site will not have time to react dynamically to the ongoing photochemistry. In contrast later stages of the photocycle involve broad rearrangement of the protein, making them susceptible to influences from the membrane matrix or detergent surroundings.

Conclusions This work presents an ultrafast photochemistry study of p-HR in the intact membrane by photoselective femtosecond pump-hyperspectral probe spectroscopy with high time resolution. These experiments were conducted on two variants of the protein; wild type retinal prosthetic groups, and the protein's reduced form, and were compared with our previous results on detergent solubilized p-HR. Results show that retinal isomerization takes place with identical kinetics in the membrane and in the solubilized sample. Selective photoexcitation of the bacterioruberin carotene, which associates to the protein in the native membrane, in wild type and reduced samples, were fitted to a model using global fit analysis, demonstrates indisputably that unlike in the XR case, the carotenoid does not act as a light harvesting antenna in this protein.



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Acknowledgment This work was supported by the James Franck program for laser matter interaction, and by the Israel Science Foundation (ISF). The James Franck program is supported by the Minerva Gesellschaft, GmbH, Munich, Germany. The ISF is administered by the Israel Academy of Sciences and the Humanities and the Lester Aronberg in Chemistry at the Hebrew University.

Supporting Information. Comparison of the Brief retinal emission dynamics, both in the native protein in membrane and isolated protein in detergent, measurement of the S1 state oscillation of the pHR at two probing wavelengths, and K product spectra measured at different pump-probe polarization geometries after excitation of HR at 505nm are supplied as Supporting Information.

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