Photoreactions of the histidine kinase rhodopsin Ot-HKR from the

4 days ago - The tiny picoalga, Ostreococcus tauri, originating from the Thau Lagoon is a member of the marine phytoplankton. Because of its highly ...
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Photoreactions of the histidine kinase rhodopsin OtHKR from the marine picoalga Ostreococcus tauri Meike Luck, Francisco Velázquez Escobar, Kathrin Glass, Mareike-Isabel Sabotke, Rolf Hagedorn, Florence Corellou, Friedrich Siebert, Peter Hildebrandt, and Peter Hegemann Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01200 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

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Biochemistry

Photoreactions of the histidine kinase rhodopsin Ot-HKR from the marine picoalga Ostreococcus tauri Meike Luck1*, Francisco Velázquez Escobar2, Kathrin Glass1, Mareike-Isabel Sabotke1, Rolf Hagedorn1, Florence Corellou3, Friedrich Siebert2,4, Peter Hildebrandt2 and Peter Hegemann1 From the 1Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany; 2Institute of Chemistry, Technische Universität Berlin, 10623 Berlin, Germany; 3Laboratoire de Biogenèse Membranaire, Unité Mixte de Recherche 5200, Université de Bordeaux, 33882 Villenave D'Ornon, France; 4Institut für Molekulare Medizin und Zellforschung, Sektion Biophysik, AlbertLudwigs-Universität Freiburg, 79104 Freiburg, Germany *To whom correspondence should be addressed: Meike Luck, Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Invalidenstraße 42, 10115 Berlin, Germany; e-mail: [email protected]; phone: +49(0)30-2093 8686

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ABSTRACT The tiny picoalga, Ostreococcus tauri, originating from the Thau Lagoon is a member of the marine phytoplankton. Because of its highly reduced genome and small cell size, while retaining the fundamental requirements of a eukaryotic photosynthetic cell, it became a popular model organism for studying photosynthesis or circadian clock-related processes. We analyzed the spectroscopic properties of the photoreceptor domain of the histidine kinase rhodopsin Ot-HKR that is suggested to be involved in the light-induced entrainment of the Ostreococcus circadian clock. We found that the rhodopsin, Ot-Rh, dark state absorbs maximally at 505 nm. Exposure to green-orange light led to the accumulation of a blueshifted M-state-like absorbance form with deprotonated Schiff base. This Ot-Rh P400-state had an unusually long life-time of several minutes. A second long-living photoproduct with red shifted absorbance, P560, accumulated upon illumination with blue/UVA light. The resulting photochromicity of the rhodopsin is expected to be advantageous to its function as molecular control element of the signal transducing HKR-domains. The light-intensity and the ratio of blue vs. green light are reflected by the ratio of rhodopsin molecules in the long-living absorbance forms. Furthermore dark state absorbance and the photocycle kinetics vary with the salt content of the environment substantially. This observation is attributed to anion binding in the dark state and a transient anion release during the photocycle indicating that the salinity affects the photoinduced processes.

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INTRODUCTION Since the discovery of Bacteriorhodopsin in 19711 microbial rhodopsins have been the subject of intensive scientific studies. Besides deciphering their physiological roles, the analyses of the molecular mechanism of ion transport or signal transduction are central objectives of research. These studies are directed to elucidate the relationship between light absorption, the subsequent thermal relaxation processes of the photoreceptor, and the functional and structural changes. In the past, two main groups of microbial rhodopsins were classified according to their functions. Light-driven ion pumps 2–5 and light-gated channelrhodopsins 6–8, on the one hand, trigger the active and passive ion transport across the membrane, respectively, on the other hand, rhodopsins with sensoric function 9,10 control a specific output reaction in terms of signal transduction or enzymatic activity. The downstream reactions can be accomplished by a separate transducer protein, as is the case in the archaeal sensory rhodopsins where the rhodopsin activates the transducer by light-induced modification of intermembrane contacts 11,12, or by shaping the interaction of a covalently bound enzymatic protein subunit as found in the enzyme rhodopsins 13,14. The photoreactions and the dynamics of the photoinduced intermediates are usually adapted to the respective rhodopsin function. Accordingly, the ion pumps employ fast photocycle kinetics (≈10 ms) to achieve a high turnover of the transported ions, especially at high light intensities. On the contrary, sensoric rhodopsins depend on long-living (up to several 100 ms) active states to optimize the signal transduction efficiency, preferentially at low light intensities 15. Members of a new family of enzyme-rhodopsins, the histidine kinase rhodopsins (HKRs), were identified by genome mining in very different organisms, such as unicellular algae, fungi, and amoeba 16,17. HKRs are characteristically organized in a modular fashion (Fig. 1). The N-terminal rhodopsin domain (Rhod) is covalently bound to a dimerization and phosphorylation domain (DHp) and a catalytic domain (CA), both of which have strong homologies to the equivalent parts in histidine kinases of microbial Two Component Systems (TCS; Fig. 1A) 18,19. The CA-domain is generally linked to a Cterminal response regulator-like receiver domain (Rec) in the HKRs. Therefore, HKRs are nothing else than specific hybrid histidine kinases, where the sensoric part is a rhodopsin (Fig. 1B). According to this architecture, light-induced phosphorylation of the signal-transducing subunits is assumed to occur in HKRs. In some HKRs, an additional C-terminal domain with high homology to guanylyl cyclases exists (Cyc). In these cases, a light-activated cGMP-production is expected (Fig. 1C).

Figure 1. Schematic overview of the phosphorelay processes after sensor domain activation. (A) The respective stimulus initiates the autophosphorylation of the conserved histidine-residue in hybrid histidine kinases followed by the phosphorelay to the conserved aspartate in the receiver domain (Sensor: sensor domain, DHp: dimerization and phosphorylation domain, CA: catalytic domain, Rec: response regulator-like receiver domain). The signal transduction implies an additional phosphoryl group transfer to an HPt-protein (HPt: histidine phosphotransferase) and a response regulator-like receiver domain (Rec: receiver domain, Output:

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Biochemistry

output-domain) controlling the output reaction. In histidine kinase rhodopsins (B and C), the rhodopsin-domain (Rhod) presumably initiates the equivalent phosphorelay processes in the hybrid histidine kinase-homologue protein domains. (B) In case of HKRs without the C-terminal effector domain, interaction with a separate protein and its activation by phosphorylation is very likely (C) In HKRs with a C-terminal cGMP-cyclase (Cyc) as effector, a light-induced cGMP-production is assumed.

The only histidine kinase rhodopsin that has been characterized spectroscopically so far is the Cr-HKR1 from Chlamydomonas reinhardtii. The isolated photoreceptor fragment revealed a highly photochromic character as a consequence of the thermally stable UV-absorbing form (Rh-UV), a photoproduct with a deprotonated retinal Schiff base (RSB) 17. Furthermore, parallel photoreactions emanate from two coexisting dark states of the blue-absorbing form (Rh-Bl), with either all-trans,15-anti- or 13-cis,15-synretinal, as characterized by UV/vis and Resonance Raman spectroscopy 20,21. These unusual properties concerning the photochemistry spurred studies on the isomeric composition in other microbial rhodopsins like channelrhodopsin 2 22. However, because of the large size of the HKRs and the existence of multiple HKR-encoding sequences in the genome of C. reinhardtii, the identification of the physiological function was not successful, thus far. The involvement of Cr-HKR1 in phototactic reactions is unlikely, owing to the high stability of the photoproduct, Rh-UV. However, the spectroscopic features enable the protein to quantify the UV/blue light ratio in the environment and is expected to be used by the cells to adapt to UVA-radiation. HKR-sequences were found in the genomes of several members of the marine phytoplankton, including the picoeukaryote Ostreococcus tauri. Unlike the unclear situation in the case of Chlamydomonas, only one HKR-encoding gene is present in the Ostreococcus genome. Ostreococcus is characterized by an extremely small cell-size and was described as the smallest free-living eukaryotic organism, reduced to the fundamental requirements of a free-living cell 23. Because the reduction in the size of cells is accompanied by a reduction in its genome size 24, as a result of evolutionary elimination of both the redundant genes and regulatory DNA, the mere existence of a single HKR strongly suggests its significance in the living organism. The Ostreococcus histidine kinase rhodopsin (Ot-HKR) shares the HKR-common modular design as hybrid histidine kinases, as mentioned for C. reinhardtii HKRs, which possess rhodopsin, histidine kinase, and receiver domains (Fig. 2A); however, it lacks the C-terminal enzymatic effector domain. Therefore, the interaction with a separate, not yet identified protein, would be essential to provoke an output reaction. In previous studies, the simple version of a green lineage-like circadian clock was thoroughly described for O. tauri 25–27. The core of this circadian rhythmicity is formed by the two proteins, Timing of CAB Expression 1 (TOC1) and Circadian Clock Associated 1 (CCA1), whose expression is controlled by a negative feedback-loop. Additionally, a regulatory influence of the environmental light on the circadian clock proteins was detected. Accordingly, Ot-HKR as putative light-induced histidine-kinase, might be involved in the light-dependent control of TOC1-expression. We studied the light-absorbing characteristics of the rhodopsin domain of Ot-Rh and showed that this photoreceptor can serve as a useful tool for sensing the daylight conditions. MATERIALS AND METHODS Protein expression and purification - The humanized gene encoding the 40 kDa fragment of OtHKR-rhodopsin (Ot-Rh: AA 1–341 from XP_003083892.1), with a C-terminal myc-tag and a 12x-His-tag was expressed in the methylotrophic yeast, Pichia pastoris (Fig. 1). Transformation, selection of clones and protein purification was executed as previously described 17,28,29. In short, the protein was solubilized at 4 °C overnight in 1% (w/v) DDM with 5 mg total protein per milliliter. The protein was purified by affinity chromatography in 20 mM HEPES, 0.1 M NaCl, 10% (v/v) glycerol, and 0.03% (w/v) DDM, pH 7.8. The buffer-exchange was achieved by using centrifugal filter units (Amicon Ultra-15, 30 kDa, Merck, Germany). The standard buffer was exchanged by using either salt-free buffer or by using buffer containing the desired end-concentration of the respective salt (each in 20 mM HEPES, 10% (v/v) glycerol, 0.03% DDM, pH 7.8). UV/visible absorption spectroscopy - The steady state absorption spectra and slow kinetics were obtained using a Cary 300 Bio spectrophotometer (Agilent, Waldbronn, Germany/Varian Inc., Darmstadt, 3 ACS Paragon Plus Environment

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Germany). All the data were recorded at 20 °C with a spectral resolution of 1 nm. The dark samples were exposed to dim red light (> 600 nm) only and measurements were started after a dark adaptation period of at least 10 min. The light spectra correspond to the absorption spectra detected directly after 30 s illumination of the sample to the respective LED (500 nm, 530 nm, 580 nm, 400 nm, all Luxeon, Phillips, Amsterdam, Netherlands/Nichia Corporation, Tokushima, Japan). In the single-wavelength kinetic studies, the absorbance differences for a fixed wavelength were recorded over time after a light stimulus (30 s with the respective LED) or after salt addition. The absorbance differences represent the formation or decay of the (photo-) intermediates within a recovery period of 20–30 min. The data were analyzed using the software package Origin (version 8.5.1). The time constants (τ) were determined by fitting mono- or bi-exponential functions to the traces. Determination of chloride affinity - The absorbance spectra were obtained before and after the addition of NaCl and the respective difference spectra were calculated. Because of slow chloride incorporation at low salt concentrations, we used an equilibration time of 24 h waiting prior to the recording of the spectra. The increase in absorbance at 490 nm was plotted against the respective NaCl concentration. A Michaelis Menten function was fitted to the data points and accordingly the dissociation constant Kd(Cl-) as measure for the chloride affinity was determined. Time-resolved absorbance spectroscopy - The transient changes in the absorbance were recorded using a LKS.60 flash-photolysis setup (Applied Photophysics Ltd., Leatherland, UK) equipped with a Rainbow OPO/Nd:YAG laser (BrilliantB, Quantel, Les Ulis Cedex, France) and an Andor iStar ICCD camera (Andor Technology Ltd, Belfast, Ireland) at 20 °C. The excitation and probing of the sample (OD = 0.3, at the chromophore absorption maximum) was performed in HEPES buffer at pH 7.8, as previously described 7,17,30. The excitation wavelength was 505 nm for sample in buffer containing 0.1 M and 1 M NaCl and 550 nm for sample in salt-free buffer. In the slow time-resolved laser flash photolysis mode, the changes in the absorbance were detected continuously after a laser flash in the time range from 300 ms to 53 min. In the short time regime, covering the time range between 0.1 µs and 10 s after excitation, the spectral changes at a single time point in each single measurement were recorded. Subsequently, photochemical reconversion of P400 into P560 was done by LED illumination (400 nm for 10 s) to accelerate monitoring of repetitive spectra of the early photocycle intermediates. In case of 0 M NaCl the photoconversion directly recovered the D560 dark state. In case of 1 M NaCl an extra delay period of 3 minutes after P400-to-P560 conversion was included for the completion of the thermal conversion of P560 to the D505 in order to prepare the protein for the next excitation cycle. The consecutive time points were arranged on a log scale to cover the above-mentioned time range. These single time point spectra were combined in the time resolved data set. 10-20 data sets were averaged for further analysis. The global analysis of these time resolved data was performed with the Glotaran software package using a simple unidirectional sequential model including three to four kinetic components 31. The resulting EADS represents an approach to display the involved spectral components. FT-IR spectroscopy - The IR measurements were performed with an IFS-28 FT-IR spectrometer or a Tensor27 (Bruker Optics), equipped with an MCT detector and a home-built sample holder. The sample solution was concentrated under a moderate N2 flow on a CaF2 window with a 2 µm deepening. The concentration of the protein microfilm was around 0.5 to 1 mM. Subsequently, the sample was covered with a plain CaF2 window. To avoid dehydration during the measurement, the sample sandwich was sealed with silicon grease. The IR measurements were done at room temperature. Following the parameters derived from the UV/vis spectroscopic analysis, photoconversion between the two photostates was achieved by irradiation, either with green (530 nm) or blue (430 nm) light, from an array of LEDs. Further IR-experimental details are given elsewhere 32. In the measured P560-minus-D505 FT-IRdifference spectrum residual P400-D505 difference-signals were observed due to contribution of the longliving P400-state. To obtain a widely pure P560-minus-D505 difference spectrum a portion (0.4*) of the P400-minus-D505-difference spectrum was subtracted from the originally measured data to eliminate residual signals that originated from D505-to-P400-transition. Accession numbers - Sequences used during this study were derived from 2AT9_A, AM774415.1, BAA07823.1, AAQ1627, and XP_003083892 4 ACS Paragon Plus Environment

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RESULTS Stationary and transient absorption spectroscopy Under moderate salt conditions (0.1 M NaCl), recombinant dark adapted Ot-HKR-rhodopsin fragment (Ot-Rh; Fig. 2A and B) absorbs maximally at 505 nm (D505), with a weak shoulder at 470 nm and a broad absorbance extension between 550 and 600 nm (Fig. 2C). Irradiation with 500 nm light for 30 s induces D505 bleaching and the concomitant rise of two absorption bands with maxima at 396 and 560 nm, respectively, and life-times in the range of minutes. We, thus, assume the formation of two longliving transient absorbance states denoted as P400 and P560 (vide infra). The blue-shifted P400 species resembles the M-states of other rhodopsins, with a deprotonated retinal Schiff base chromophore and the Rh-UV-form in Cr-HKR1. Compared to the Rh-UV-form, the P400 state of Ot-Rh is thermally more labile but its decay is much slower compared to that of the M-intermediates of almost all other microbial rhodopsins described till date 15,33. In contrast, the nature of the P560 formation was unclear. During a dark period of several minutes the D505 absorption is regenerated.

Figure 2. Modular architecture of the full-length Ostreococcus tauri Ot-HKR as a photo-sensoric hybrid histidine kinase (A) comprising a rhodopsin domain (Rhod), a histidine kinase domain (DHp: dimerization and phosphorylation domain, CA: catalytic domain) and a response regulator-like receiver domain (Rec). The recombinant purified rhodopsin fragment Ot-Rh (amino acids 1–341) results in (B) a 40-kDa band in the SDS-gel and a minor second band originating from non-homogeneous glycosylation. Ot-Rh is characterized (in 0.1 M NaCl and 20 mM HEPES pH 7.8) by (C) an absorbance maximum at 505 nm in the dark and by two long-lived absorbance forms, a blue- (396 nm) and a red-shifted (560 nm) meta-stable state.

Due to the life-times of P400 and P560, both on the minutes time scale, we studied the transient absorption kinetics of Ot-Rh by laser-flash photolysis (Fig. 3). These experiments offered a more precise view to the light-induced processes and avoided photoexcitation of semi-stable intermediates to decipher the time-correlation of the two long-living intermediate states (P400 and P560) appearing after the excitation of D505. Within a given time window (300 ms to 50 min; Fig. 3A and B), global analysis revealed the spectral components in terms of Evolution Associated Difference Spectra (EADS) and the associated kinetic parameters. The first EADS is characterized by a broad ground state bleach (GSB), with a minimum at 505 nm and positive absorbance at 400 nm (P400). This EADS1 evolves with a kinetic constant of ca. 5 s to EADS2, with similar absorbance properties but higher amplitude, indicating that the development of P400 and the concomitant deprotonation of the Schiff base are incomplete at 300 ms after excitation. The transition of EADS2 to EADS3 is associated with a partial loss of absorbance at 400 nm and an increased amplitude at 560 nm, indicating the conversion from P400 to the late red-shifted P560state on a time scale of ca. 1 min. EADS3 decays with an apparent time constant of ca. 5-6 min. This 5 ACS Paragon Plus Environment

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EADS3 signal displays the rise of the late 560 nm species as well as an increment of the P400 intermediate (Fig. 3C). In this respect, global analysis does not afford a clear picture about the P400-toP560-kinetics, which presumably includes a second time constant in the minute time range.

Figure 3. Laser-flash photolysis (505 nm laser flash, 300 ms to 50 min after excitation) reveals the sequential appearance of the two long-living intermediates in 0.1 M NaCl and 20 mM HEPES pH 7.8. (A) Three-dimensional data plot of the time resolved difference spectra and global analysis uncover the bi-exponential decay of the blue-shifted P400-intermediate on a minute-time scale to the red-shifted late P560 state before the D505 dark state is recovered in a time range of several minutes, as represented by the (B) absorbance differences at selected wavelengths (398, 575, 505 nm) and (C) the Evolution Associated Difference spectra (EADS).

The slow processes after photoexcitation are summarized in a preliminary model (Fig. 4A), as follows: The P400-state arises bi-exponentially and involves deprotonation of the RSB, with time constants < 300 ms and ca. 5 s. The equally biphasic transition of the P400-state to the reprotonated P560 state occurs on a time scale of one to several minutes, followed by the decay of P560 and the regeneration of the D505 dark-state with an apparent time constant of 5–6 min. The unusually long lifetimes of both P400 and P560 implicate a possible photoreactivity of these photocycle intermediates. Indeed, blue light (400–450 nm) irradiation converted the previously accumulated P400 state to P560 within less than a second (Figure S1, Fig.4B). Conversely, green-orange light (530–580 nm) exposure of P560, or more precisely of the P560–D505 equilibrium, yielded P400 enrichment (Fig. 4B). However, direct photorecovery of the D505 dark-state was not possible. The D505 form can solely be regenerated thermally through prolonged dark periods of 5–10 min.

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Figure 4. Variation of the irradiation conditions results in the interconversion between the two long-lived intermediates, P400 and P560, of Ot-Rh. (A) A simplified photocycle model involving the photochemical and slow relaxation processes at moderate salt conditions (0.1 M NaCl, 20 mM HEPES pH 7.8) summarizes these observations. (B) Long-wavelength (530–590 nm) LED exposure (30 s) leads to P400 accumulation, whereas shorter wavelengths (400–450 nm; 30 s) induce the predominant formation of the P560.

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The role of halides As marine alga, the concentration of inorganic ions is expected to play a key role in the physiology of Ostreococcus. Therefore, the halide dependence of the spectroscopic properties of the HKR was tested. Although the presence of salt is expected to be a given fact in the natural marine environment of the algae, the non-physiologic investigations under salt-free conditions delivered important information about the nature of the late P560-state. Intriguingly, in the absence of salt, we observed a distinctly redshifted dark state absorption maximum of 560 nm (D560; Fig. 5A), whereas addition of NaCl recovered the D505 absorption (Fig. 5B). Under salt-free conditions, photo-excitation converted the D560 efficiently into a blue-shifted P400 intermediate similar to the P400 previously observed under moderate salt conditions (Fig. 5, A and C). Furthermore, under salt-free conditions, P400 was thermally converted to D560 with no evidence for the formation of D505. We, therefore, assume that the absence of salt simply arrests the photocycling in the D560 state. The photo-activation of the salt-free P400 state again leads to the rapid formation of D560, as observed under moderate NaCl concentrations (Fig. 4B). The P560 absorption in the presence of 0.1 M NaCl reflects the D560 state in 0 M NaCl but with broader band-width originating from the coexistence of the D505 and D560 states (Fig. 5D).

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Figure 5. Properties of dark-adapted Ot-Rh absorbance depend on the presence of NaCl. (A) Absorbance spectra show a red-shift from 505 nm to 560 nm upon the removal of salt (0 M NaCl, 20 mM HEPES pH 7.8). After light-induced (green-orange light, 30 s) P400 accumulation, the recovery of D560 occurs photochemically (UVA-blue light, 30 s) or thermally. (B) The D505 dark state is regenerated upon the addition of NaCl (0.1 M). (A, C) Illumination of the salt-free sample (530 nm LED, 30 s) induces the formation of a P400 form similar to that in the presence of NaCl. (D) Comparison of the light-induced accumulated P560 state (0.1 M NaCl, 20 mM HEPES pH 7.8) and the salt-free D560 dark state.

Different sodium salts (0.1 M) were added to the ion-free Ot-Rh solution to gain insights into the ion-selectivity and binding affinity of the protein. The HKR absorption shifted to shorter wavelengths in presence of all the halides, whereas after Na2SO4 addition, the D560-absorbance remained unaffected and no rise was detected in the kinetic measurements at 505 nm (Fig. 6B). Consequently, the large hypsochromic shift can be attributed to the specific interactions with monovalent anions. Small differences were found for the various halides in as much as the absorption maximum seemed to vary with the halide size from 505 nm (chloride) through 507 nm (bromide) to 511 nm (iodide) (Fig. 6A) because of the charge delocalization. Similarly, the thermal recovery of the D505 state was accelerated in the same order, with apparent time constants of 5–6 min (Cl-), 1–2 min (Br-), and < 1 min (I-). The apparent D560-to-D505-conversion was found to be dependent on the chloride concentration in the range from 11 to 800 mM NaCl, with time constants changing from τ 11mM = 15 min to τ 800mM < 1 min (Fig. 6, C and D). Moreover, low chloride concentration resulted in an incomplete D560-to-D505 conversion (Inset Fig. 6E). The uptake of chloride by the protein was associated with a modification of the electronic properties of the protonated Schiff base, which points to the existence of an anion-binding site close to the Schiff base. Based on the chloride-induced absorption changes (Fig. 6E) the Cl--affinity of this binding site was calculated and resulted in a chloride dissociation constant Kd(Cl-) of 24 mM NaCl (Fig. 6F).

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Figure 6. Effect of salt on the Ot-Rh dark states (0.1 M NaCl, 0.1 M NaBr or 0.1 M NaI, 0.1 M Na2SO4). (A) D505 dark state absorbance and (inset A) halide-anion-induced hypsoochromic shift. (B) salt-dependent D505 recovery. (C) Time-dependent absorption recovery at 500 nm (D560-to-D505 thermal transition) at different NaCl concentrations from 11 to 800 mM. (D) Rate constant k for the D505-recovery as a function of the NaCl concentration. (E) Difference absorbance spectra: positive bands reflect NaCl addition, whereas negative peaks refer to the NaCl free reference (inset in C). (F) Fit of the Michaelis–Menten equation to the NaCl-induced absorbance differences at 490 nm resulting in a Kd(Cl-)-value of 24 mM. All samples were buffered in 20 mM HEPES pH 7.8.

In contrast to the dark equilibrium, neither the P400 absorption nor the reprotonation kinetics were dependent on halides (Figure S2, Fig. 7, A and B). The P400-to-P/D560-conversion kinetics was biexponential and could be described by time constants τ1 = 60 s and τ2 = 6-8 min, both in the presence and in the absence of the tested halide anions. These findings suggest that the chloride is released during the formation of P400. It remained unclear whether the relaxation processes of the P560-to-D505 transition displayed a salt-dependence like that for the D560-to-D505-conversion. Despite the similar absorption properties, a possible structural correlation between the D560 state observed under salt-free conditions and the P560 intermediate observed under moderate salt awaits further proof. To answer this question, we accumulated P560 by illuminating the sample with 400 nm light to excite the dark-state and to induce the conversion of the long-lived intermediate P400 to P560. The subsequent thermal decay of the accumulated P560 was recorded in different sodium salts (Fig. 7C) and, under different NaCl concentrations, in particular (Fig. 7D). Interestingly, the kinetics was observed to depend, as in the case of the D560-toD505-conversion, on the size of the anion (Fig. 7C) and, as shown for chloride, on the halide 9 ACS Paragon Plus Environment

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concentration (Fig. 7D). The P560 decay was slow (>20 min) at low Cl--concentration (11 mM) and was accelerated (