Volume 93, Issue 3 pp. 762-771

Special Issue Research Article

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Proteorhodopsin Photocycle Kinetics Between pH 5 and pH 9^†

Thomas Köhler,

Thomas Köhler

Institute of Physical and Theoretical Chemistry, Goethe Universität Frankfurt am Main, Frankfurt, Germany

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Ingrid Weber,

Ingrid Weber

Institut für Biophysikalische Chemie, Goethe Universität Frankfurt am Main, Frankfurt, Germany

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Clemens Glaubitz,

Clemens Glaubitz

Institut für Biophysikalische Chemie, Goethe Universität Frankfurt am Main, Frankfurt, Germany

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Josef Wachtveitl,

Corresponding Author

Josef Wachtveitl

[email protected]

Institute of Physical and Theoretical Chemistry, Goethe Universität Frankfurt am Main, Frankfurt, Germany

Corresponding author email: [email protected] (Josef Wachtveitl)Search for more papers by this author

Thomas Köhler,

Thomas Köhler

Institute of Physical and Theoretical Chemistry, Goethe Universität Frankfurt am Main, Frankfurt, Germany

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Ingrid Weber,

Ingrid Weber

Institut für Biophysikalische Chemie, Goethe Universität Frankfurt am Main, Frankfurt, Germany

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Clemens Glaubitz,

Clemens Glaubitz

Institut für Biophysikalische Chemie, Goethe Universität Frankfurt am Main, Frankfurt, Germany

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Josef Wachtveitl,

Corresponding Author

Josef Wachtveitl

[email protected]

Institute of Physical and Theoretical Chemistry, Goethe Universität Frankfurt am Main, Frankfurt, Germany

Corresponding author email: [email protected] (Josef Wachtveitl)Search for more papers by this author

First published: 13 May 2017

https://doi.org/10.1111/php.12753

Citations: 6

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This article is a part of the Special Issue dedicated to Dr. Wolfgang Gärtner on the occasion of his 65^th birthday.

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Abstract

The retinal protein proteorhodopsin is a homolog of the well-characterized light-driven proton pump bacteriorhodopsin. Basic mechanisms of proton transport seem to be conserved, but there are noticeable differences in the pH ranges of proton transport. Proton transport and protonation state of a carboxylic acid side chain, the primary proton acceptor, are correlated. In case of proteorhodopsin, the pK_a of the primary proton acceptor Asp-97 (pK_a ≈ 7.5) is unexpectedly close to environmental pH (pH ≈ 8). A significant fraction of proteorhodopsin is possibly inactive at natural pH, in contrast to bacteriorhodopsin. We investigated photoinduced kinetics of proteorhodopsin between pH 5 and pH 9 by time resolved UV/vis absorption spectroscopy. Kinetics is inhomogeneous within that pH region and can be considered as a superposition of two fractions. These fractions are correlated with the Asp-97 titration curve. Beside Asp-97, protonation equilibria of other groups influence kinetics, but the observations do not point toward major differences of primary proton acceptor function in proteorhodopsin and bacteriorhodopsin. The pK_a of proteorhodopsin and some of its variants is suspected to be an example of molecular adaptation to the physiology of the original organisms.

Introduction

Proteorhodopsin (PR) belongs to the large family of type-I rhodopsins 1. Members of that membrane protein family (also known as microbial rhodopsins) bind the chromophore retinal. Photoexcitation induces an all-trans to 13-cis chromophore isomerization followed by relaxation of the system which passes a series of metastable states before it reaches the initial equilibrium state (resting state) again. Protein function is coupled to the relaxation process.

Function and structure of the proton pump bacteriorhodopsin (BR), the first known type-I rhodopsin found in the archaeon Halobacterium salinarum, have been studied intensively 2. After photoexcitation, a proton is transported from the cytosolic side of the plasma membrane to the organism's environment on a timescale of milliseconds 3. The transport process is a series of proton transfer steps between pairs of proton donors and proton acceptors 2. Several of these potentially charged amino acids and the Schiff-base, an imine formed between retinal and a lysine side chain, are located in a narrow channel spanning the protein. Four of the seven transmembrane α-helices of BR line that channel (helix B, C, F, G), which is divided into a cytosolic and extracellular part by the chromophore 4. A proton transfer step can be coupled to a transition of one metastable state (intermediate) to another, and the complete sequence is referred to as the photocycle 5.

BR generates a proton gradient used by the organism to drive endergonic processes 6. Different systems for light energy conversion exist in nature, but BR is considerably smaller than the photosystems of plants, algae or cyanobacteria and represents a well-established model for proton pumps.

Béjà et al. discovered proteorhodopsin (PR) in a marine γ-proteobacterial species in the year 2000 7. Meanwhile, thousands of PR variants have been identified in bacteria, archaea and eukaryotes 8, 9. PR and BR share only about 30% of their amino acids, but PR adopts the typical seven transmembrane helix structure and binds all-trans retinal via a Schiff-base linkage to Lys-231 7, 10. Most of the amino acids involved in proton transport of BR are conserved in PR, including primary proton acceptor Asp-97 (Asp-85 in BR) and primary proton donor Glu-108 (Asp-96 in BR) 7, 11, 12. However, no homolog of a Asp-194/Asp-204 dyad, the proton release complex of BR, is present in PR 13. Light-driven proton transport has been demonstrated for PR and its photocycle between pH 9 and pH 10 resembles the BR photocycle with some exceptions 7, 11, 12, 14, 15.

Both proteins differ in the pH range of proton transport. BR pumps protons from the cytosolic to the extracellular side between ca. pH 2 and pH 12 with a broad range of close to maximum activity between pH 4 and pH 8 11, 16. Proton transport has been related to the protonation state of the primary proton acceptor in equilibrium 16, 17. Protons can be exchanged between the proton binding site of the primary proton acceptor (its carboxyl group) and the aqueous environment through the extracellular half channel in equilibrium 18. After photocycle initiation, the Schiff base is the proton donor of the first transfer step 19. The carboxyl group can act as the proton acceptor of the first step if it was deprotonated prior to that stage of the photocycle. If the carboxyl group is protonated at that stage already, it cannot function as a proton acceptor. This results in a truncated photocycle observed at pH values < 2 20. BR does not pump protons under that condition 21. The lower pH limit of proton transport by BR is related to the primary proton acceptor's pK_a value that is close to 2.5 16, 22.

The ability of PR to transport protons from the cytosolic side to the extracellular side peaks in the alkaline range (presumably between pH 9 and pH 10) and decreases substantially at lower pH values 13, 14, 23, 24. The reported pK_a values of Asp 97 vary between ca. 7 and 8 11, 12, 25, 26. (This applies to in vitro PR-samples in detergent or lipid environment, but also to PR in living Escherichia coli cells (pK_a = 7.6).) In analogy to the conclusions drawn for BR, the high pK_a value of PR provides an explanation for its limited range of proton transport.

However, pH dependence of the activity is still under discussion. It was claimed that the proton transport direction of PR reverses when the primary proton acceptor is protonated in the initial state 12, 13, 27, 28. Reversed active transport was described below ca. pH 6, a phenomenon which is not known for the BR wild type. This was questioned in several respects: Some authors argue PR does not transport protons when the primary proton acceptor is protonated, accordingly PR is inactive at pH < 6 14, 24, 29. Others consider reverse transport as a purely passive proton flux driven by pH and potential gradients 30.

The high pK_a of PR raises further questions. Surface seawater, the living environment of many bacteria containing PR variants, has a pH of ca. 8 31. Hence, the primary proton acceptors of a significant fraction of PR molecules are assumed to be protonated in the initial state. These PR's are inactive in light energy conversion, as they do not transport protons from the cytosolic to the extracellular side (periplasmatic space) actively. However, a growing number of evidence supports the idea that PR and some PR variants are indeed systems for light energy conversion 32-35. Astonishingly PR does not seem to be optimized for that purpose in every respect.

Potentially, an unknown mechanism of proton transport gives rise to the high pK_a of PR. Deviations of the proton transport mechanisms of PR and BR may be related to the amino acid composition of the extracellular half channels. PR lacks a proton release complex and has a histidine at position 75, which is highly conserved among PR-variants 36, 37. Histidine forms a pH-dependent cluster with the primary proton acceptor and might be involved in proton transport 36, 37. No homolog of His-75 is present in BR, which has an uncharged amino acid (Met) at the corresponding position 36, 37.

The mechanism of proton transport is related to photocycle kinetics. Most studies of the latter topic focus on in vitro PR wild-type samples under alkaline conditions (pH ≥ 9) or acidic conditions (pH ≤ 6) 11, 12, 14, 15, 28, 29, 38, 39. We investigate photocycle kinetics at various pH values including natural pH (~ 8) by time resolved difference spectroscopy in the visible spectral region. As such measurements do not allow for direct conclusions regarding the pH-dependence of proton pumping activity, emphasis is put on the discussion of kinetics.

In the pH-range of interest (pH 5 to pH 9), the sample is mostly inhomogeneous with respect to the protonation state of the primary proton acceptor. Taking BR as a reference system, different photocycle kinetics is expected to be related to the protonated and deprotonated form of the primary proton acceptor respectively 20, 40. Considerable deviations of PR pH-dependent photocycle kinetics from that dependency would imply the existence of an unknown mechanism that may provide explanations for the high pK_a.

PR wild type (it is a green absorbing PR variant according to the classification of Beja et al. 41) and H75N mutant samples were examined. His 75 is known to stabilize the pK_a of the particular PR variant investigated here at its high value 37. Replacing His by Asn lowers the pK_a. In that respect, the mutant is closer to the reference system BR than the PR wild type. We therefore validate whether the relationship of primary proton acceptor protonation state and photocycle kinetics of the PR wild type is preserved in that mutant.

Materials and Methods

Sample preparation

The PR variant (GeneBank accession number AF279106) first described by Béjà et al. was used 7. PR wild type was cloned in the pET-27b(+) plasmid. The H75N mutation was created by PCR using the PR wild-type plasmid and mismatch primers as described in Pfleger et al. 27.

Expression and purification were essentially carried out as described in Neumann et al. 42. Escherichia coli strain C43 (DE3) was used for transformation. The cells grew in lysogeny broth (LB) medium with 50 μg mL⁻¹ kanamycin. Protein expression was induced by 1 mm isopropyl ß-D-1-thiogalactopyranoside (IPTG), and 10 μm all-trans retinal was added at the same time. After 3 h, the cells were resuspended in buffer (50 mm tris(hydroxymethyl)aminomethane (TRIS), 5 mm MgCl₂, pH 8) and disrupted. The membranes were sedimented and solubilized in buffer (1.5% w/v n-Dodecyl-β-D-maltopyranoside (DDM), 300 mm NaCl, 5 mm imidazole, pH 6) for 16 h at 4 °C. Solubilized PR was left in the supernatant after centrifugation. The supernatant was incubated with Ni-nitrilotriacetic acid agarose for 1 h at 4 °C. The column was washed with buffer (0.15% w/v DDM, 300 mm NaCl, 50 mm 2-(N-morpholino)ethanesulfonic acid (MES), 50 mm imidazole, pH 7.5). Buffer (0.5% w/v DDM, 300 mm NaCl, 50 mm MES, 200 mm imidazole, pH 7.5) was used for elution of hexa-His tagged PR wild type and the hexa-His tagged H75N mutant. Buffer composition was changed by ultrafiltration or dialysis as specified. Protein concentration was chosen in the range of 1.1 × 10⁻⁵–2.3 × 10⁻⁵ mol L⁻¹ for laser flash photolysis experiments.

Photometric pH titration and spectral decomposition

An Analytik Jena spectrophotometer (Specord S100) was used. The proteins were transferred in a solution that contained 0.1% w/v DDM and 300 mm NaCl. pH was changed by addition of small amounts of NaOH or HCl under constant pH control with a Hamilton Bonaduz AG (Biotrode) electrode.

Contribution of Rayleigh light scattering to extinction was approximated by a power function A + B/λ^c. Each PR wild-type absorption spectrum at pH 5.5 and at pH 9.2 (spectral range: 700–320 nm) was described by a sum of two Gaussian functions and one skewed Gaussian function. The latter is shown here:

$urn:x-wiley:00318655:media:php12753:php12753-math-0001$ (1)

$urn:x-wiley:00318655:media:php12753:php12753-math-0002$ is the wavenumber at maximum amplitude, A is the amplitude at $urn:x-wiley:00318655:media:php12753:php12753-math-0003$ , ω is the full-width at half-maximum and p is the skewness 12, 43. One Gaussian function (Abs_uv) describes a band of uncertain origin in the near UV-region (λ_max ≈ 390 nm). A sum of a Gaussian function and a skewed Gaussian function describes the main band in the visible spectral range.

At pH 9.2, this sum ( $urn:x-wiley:00318655:media:php12753:php12753-math-0004$ ) represents the main band of the sample being in state D⁻ completely. At pH 5.5, another sum ( $urn:x-wiley:00318655:media:php12753:php12753-math-0005$ ) represents the main band of the sample being in state DH completely. The fit function for PR wild-type absorption spectra between pH 9.2 and pH 5.5 (Abs_pH) is composed of three terms.

$urn:x-wiley:00318655:media:php12753:php12753-math-0006$ (2)

Central wavenumber, full width at half maximum and amplitude of Abs_uv as well as α (constraint: 0 ≤ α ≤ 1) were free parameters of the fit, whereas the parameters of $urn:x-wiley:00318655:media:php12753:php12753-math-0007$ and $urn:x-wiley:00318655:media:php12753:php12753-math-0008$ were kept constant. According to Lambert–Beer's law, the weighting factor α is equivalent to a relative specification of state DH concentration (C_DH/(C_DH + C_D−)). Figure 1 shows an example for spectral decomposition of an absorption spectrum (analysis software: Igor Pro, version: 6.0.3.0).

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Spectral decomposition of a PR wild-type absorption spectrum at pH 6.4 (α = 0.82).

The graph of α and λ_max as functions of pH (insets of Figs 2 and 6) were fitted with a logistic function:

$urn:x-wiley:00318655:media:php12753:php12753-math-0009$ (3)

P is an inflection point and n can be regarded as the Hill coefficient 12.

Laser flash photolysis setup

The laser flash photolysis photometer setup was essentially the same as described in Scaiano 44. A Xenon Arc lamp (Hamamatsu Photonics, LC8) was the source of continuous detection light. Detection wavelength was selected by a monochromator (Photon Technology International, type 101, grating: 1200 g mm⁻¹). The monochromatic detection light passed the sample and was focused in an identical second monochromator to keep scattered excitation light away from the photomultiplier tube (Hamamatsu Photonics, H5784-02). A Nd-YAG laser (InnoLas GmbH, SpitLight 600) was the source of excitation pulses (repetition rate 1 Hz or 0.5 Hz, pulse length 5 ns). The wavelength of frequency tripled Nd-YAG laser pulses was converted by an optical parametric oscillator (GWU Lasertechnik, preciScan/MB). Datasets were recorded with a digital storage oscilloscope (Lecroy Corporation, WaverunnerXi) in real-time measurement mode (Time resolution is given by the fixed data point separation of 50 ns or 200 ns.). Each measurement was carried out at room temperature and was repeated between 1000 and 10 000 times to increase signal to noise ratio by averaging.

Intermediate spectra

Transient data (see Figure S2) were measured from 410 nm to 630 nm detection wavelength in steps of 20 nm. Average excitation energy was 4.4 mJ cm⁻² at 520 nm for pH 9 and 3.2 mJ cm⁻² at 540 nm for pH 5.75. Intermediate spectra were obtained by model based transient data (raw data) analysis with the TIMP program and its graphical user interface Glotaran 45. For high pH, the kinetic model (reaction scheme) published by Váró et al. was chosen (see results and discussion) 15. A truncated version of the reaction scheme published by Lakatos et al. was used for analysis of low pH transient data 29 (Scheme 1):

pH dependent transient data

The buffer contained 300 mm NaCl, 0.05% w/v DDM, 20 mm MES and 20 mm 1,3-bis(tris(hydroxymethyl)methylamino)-propane (Bis-Tris propane). pH was adjusted by addition of small amounts of NaOH or HCl. Excitation wavelength was chosen close to the respective absorption maximum between 520 nm and 545 nm. The average excitation energy was 1 mJ cm⁻².

Excitation wavelength variation

PR wild-type buffer contained 300 mm NaCl, 0.1% w/v DDM and 5 mm MES at pH 5.8. Average excitation energy was 1.4 mJ cm⁻² at 470 nm and 580 nm. The buffer for the H75N mutant contained 300 mm NaCl, 0.05 % w/v DDM, 50 mm MES, 50 mm imidazole at pH 5.6. Average excitation energy was 4 mJ cm⁻² at 470 nm and 590 nm.

Data processing

Data preparation for fitting: Data points with an exponentially increasing separation in time were extracted from absorbance change raw data to reduce the number of values to be fitted.

Data preparation for presentation: Absorbance change raw data were smoothed using a Kaiser low-pass filter with a cutoff frequency of 10⁵ Hz. Afterwards, the number of data points was reduced as described above.

Results

Spectral decomposition of pH-dependent PR wild-type absorption spectra

A major absorption band in the visible spectral range is characteristic for absorption spectra of type-I rhodopsins. Its spectral position and shape depends (inter alia) on the pH value (Fig. 2) 29, 46. A reversible spectral shift takes place between pH 5.5 and pH 9.2. From pH 4.5 to pH 5.5, the absorption maximum (λ_max) is located at ca. 544 nm (see Figure S1) and shifts to ca. 516 nm at pH 9.2. The position of the maximum persists up to pH 9.9 (see Figure S1).

An isosbestic point is observed at 529 nm. This is in disagreement with a putative shift of a single homogeneous absorption band, but rather indicates two spectrally overlapping absorption bands whose amplitudes are pH dependent. Each measured spectrum was decomposed into two absorption bands by a fitting procedure described in 2 section. Figure 2 (see inset) shows the value α as a function of pH. α can be regarded as the normalized amplitude of a band at λ_max = 544 nm. The graph was parameterized using a logistic function (see 2 section Eq. 3). An inflection point P is located at pH 7.3, and the value of the exponent n is 0.53. These values correspond to published values for P (P is often regarded as a pK_a value in the literature) and n obtained by other methods of absorption spectra analysis 11, 12.

pH dependence of PR wild-type photocycle kinetics

PR and BR photocycle kinetics can be described by sequential reaction schemes 15, 47. Photocycle intermediates of PR are often termed in analogy to the BR photocycle intermediates K, L, M, N and O (some intermediate substates postulated in literature are not listed 2). K (or a K-like precursor) emerges on a timescale of picoseconds after photoexcitation 48. The reaction sequence is finished by the transition of the O intermediate to the initial state on a timescale of milliseconds 49. For the high pH (≥ 9) PR photocycle, K-, M-, N- and O-like/PR’ intermediates have been identified 12, 15. The low pH (<6) PR-photocycle is different from the former one and comprises K-, L-, N- and O-like/PR’ intermediates (some authors found evidence for the existence of an M-like intermediate in the low pH photocycle kinetics, but it probably does not accumulate for kinetic reasons at room temperature 13) 12, 29.

Figure 3 shows intermediate spectra calculated on the basis of time dependent difference absorbance spectra (see Figure S2) of solubilized PR wild-type samples at high and low pH (see Tables S1 and S2 for corresponding rate constants). These spectra resemble those published for reconstituted PR samples with two exceptions 15, 29. No N-like intermediate was identified at low pH. At high pH, no PR’ intermediate could be detected. Váró et al. described the PR’ absorption spectrum to be indistinguishable from the resting state absorption spectrum 15. In case of DDM solubilized PR at high pH, the absorption spectrum of the last intermediate is red shifted with respect to the resting state absorption. This property was reported for the spectrum of the last intermediate O in the BR photocycle too 50. Consequently, the last intermediate of the PR photocycle is termed O-like.

Photoinduced transient absorbance changes were detected by difference spectroscopy. Excitation wavelength was adjusted to the respective absorption maximum (to achieve maximal absorbance changes) in the range from 520 nm to 545 nm. Three representative detection wavelengths were chosen (Fig. 4): 600 nm, 500 nm and 400 nm (400 nm was chosen due to the comparatively low radiant sensitivity of the existent photomultiplier tube in the near UV region.). At 600 nm positive contributions of K-, N- and O-like intermediates dominate the signal, small negative contributions of resting state depletion and small positive contribution of the low pH PR’ intermediate are also present. Resting state depletion, L-like and low pH PR’ intermediate dynamics are observable at 500 nm; furthermore, K- and O-like intermediates contribute to a minor extent. At 400 nm, the dominating M-like intermediate absorbance superimposes small contributions of resting state depletion in most time domains (see Fig. 4c, transient at pH 7.5 and 0.02 s).

PR photocycle kinetics have been investigated at various pH values between pH 5 and pH 9. At 600 nm detection wavelength and pH 9, the decay of a K-like intermediate is observed on a timescale of microseconds (Fig. 4a) (no time dependent measurements were carried out at pH values above 9 to avoid accumulation of significant fractions of a long-lived photoproduct 51). Absorbance changes of N-like and O-like intermediates build up within a few milliseconds (Kinetics of these intermediates can be separated by quantitative analysis methods.). A maximum is reached at ca. 30 ms. Decay of these intermediates is nearly completed after one-second. At pH 5, the decay of a K-like intermediate is also observed, but only minor contributions of late intermediates are present. The accumulated amounts of N- and O-like intermediates decrease continuously, when the pH value is lowered.

Figure 4b depicts transient absorbance changes at 500 nm detection wavelength. At pH 9, the decay of the K-like intermediate is observed on a timescale of microseconds. It converts into the M-like intermediate which has no spectral contribution at 500 nm 15. Repopulation of the resting state from the O-like intermediate is not completely finished after 1 s. At pH 5, positive contributions of a K-like intermediate are present on a timescale of microseconds as well, but they convert to the L-like intermediate which has a substantial positive contribution at 500 nm. Thus, no local minimum emerges. A continuous conversion of high pH progression to the low pH progression is observed.

Figure 4c shows transient absorbance changes at 400 nm detection wavelength. The amount of accumulated M-like intermediate decreases, when the pH value is decreased. At pH 5, no M-like intermediate can be detected at room temperature, as already reported elsewhere 12, 14, 29. Furthermore, it is impossible to superimpose the transients obtained at 400 nm and different pH values by scaling. The M-like intermediate decay seems to be pH dependent. It persists more than 100 ms at pH 9, but disappears after 10 ms at pH 7.5.

Table 1 shows results of a global lifetime analysis of the transient traces recorded at pH 9 and pH 5 with sums of exponential terms. At pH 9, six terms are needed, whereas four terms are sufficient at pH 5. The low pH photocycle is faster than the high pH photocycle, similar to what was published elsewhere for PR in lipid environment (compare Lakatos et al. and Váró et al. 15, 29). Datasets between pH 5 and pH 9 indicate contributions of both photocycle kinetics; however, global lifetime analysis with more than six terms is not feasible in that pH range.

Table 1. Time constants τ obtained by global lifetime analysis of time-dependent data (raw data) at pH 9 and pH 5 presented in Fig. 4

	τ₁ [μs]	τ₂ [μs]	τ₃ [ms]	τ₄ [s]	τ₅ [s]	τ₆ [s]
pH 9	4	180	2.4	0.011	0.17	0.75
pH 5	8	100	14	0.072	–	–

Excitation wavelength dependence of PR wild-type photocycle kinetics at pH 5.8

Transient absorbance changes of a PR sample excited at 470 nm or 580 nm are shown in Fig. 5. Almost no M-like intermediate accumulates after excitation at 580 nm as observed at 400 nm detection wavelength. At 470 nm excitation wavelength, small amounts of M-like intermediate are present. Furthermore, the transient at 600 nm detection wavelength and 470 nm excitation wavelength indicates higher amounts of N- and O-like intermediates than at 600 nm detection wavelength and 580 nm excitation wavelength.

pH dependence of PR H75N absorption

For solubilized H75N, a spectral shift is observed between pH 7.9 and pH 3.5 (Fig. 6). λ_max is 510 nm at pH 7.9 and 547 nm at pH 3.5. No clear isosbestic point can be identified. Therefore, a decomposition of the absorption spectra in two bands, as described for PR wild-type data, was not applied. An alternative (approximate) evaluation method is to plot the absorption maxima as a function of pH value (see inset of Fig. 6) 52. That graph was parameterized with a logistic function (see 2 section Eq. 3). An inflection point P is located at pH 5.6, and the value for n is 0.8. Similar values have been published for the H75N mutant elsewhere 37.

Excitation wavelength dependence of PR H75N photocycle kinetics at pH 5.6

Figure 7 shows transient absorbance changes of H75N excited at 470 nm, 530 nm or 590 nm. At 410 nm detection wavelength, an M-like intermediate rise is observed within ca. 50 μs and its decay is nearly finished after 1 ms (Fig. 7a). Small contributions of the resting state depletion persist between 1 ms and ca. 100 ms. As already observed for the wild type, the accumulated amount of M-like intermediate decreases with increasing excitation wavelength. At 580 nm detection wavelength, the trends resemble those observed for PR wild type (Fig. 7b). Higher amounts of N- and O-like intermediates accumulate at lower excitation wavelength.

Discussion

PR wild-type absorption spectra (visible spectral range, pH 5.5 to pH 9.2) are regarded as a superposition of two absorption bands with maxima at 544 nm and 516 nm. Each band represents a protein state, termed DH (λ_max = 544 nm) and D⁻ (λ_max = 516 nm), which are in pH dependent equilibrium. The primary proton acceptor is assumed to be protonated in state DH and deprotonated in state D⁻. Conversion of state DH to state D⁻ introduces a negative charge in close vicinity to the retinal Schiff-base. The presence of that charge increases the energy of the chromophore's S₀ → S₁ electronic transition 53. (The chromophore undergoes a S₀ → S₁ transition in protein states DH as well as D⁻, but due to the difference in excitation energy, the transition is referred to as two absorption bands.) This explanation is based on conclusions drawn from pH titrations of BR (purple to blue transition) and has already been applied to a variety of type-I rhodopsins including PR 12, 17, 54, 55. The interpretation could be verified by photometric titration of a PR mutant that lacks the primary proton acceptor (see Figure S1), but it has not yet been confirmed by experimental methods which directly detect the protonation state of the primary proton acceptor.

The value α shown in the inset of Fig. 2 is equal to the relative concentration (C_DH/(C_DH + C_D−), see 2 section) of state DH. Hence, the graph represents a titration curve of the primary proton acceptor. Parametrization with the Henderson–Hasselbalch equation gives poor results, but analogous photometric titrations of PR have been described by the Hill-equation in the literature 12, 29. The Hill coefficient n was found to be smaller than 1, and the data may be discussed in terms of negative cooperativity. However, the data presented in Fig. 2 can be treated as a titration of two independent PR fractions which differ regarding the pK_a values of their primary proton acceptor as well. Due to this ambiguity, the titration curve is not discussed in terms of a specific chemical model. Nevertheless, the inflection point P = 7.3 can be regarded as the apparent pK_a of the primary proton acceptor.

Solubilized PR photocycle intermediate spectra and photocycle kinetics at pH values considerably higher than the pK_a (pH 9) and below the pK_a (pH < 6) resemble those of PR samples in lipid environment presented in the literature 12, 15, 29. We refer to the authors discussions of the corresponding datasets in terms of homogeneous high pH photocycle kinetics and homogeneous low pH photocycle kinetics 12, 15, 29. To avoid confusion in the discussion below, the high pH photocycle kinetics is termed “M-photocycle kinetics” and low pH photocycle kinetics is termed “L-photocycle kinetics.”

The amplitudes of absorbance changes decrease when the pH value is lowered. To some extent that might be due to different spectral characteristics of the L- and M-photocycle kinetics intermediate spectra. Changes of the quantum yield of K-like intermediate formation cannot be excluded as well. This has been discussed with respect to the protonation state of the primary proton acceptor in the literature 42.

Changes of M-, N- and O-like intermediates accumulation indicate continuous reduction in the M-photocycle kinetics contribution when the pH value is lowered. The lower the pH value, the higher the contribution of L-photocycle kinetics. This suggests coexistence of (at least) two cycling fractions.

These fractions depend on pH value but also on excitation wavelength. The excitation wavelength dependence is no artifact caused by detergent solubilization. In case of an lipid environment, M-like intermediate accumulation also changes when different excitation wavelengths are chosen (see Figure S3).

It is assumed that excitation of state D⁻ induces M-photocycle kinetics and excitation of state DH induces L-photocycle kinetics. The absorption band of state D⁻ is shifted to lower wavelengths with respect to the absorption band of state DH. Therefore, the relative probability of M-photocycle kinetics induction is higher at 470 nm excitation wavelength than at 580 nm excitation wavelength.

The same argumentation holds for the mutant H75N. Its photocycle kinetics has already been described elsewhere 37. The kinetics resemble L- and M-photocycle kinetics qualitatively, and the contribution of the latter vanishes at low pH. However, it is not yet completely elucidated why the H75N photocycle proceeds faster than the one of the PR wild type.

As observed for the wild type, the contribution of the H75N M-photocycle kinetics decreases, when the excitation wavelength increases. But in contrast to the PR wild type at pH 5.8 and 580 nm excitation wavelength, a significant fraction of molecules pass the M-photocycle kinetics in the H75N sample at pH 5.6 and 590 nm excitation wavelength. The mutant's D⁻ fraction is bigger at low pH as its pK_a is lower than the pK_a of PR wild type. The inflection point P = 5.6 of the graph presented in the inset of Fig. 6 provides an estimation for the pK_a of the mutant.

Accordingly, photocycle kinetics and the titration curve of PR are correlated. That correlation is preserved in the absence of His 75. As a preliminary approximation, PR wild-type photocycle kinetics observed between pH 9 and pH 5.5 is considered to be a linear superposition of photocycle kinetics at pH 9 and at pH 5.5. Their ratio depends (excitation at, or rather close to 529 nm is assumed) on the fraction of D⁻ and DH state given by the titration curve. Figure 8 shows absorbance change data in dependence of the pH value at a fixed time after excitation. Absorbance change values and titration curve follow the same trend, but in disagreement with the approximation above, it is impossible to superimpose these values by linear scaling.

Differences of the titration curve and absorbance change data might be associated with contributions of additional photoinduced kinetics. A potential candidate is a 13-cis photocycle known from BR 56. However, the 13- cis retinal content of PR samples in thermal equilibrium was found to be negligible by spectroscopic techniques 27. A 13-cis photocycle kinetics is thus not taken into consideration. Another possible reason for the observed differences are changes of excitation wavelength. The data presented in Fig. 8 originate from flash photolysis experiments at excitation wavelengths between 520 nm and 545 nm. These deviations from the isosbestic point at 529 nm are neglected too.

The fact that M-like intermediate kinetics depends on pH (Fig. 4c) is inconsistent with the assumption of a linear superposition of photocycle kinetics at pH 9 and at pH 5.5. To account for that dependency, at least one of the transitions in M-photocycle kinetics has to be related to proton concentration in the aqueous phase. M-intermediate decay is not directly related to proton concentration of the aqueous phase, but rather to the protonation state of the primary proton donor (Glu-108). Reprotonation of the latter from cyctosolic side is likely to be pH dependent. That step has been assigned to a putative transition of two N-intermediate substates by Dioumaev et al. 11. Alternatively, the primary proton donor might be reprotonated during the N- to PR’(O)-like intermediate transition, similar to what was observed for BR 57. Proton uptake (and release) is indicated in a M-photocycle reaction scheme published by Váró et al. (protons were added by the authors) 15 (Scheme 2).

The reaction scheme is extended by considering k₄ as a pseudo-first-order rate constant, which is a product of (time independent) proton concentration and a rate constant k. k₄ increases when the pH is lowered. Therefore, the accumulated amount of PR’(O) increases. That promotes irreversible decay of the system to the initial state PR. k₄ is low at high pH. The accumulated amount of N increases, and therefore, the rate of the back reaction N to M₂ increases as well. That promotes formation of a “transient equilibrium” of intermediates. In turn, decay of the M-like intermediate is delayed at high pH with respect to low pH.

Delayed decay of the M-like intermediate at pH values above 9 has been observed by other groups for PR in lipid environment too, but the magnitude of the effect varies considerably 12, 15, 39, 58. This may depend on sample composition or preparation.

At pH 9 M-like intermediate absorbance change exhibits a maximum at ca. 1 ms (Fig. 4c). At pH values below 9, the M-like intermediate absorbance change decreases at 1 ms already. Hence, some of the absorbance change values at 400 nm detection wavelength presented in Fig. 8 are lower than expected. As the signal related to the M-like intermediate decays earlier at low pH, N- and O-like intermediate accumulation reaches a maximum earlier as well. Therefore, some of the absorbance change values at 600 nm detection wavelength presented in Fig. 8 are higher than expected.

Time-dependent data between pH 9 and pH 5.5 are finally regarded as a linear superposition of M- and L-photocycle kinetics at the actual pH value of the measurement. pH affects photocycle kinetics in several respects: Beside the protonation state of the primary proton acceptor, “transient equilibria” of other titratable groups (such as Schiff-base and primary proton donor) seem to be relevant too. The effects of additional groups have been discussed for PR photocycle kinetics at pH 5.5 and pH 5.1 in Verhoefen et al. 28.

Conclusion

Delay of M-intermediate decay at high pH is known from BR 59. This phenomenon had been interpreted in terms of an M-/N-intermediate equilibrium analogous to the explanation of the M-like intermediate pH dependence given for PR here 60. Furthermore, static PR absorption spectra can be discussed on the basis of BR data. The concept of superposition of two different PR photocycle kinetics between pH 5 and pH 9 is compatible with established facts for BR as well.

Therefore, no unknown relationship between the protonation state of the primary proton acceptor and photocycle kinetics could be identified for PR. Although His-75 influences pK_a and photocycle kinetics, the aforesaid relationship persists basically, when this amino acid is replaced. Regarding primary proton acceptor function, there is no evidence for a unique mechanism of PR action, which necessitates a high pK_a.

Moreover, a high pK_a does not seem to be essential for PR function. Replacing His-75 by Asn lowers the pK_a. As expected, the mutant has been shown to be active in proton transport from the cytosolic to the extracellular side down to pH 4 37. Furthermore, type-I rhodopsin proton pumps which lack the proton release complex of BR and possess a homolog of His-75 were found in a marine eukaryote (Oxyrrhis marina rhodopsin), a terrestrial bacterium (Exiguobacterium sibiricum rhodopsin) and a thermophilic bacterium (Thermus thermophilus rhodopsin) recently 61-63. The pK_a is < 3 for Oxyrrhis marina rhodopsin, 2.3 for Exiguobacterium sibiricum rhodopsin (beside pK_a 2.3 two more transitions at pK_a 6 and at pK_a 9.1 were reported) and 3.4 for thermophilic rhodopsin 61-63. Oxyrrhis marina rhodopsin and Thermus thermophilus rhodopsin belong to the PR family, whereas Exiguobacterium sibiricum rhodopsin was classified as an “unusual PR” 64.

To account for the elevated pK_a of PR, a speculation based on the aforesaid facts is as follows: High pK_a values might be characteristic for PR variants of marine bacterial origin but do not seem to be an intrinsic property among all proton pumping PR variants. Provided proton transport is based on similar mechanisms within that group, other factors than a special transport mechanism need to induce the elevated pK_a of PR. These factors could be related to marine bacteria physiology and environmental conditions.

The PR variant investigated here resides in the inner membrane of gram negative bacteria living in the photic zone at ca. pH 8. A fraction of the protons pumped to the extracellular (periplasmatic) side passes the outer membrane (the outer membrane is permeable for protons 65) and disappears into the environment 7. In case of an alkaline environment, such as surface seawater, the local pH value on the outer side of the inner membrane is assumed to be decreased only moderately below ambient pH by proton pump activity.

This might have consequences for PR regulation. BR is regulated by negative feedback. At a certain membrane potential and low extracellular pH, the primary proton acceptor is protonated via the extracellular half channel 66, 67. Provided that the local extracellular (periplasmatic) pH cannot drop substantially below pH 8 in the case of PR, the activity could hardly be downregulated, when PR had a low pK_a. A high pK_a allows the protein activity to be regulated at the expense of proton pumping efficiency.

By contrast, Oxyrrhis marina rhodopsin, Thermus thermophiles rhodopsin and Exiguobacterium sibiricum rhodopsin possibly generate higher local proton concentrations than PR. Oxyrrhis marina rhodopsin resides in an endomembrane system of the marine eukaryote 63. That localization inhibits proton losses into environmental seawater. The environmental pH-values of Exiguobacterium sibiricum and Thermus thermophiles are close to 7, so proton losses due to neutralization reactions are smaller in those cases 61, 62. According to the speculation above, high local proton concentrations favor a low pK_a and therefore allow for a higher proton pumping efficiency.

The pK_a of the primary proton acceptor might be an example for molecular adaptation. Its value may depend on environmental pH as well as on the different physiology of those microorganisms which express PR variants. However, measurements of PR photocycle kinetics and PR activity within living original organisms are necessary to confirm and specify these proposals.

Acknowledgements

This work has been supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich (SFB) 807.

Supporting Information

References

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Citing Literature

Volume93, Issue3

May/June 2017

Pages 762-771

Proteorhodopsin Photocycle Kinetics Between pH 5 and pH 9^†

Abstract

Introduction