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Protonation/Deprotonation Reactions Triggered by Photoactivation of Photoactive Yellow Protein from Ectothiorhodospira halophila *

  • Johnny Hendriks
    Affiliations
    From the Laboratory for Microbiology, E. C. Slater Institute, BioCentrum, University of Amsterdam, 1018 WS Amsterdam, The Netherlands
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  • Wouter D. Hoff
    Footnotes
    Affiliations
    From the Laboratory for Microbiology, E. C. Slater Institute, BioCentrum, University of Amsterdam, 1018 WS Amsterdam, The Netherlands
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  • Wim Crielaard
    Affiliations
    From the Laboratory for Microbiology, E. C. Slater Institute, BioCentrum, University of Amsterdam, 1018 WS Amsterdam, The Netherlands
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  • Klaas J. Hellingwerf
    Correspondence
    To whom correspondence should be addressed: Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands. Tel.: 31-20-5257055; Fax: 31-20-5257056;
    Affiliations
    From the Laboratory for Microbiology, E. C. Slater Institute, BioCentrum, University of Amsterdam, 1018 WS Amsterdam, The Netherlands
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  • Author Footnotes
    * This research was supported by the Netherlands Foundation for Chemical Research and the Netherlands Foundation for Life Science, with financial assistance from the Netherlands Organization for Scientific Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Present address: Dept. of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637.
Open AccessPublished:June 18, 1999DOI:https://doi.org/10.1074/jbc.274.25.17655
      Light-dependent pH changes were measured in unbuffered solutions of wild type photoactive yellow protein (PYP) and its H108F and E46Q variants, using two independent techniques: transient absorption changes of added pH indicator dyes and direct readings with a combination pH electrode. Depending on the absolute pH of the sample, a reversible protonation as well as a deprotonation can be observed upon formation of the transient, blue-shifted photocycle intermediate (pB) of this photoreceptor protein. The latter is observed at very alkaline pH, the former at acidic pH values. At neutral pH, however, the formation of the pB state is not paralleled by significant protonation/deprotonation of PYP, as expected for concomitant protonation of the chromophore and deprotonation of Glu-46 during pB formation. We interpret these results as further evidence that a proton is transferred from Glu-46 to the coumaric acid chromophore of PYP, during pB formation. One cannot exclude the possibility, however, that this transfer proceeds through the bulk aqueous phase. Simultaneously, an amino acid side chain(s) (e.g. His-108) changes from a buried to an exposed position. These results, therefore, further support the idea that PYP significantly unfolds in the pB state and resolve the controversy regarding proton transfer during the PYP photocycle.
      PYP
      The abbreviations used are: PYP, photoactive yellow protein; pG, ground state of PYP; pB, transient, blue-shifted photocycle intermediate of PYP; pR, transient red-shifted photocycle intermediate of PYP; MES, 2-(N-morpholino)ethanesulfonic acid.
      1The abbreviations used are: PYP, photoactive yellow protein; pG, ground state of PYP; pB, transient, blue-shifted photocycle intermediate of PYP; pR, transient red-shifted photocycle intermediate of PYP; MES, 2-(N-morpholino)ethanesulfonic acid.
      (
      • Meyer T.E.
      ,
      • Sprenger W.W.
      • Hoff W.D.
      • Armitage J.P.
      • Hellingwerf K.J.
      ) presumably is a blue-light photoreceptor from the purple sulfur bacteriumEctothiorhodospira halophila (2), which shows many similarities with the archaeal sensory rhodopsins (
      • Meyer T.E.
      • Yakali E.
      • Cusanovich M.A.
      • Tollin G.
      ,
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Van Ramesdonk J.
      • Van Brederode M.E.
      • Brouwer A.M.
      • Fitch J.C.
      • Meyer T.E.
      • Van Grondelle R.
      • Hellingwerf K.J.
      ), although PYP contains 4-hydroxycinnamic acid as its chromophore (
      • Hoff W.D.
      • Düx P.
      • Hård K.
      • Devreese B.
      • Nugteren-Roodzant I.M.
      • Crielaard W.
      • Boelens R.
      • Kaptein R.
      • van Beeumen J.
      • Hellingwerf K.J.
      ,
      • Baca M.
      • Borgstahl G.E.O.
      • Boissinot M.
      • Burke P.M.
      • Williams W.R.
      • Slater K.A.
      • Getzoff E.D.
      ) and is water-soluble. Activation of PYP function proceeds through light-induced trans/cis isomerization of the 7,8-vinyl bond of its chromophore (
      • Kort R.
      • Vonk H.
      • Xu X.
      • Hoff W.D.
      • Crielaard W.
      • Hellingwerf K.J.
      ,
      • Genick U.K.
      • Borgstahl G.E.
      • Ng K.
      • Ren Z.
      • Pradervand C.
      • Burke P.M.
      • Srajer V.
      • Teng T.Y.
      • Schildkamp W.
      • McRee D.E.
      • Moffat K.
      • Getzoff E.D.
      ). This ultimately leads to a modulation of the direction of rotation of the flagella of the photoreceptor-producing cell. The chromophore is present in the anionic form in the ground state (pG) of PYP (
      • Borgstahl G.E.O.
      • Williams D.R.
      • Getzoff E.D.
      ,
      • Kim M.
      • Mathies R.A.
      • Hoff W.D.
      • Hellingwerf K.J.
      ). The anionic phenolate is buried within the hydrophobic core of PYP and is stabilized via a hydrogen-bonding network involving the amino acids Tyr-42, Thr-50, and (protonated) Glu-46 (
      • Borgstahl G.E.O.
      • Williams D.R.
      • Getzoff E.D.
      ). Photoactivation of PYP initiates a photocycle containing several transient intermediate states. In a very short time, red-shifted intermediates are formed, of which pR has the longest lifetime. It decays bi-exponentially (with rate constants of 4·103 and 8·102 s−1 (
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Van Ramesdonk J.
      • Van Brederode M.E.
      • Brouwer A.M.
      • Fitch J.C.
      • Meyer T.E.
      • Van Grondelle R.
      • Hellingwerf K.J.
      )) into a blue-shifted intermediate (pB). The latter is the longest living intermediate of this cycle (τ = 0.15 s (
      • Meyer T.E.
      • Yakali E.
      • Cusanovich M.A.
      • Tollin G.
      ,
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Van Ramesdonk J.
      • Van Brederode M.E.
      • Brouwer A.M.
      • Fitch J.C.
      • Meyer T.E.
      • Van Grondelle R.
      • Hellingwerf K.J.
      ,
      • Meyer T.E.
      • Tollin G.
      • Hazzard J.H.
      • Cusanovich M.A.
      ,
      • Ujj L.
      • Devanathan S.
      • Meyer T.E.
      • Cusanovich M.A.
      • Tollin G.
      • Atkinson G.H.
      )) and is also referred to as I2 (e.g. see Refs.
      • Meyer T.E.
      • Yakali E.
      • Cusanovich M.A.
      • Tollin G.
      and
      • Ujj L.
      • Devanathan S.
      • Meyer T.E.
      • Cusanovich M.A.
      • Tollin G.
      • Atkinson G.H.
      ). The photocycle of PYP can be observed in a wide range of pH values, although significant effects on the rates of the transitions are observed (
      • Genick U.K.
      • Devanathan S.
      • Meyer T.E.
      • Canestrelli I.L.
      • Williams E.
      • Cusanovich M.A.
      • Tollin G.
      • Getzoff E.D.
      ,
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Gural J.
      • Hellingwerf K.J.
      ).
      ApoPYP can be produced heterologously in Escherichia coli(
      • Kort R.
      • Hoff W.D.
      • Van West M.
      • Kroon A.R.
      • Hoffer S.M.
      • Vlieg K.H.
      • Crielaard W.
      • Van Beeumen J.J.
      • Hellingwerf K.J.
      ) and can subsequently be converted to functional holoprotein through reconstitution with activated derivatives of its chromophore (
      • Imamoto Y.
      • Ito T.
      • Kataoka M.
      • Tokunaga F.
      ,
      • Kroon A.R.
      • Hoff W.D.
      • Fennema H.
      • Gijzen J.
      • Koomen G.-J.
      • Verhoeven J.W.
      • Crielaard W.
      • Hellingwerf K.J.
      ). This capability has made PYP available in sufficient amounts to allow detailed analysis of its photocycle using biophysical techniques like x-ray diffraction, Fourier transform infrared, Raman, NMR, transient absorption, and fluorescence spectroscopy (seee.g. Refs.
      • Genick U.K.
      • Borgstahl G.E.
      • Ng K.
      • Ren Z.
      • Pradervand C.
      • Burke P.M.
      • Srajer V.
      • Teng T.Y.
      • Schildkamp W.
      • McRee D.E.
      • Moffat K.
      • Getzoff E.D.
      ,
      • Kim M.
      • Mathies R.A.
      • Hoff W.D.
      • Hellingwerf K.J.
      ,
      • Ujj L.
      • Devanathan S.
      • Meyer T.E.
      • Cusanovich M.A.
      • Tollin G.
      • Atkinson G.H.
      , and
      • Imamoto Y.
      • Ito T.
      • Kataoka M.
      • Tokunaga F.
      ,
      • Kroon A.R.
      • Hoff W.D.
      • Fennema H.
      • Gijzen J.
      • Koomen G.-J.
      • Verhoeven J.W.
      • Crielaard W.
      • Hellingwerf K.J.
      ,
      • Xie A.
      • Hoff W.D.
      • Kroon A.R.
      • Hellingwerf K.J.
      ,
      • Baltuska A.
      • van Stokkum I.H.M.
      • Kroon A.
      • Monshouwer R.
      • Hellingwerf K.J.
      • van Grondelle R.
      ,
      • Chosrowjan H.
      • Mataga N.
      • Nakashima N.
      • Imamoto Y.
      • Tokunaga F.
      ,
      • Changenet P.
      • Zhang H.
      • van der Meer M.
      • Hellingwerf K.J.
      • Glasbeek M.
      ,
      • Imamoto Y.
      • Mihara K.
      • Hisatomi O.
      • Kataoka M.
      • Tokunaga F.
      • Bojkova N.
      • Yoshihara K.
      ), and it has led to detailed insight into the photocycle characteristics of PYP.
      One aspect that has not yet been clearly resolved, however, is the involvement of net proton uptake/release during the transitions of the PYP photocycle. In an early study (
      • Meyer T.E.
      • Cusanovich M.A.
      • Tollin G.
      ) it was reported that during the lifetime of pB, PYP takes up a proton from solution. In a subsequent paper (
      • Genick U.K.
      • Devanathan S.
      • Meyer T.E.
      • Canestrelli I.L.
      • Williams E.
      • Cusanovich M.A.
      • Tollin G.
      • Getzoff E.D.
      ) the observation of proton uptake in parallel with pB formation was confirmed. The proton uptake was interpreted as the result of protonation of the chromophore after its exposure to solvent (
      • Genick U.K.
      • Borgstahl G.E.
      • Ng K.
      • Ren Z.
      • Pradervand C.
      • Burke P.M.
      • Srajer V.
      • Teng T.Y.
      • Schildkamp W.
      • McRee D.E.
      • Moffat K.
      • Getzoff E.D.
      ).
      In pR, at 77 K, the hydrogen bond between the chromophore and Glu-46 remains intact, as has been demonstrated with Fourier transform infrared (
      • Xie A.
      • Hoff W.D.
      • Kroon A.R.
      • Hellingwerf K.J.
      ) and x-ray diffraction (
      • Genick U.K.
      • Soltis S.M.
      • Kuhn P.
      • Canestrelli I.L.
      • Getzoff E.D.
      ). This finding led to the proposal (
      • Xie A.
      • Hoff W.D.
      • Kroon A.R.
      • Hellingwerf K.J.
      ) that isomerization of the 4-hydroxycinnamic acid chromophore occurs through a two-bond isomerization reaction, which is characterized by a rotation of the carbonyl group of the chromophore around the long axis of the 4-hydroxycinnamic acid. In addition, it was proposed that during pB formation, proton transfer occurs from Glu-46 to the chromophore. Subsequent extension of these Fourier transform infrared analyses, using isotope enrichment and variants of PYP obtained through site-directed mutagenesis, provided further evidence supporting this proposal (
      • Imamoto Y.
      • Mihara K.
      • Hisatomi O.
      • Kataoka M.
      • Tokunaga F.
      • Bojkova N.
      • Yoshihara K.
      ). However, time-resolved x-ray crystallography of PYP in the nanosecond time domain indicates that at room temperature the hydrogen bond between the side chain of Glu-46 and the phenolic oxygen of the chromophore is already disrupted in the pR intermediate (
      • Perman B.
      • Srajer V.
      • Ren Z.
      • Teng T.
      • Pradervand C.
      • Ursby T.
      • Bourgeois D.
      • Schotte F.
      • Wulff M.
      • Kort R.
      • Hellingwerf K.J.
      • Moffat K.
      ). Therefore, the pathway of proton transfer during the PYP photocycle still is an unresolved issue. The two most extreme views are that this proton transfer may take place directly, within the chromophore binding pocket inside the protein, or indirectly, through the bulk solvent.
      As the chromophore of PYP during pB formation is protonated (
      • Genick U.K.
      • Borgstahl G.E.
      • Ng K.
      • Ren Z.
      • Pradervand C.
      • Burke P.M.
      • Srajer V.
      • Teng T.Y.
      • Schildkamp W.
      • McRee D.E.
      • Moffat K.
      • Getzoff E.D.
      ) and Glu-46 is deprotonated (
      • Xie A.
      • Hoff W.D.
      • Kroon A.R.
      • Hellingwerf K.J.
      ,
      • Imamoto Y.
      • Mihara K.
      • Hisatomi O.
      • Kataoka M.
      • Tokunaga F.
      • Bojkova N.
      • Yoshihara K.
      ), net proton uptake by the protein while it progresses through the photocycle is difficult to understand. Therefore, we have investigated these reversible (de)protonation reactions, using not only transient absorption spectroscopy, in combination with pH indicator dyes, but also transient pH measurements with a sensitive combination pH electrode. The latter technique has been combined with simultaneous absorption measurements. Deprotonation of bacteriorhodopsin, present in the form of purple membranes, served as a control to test the sensitivity of the set-up for pH measurements.

      EXPERIMENTAL PROCEDURES

      Materials

      Recombinant apoPYP and two variants obtained through site-directed mutagenesis were produced heterologously inE. coli, as described previously (
      • Kort R.
      • Hoff W.D.
      • Van West M.
      • Kroon A.R.
      • Hoffer S.M.
      • Vlieg K.H.
      • Crielaard W.
      • Van Beeumen J.J.
      • Hellingwerf K.J.
      ). ApoPYP was reconstituted with the anhydride derivative of 4-hydroxycinnamic acid according to Imamoto et al. (
      • Imamoto Y.
      • Ito T.
      • Kataoka M.
      • Tokunaga F.
      ). The obtained PYP was used after removal of its polyhistidine tail. Purple membranes were kindly provided by D. Oesterhelt (MPI, Martinsried, Germany). 4-Hydroxycinnamic acid was obtained from Sigma. All other materials were reagent grade and were obtained from commercial sources.

      Site-directed Mutagenesis

      The PYP variants E46Q and H108F were made by site-directed mutagenesis using the mega-primer method (
      • Landt O.
      • Grunert H.-P.
      • Hahn U.
      ). The gene products were first screened, using restriction analyses, and mutagenesis was subsequently confirmed by DNA sequencing.

      Spectroscopy

      UV/Vis static and transient absorption spectra were recorded with a model 8453 Hewlett Packard diode array spectrophotometer (Portland, OR), which has a time resolution of 0.1 s. Typically UV/Vis spectra from 250 to 550 nm were recorded every 0.1 s.

      Nanosecond Time-resolved Absorption Spectroscopy

      Laser-induced transient absorption spectra were measured with a system composed of a Continuum Surelite I-10 YAG laser (output intensity 140 mJ at 355 nm), a Continuum Surelite OPO (output range 410–2200 nm, set at 446 nm), and a LP900 Spectrometer, custom made by Edinburgh Instruments Ltd. (Edinburgh, UK). The spectrometer contains a 450-watt short arc Xe lamp in combination with a pulsed power supply and a Peltier cooled CCD camera (Wright Instruments). The time resolution attainable with this set-up is 10 ns.

      pH Measurements

      pH measurements were carried out with a Mettler Toledo micro(combination)-electrode (InLab 423) connected to a Dulas Engineering amplifier (input impedance > 1013ohms). The amplified signal was fed into a linear strip-chart recorder (Kipp & Zonen, Delft, The Netherlands, type BD41). A battery driven back-off box was used to decrease the signal to the appropriate size. pH changes were converted into moles of protons by calibration with microliter amounts of 2.5 mm oxalic acid. Simultaneously, the absolute pH of the solution was visualized on the display of the pH meter. The electrode was calibrated with calibration buffers of pH 4.01, 6.98, and 9.18 (Yokogawa Europe BV, Amersfoort, The Netherlands). The electrode signal was usually recorded at 0.025–0.1 pH units full scale sensitivity.

      Simultaneous Transient Absorption and pH Measurements

      Absorption and pH signals were measured simultaneously by placing a “Kraayenhof vessel” (
      • Kraayenhof R.
      • Schuurmans J.J.
      • Valkier L.J.
      • Veen J.P.
      • Van Marum D.
      • Jasper C.G.
      ) in the sample compartment of the Hewlett Packard 8453 spectrophotometer. Two of the four available ports of the vessel were used for the measuring beam of the spectrophotometer, and a third one was used for the combination pH electrode. Measurements were carried out at room temperature (between 291 and 293 K). Although this vessel is equipped with Peltier temperature control, this was not used to reduce temperature artifacts (see “Results”). Continuous actinic illumination was provided through the fourth port of the vessel by a Schott KL1500 light source (containing a 150-watt halogen lamp). PYP was routinely used at a concentration of 33 μm in a working volume of 1.8–2 ml using an unbuffered solution containing 1 m KCl.

      RESULTS

      Laser-induced Transient Absorbance Measurements

      As a first test for the occurrence of reversible (de)protonation reactions linked to the photocycle of PYP, transient absorption spectra were recorded in an unbuffered suspension of PYP at pH 6 in the presence of the pH indicator dye bromcresol purple. In complete agreement with earlier observations (
      • Meyer T.E.
      • Cusanovich M.A.
      • Tollin G.
      ,
      • Genick U.K.
      • Devanathan S.
      • Meyer T.E.
      • Canestrelli I.L.
      • Williams E.
      • Cusanovich M.A.
      • Tollin G.
      • Getzoff E.D.
      ), it was observed that at this pH PYP caused a reversible alkalization of the medium upon actinic illumination, which in time parallels the formation and decay of the pB intermediate (Fig.1 A). Because it has been demonstrated that PYP exposes hydrophobic contact surface when it is present in the pB state (
      • Van Brederode M.E.
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Groot M.-L.
      • Hellingwerf K.J.
      ,
      • Hoff W.D.
      • Xie A.
      • Van Stokkum I.H.M.
      • Tang X.
      • Gural J.
      • Kroon A.R.
      • Hellingwerf K.J.
      ) and pH indicators might bind to such sites, similar measurements were performed in a solution buffered with 50 mm MES buffer (pH 6.0). Fig. 1 B shows that under these conditions bromcresol purple does not reveal any transient absorption signal, which implies that no pH change nor any artifact based upon nonspecific binding of the indicator to the pB form of PYP occurs. Because E. halophila is an alkaliphilic bacterium and therefore most probably has a slightly alkaline cytoplasm (
      • Booth I.R.
      ), we next tested the universality of these observations by performing similar experiments with the pH indicator dye cresol red, which has a pK of 8. The analyses given in Fig. 1, panel Cshows, however, that at pH 8 significant pH changes are not observed, but the photocycle intermediate is formed in an amount comparable with the experiment performed at pH 6 (Fig. 1 A).
      Figure thumbnail gr1
      Figure 1Reversible protonation of PYP as studied with laser-induced transient absorption spectroscopy. PYP (12 μm) in 2 ml of an unbuffered solution containing 1m KCl and either 100 μm bromcresol purple at pH 6 (A and B) or 100 μm cresol red at pH 8 (C). In B, 50 mm MES buffer (pH 6) was added. Laser-induced transient absorbance spectra were recorded in the indicated wavelength region after 61 μs (trace1), 3.9 ms (trace 2), 250 ms (trace3), and 1 s (trace 4). Theasterisk (*) refers to a calibration experiment in which the difference (bromcresol purple, ΔA595, cresol red ΔA575) spectrum is recorded, induced by the addition of 10 nmol of protons (using 2.5 mm oxalic acid).

      Simultaneous Measurements of pH and Absorbance Transients

      Because analysis of PYP (de)protonation with the use of pH indicator dyes is limited to the region around the pKof such dyes, we decided to use direct measurement with a pH electrode to investigate the pH dependence of the reversible protonation, as observed with PYP at pH 6. Initial experiments to test the sensitivity of the set-up were performed with unbuffered solutions of purple membrane (containing bacteriorhodopsin at a concentration of 130 μm, in 3 m KCl). These experiments revealed that the pH signal showed random noise (2.5·10−4 pH units), on top of a very slowly decaying drift, and confirmed the pH dependence of proton release by bacteriorhodopsin and the stimulation of this proton release by increasing salt concentrations (data not shown; see also Ref.
      • Renthal R.
      ).
      Initial experiments with PYP revealed that pH changes could also be observed in unbuffered solutions containing micromolar concentrations of PYP using the pH electrode (Fig. 2). Surprisingly, the sign of the PYP-dependent pH change was dependent on the absolute pH (compare panels A and B of Fig.2), whereas the sign of the drift in the pH, caused by the light-induced heating, was not. The latter was shown in experiments in which PYP was replaced by an equivalent amount of bovine serum albumin (data not shown). For quantitative evaluation of these experiments, it is important to distinguish between these heating artifacts by the actinic illumination and actual proton uptake/release by PYP. The Peltier temperature control of the Kraayenhof vessel responded slowly to the light-induced change in temperature of the solution in the vessel, thus causing an oscillating signal from the pH electrode (data not shown). Therefore, this temperature control system was switched off in all subsequent experiments, and the PYP-mediated pH changes were calculated from the pH recordings after correction for the light-induced heating of the sample, which caused an apparent change in the drift of the pH electrode (Fig. 2, A and B). The response time of the electrode (t 0.9) to additions of small amounts of oxalic acid is ∼10 s, because of the mixing time in the vessel. The pH response upon illumination, however, is immediate (i.e. within 1 s). The contribution of PYP to the light-induced pH change can therefore be calculated from the measured pH changes by back extrapolation to the time of switching on the light, as shown in Fig. 2, A and B. Under most conditions, however, very little correction for this light-induced heating was necessary (Fig. 2 C). The minimal amount of actinic illumination required for the maximal extent of pB formation was determined at pH 4.5 by parallel absorbance measurements (see below). The intensity of the actinic illumination was adjusted by variation of the voltage on the Schott KL1500 light source.
      Figure thumbnail gr2
      Figure 2Typical recordings in the set-up for simultaneous pH and absorbance measurements. Details of the set-up are described under “Experimental Procedures.” Panels Aand B show a typical recording of the pH signal at pH 6.07 (A) and 9.95 (B). In panel C, the pH signal is complemented with a recording of the absorbance signal. The series of spectra from which the latter recording was derived shows an authentic isosbestic point at 386 nm (and shows zero absorbance > 550 nm). 1 and 2, actinic light on and off, respectively; 3, graphical method to determine the extent of (de)protonation of PYP upon illumination by back-extrapolation;4a and 4b, addition of 10 and 20 nmol, respectively, of H+.
      Absorbance changes of PYP (from 250 to 550 nm) were recorded every 0.1 s, simultaneously with the pH measurements. There was no measurable interference of the actinic illumination (as we concluded from experiments at slightly alkaline pH (pH ± 8) with wild type PYP and in the alkaline pH range with the E46Q mutant protein (see Figs. 3 and 5)). With this set-up, rates of (de)protonation and pG bleaching and recovery can be measured that are slower than ∼5 s−1. Fig. 2 C shows the result of a typical simultaneous recording of the pH and the absorbance (of which only the value at 446 nm is plotted).
      Figure thumbnail gr3
      Figure 3Reversible (de)protonation of PYP at acidic, neutral, and alkaline pH as measured with a combination pH electrode. The pH of the unbuffered solution of PYP was adjusted with small aliquots of concentrated HCl and/or KOH. Transient pH changes were analyzed as described under “Experimental Procedures” and in the legend to Fig. . ●, extent of pB accumulation as calculated from the absorbance signal; ▴, extent of the light-induced change in the degree of protonation of PYP. Positive numbersrefer to proton uptake by PYP.
      Figure thumbnail gr5
      Figure 5PH dependence of the light-induced reversible (de)protonation of wild type PYP and the H108F and E46Q mutant forms. Data as shown in Fig. for wild type PYP were recorded for the H108F and E46Q proteins as well and plotted for all three as the calculated H+/pB molar ratio: ●, wild type PYP; ▪, E46Q; ▴, H108F. The lines through the data points for wild type PYP and H108F are fitted curves, based on the Henderson-Hasselbalch equation, using n (the number of protons involved in the transition) = 1. The pK values obtained for wild type PYP and H108F are 6.6 and 5.5 for the titration between pH 8 and 5, respectively. For the titration between pH 8 and 10.5, these values are >10.2 and >10, respectively. When the data points for wild type PYP between pH 5 and 8 are fitted without fixing the n value, the fit yields n = 1.2 and pK = 6.6.
      The pB intermediate is by far the longest living intermediate of PYP at all pH values tested so far. Therefore, to a good approximation (compare Refs.
      • Miller A.
      • Leigeber H.
      • Hoff W.D.
      • Hellingwerf K.J.
      and
      • Oesterhelt D.
      • Hess B.
      ), the photocycle of PYP can be simplified to a two-state photocycle in which only the ground state pG and the intermediate pB are taken into consideration. The amount of pB formation can then be calculated from these transient absorbance measurements by taking
      pB(%)=1A446lA446d×100


      in which the superscripts l and d refer to the steady state reading of the absorbance at 446 nm in the light and the dark, respectively.
      By analyzing the changes in pH induced by illumination of PYP in the range of pH 4–11, it was revealed that PYP transiently takes up protons at acidic pH but releases protons at alkaline pH (Fig. 3). In addition, in a wide pH range at slightly alkaline pH only a very small (de)protonation was observed. The inversion of the signal of the light-induced pH change could even be effected by having the sample (making use of the slight pH drift) go through the pH transition range (∼pH 7.8). Changes in the protonation level of PYP cannot be measured outside of the pH range from 4 to 11, because at a pH < 4.0 significant amounts of pBdarkare formed (
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Gural J.
      • Hellingwerf K.J.
      ), and at pH > 11 the thiol ester linkage in PYP starts to hydrolyze at a significant rate (
      • Hoff W.D.
      • Devreese B.
      • Fokkens R.
      • Nugteren-Roodzant I.M.
      • Van Beeumen J.
      • Nibbering N.
      • Hellingwerf K.J.
      ). The noise on the signal from the pH electrode increases slightly at alkaline pH, but it is always less than 0.05 H+/PYP in the set-up used.
      Calculation of the amount of pB formed under these conditions shows that it varies between 85 (at acidic pH) and 20% (at neutral pH). The variation in this amount is primarily because of the pH dependence of the rate of the pG recovery reaction (
      • Genick U.K.
      • Devanathan S.
      • Meyer T.E.
      • Canestrelli I.L.
      • Williams E.
      • Cusanovich M.A.
      • Tollin G.
      • Getzoff E.D.
      ,
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Gural J.
      • Hellingwerf K.J.
      ). These data can then be used to calculate the number of protons taken up or released per molecule of pB formed. This calculation leads to the conclusion that this number is ∼1 at low pH and >1 at high pH. The calculations at alkaline pH, however, are complicated by changes in the spectral characteristics of pB (see below).

      The Spectrum of pB at High pH

      One of the groups that may contribute to the inversion from reversible proton uptake (at low pH) to reversible proton release at high pH in PYP is the phenolate moiety of the chromophore. Evidence suggests that the chromophore of PYP is exposed to solvent when the protein is in the pB state (
      • Genick U.K.
      • Borgstahl G.E.
      • Ng K.
      • Ren Z.
      • Pradervand C.
      • Burke P.M.
      • Srajer V.
      • Teng T.Y.
      • Schildkamp W.
      • McRee D.E.
      • Moffat K.
      • Getzoff E.D.
      ,
      • Meyer T.E.
      • Cusanovich M.A.
      • Tollin G.
      ). Because phenolates generally have a pK around 9, one might anticipate that the chromophore in the pB state could not be protonated at pH values above 9. We therefore analyzed in detail the light-induced absorbance difference spectra of PYP in the alkaline pH range. The results obtained indeed show a transition from the known pB difference spectrum at neutral pH (with an absorbance maximum at 355 nm (
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Van Ramesdonk J.
      • Van Brederode M.E.
      • Brouwer A.M.
      • Fitch J.C.
      • Meyer T.E.
      • Van Grondelle R.
      • Hellingwerf K.J.
      )) to a difference spectrum with a component that absorbs maximally at 420 nm, which is indicative of the presence of a phenolate anion (Fig.4 and Ref.
      • Hoff W.D.
      • Devreese B.
      • Fokkens R.
      • Nugteren-Roodzant I.M.
      • Van Beeumen J.
      • Nibbering N.
      • Hellingwerf K.J.
      ). A similar transition in the spectrum of pB was also detected with laser-induced transient absorbance measurements (data not shown). The latter experiments, however, are more complicated because they require (for technical reasons) that the sample be kept at the very alkaline pH for a longer period, which causes interference with the measurements through thiol ester hydrolysis (
      • Hoff W.D.
      • Düx P.
      • Hård K.
      • Devreese B.
      • Nugteren-Roodzant I.M.
      • Crielaard W.
      • Boelens R.
      • Kaptein R.
      • van Beeumen J.
      • Hellingwerf K.J.
      ,
      • Baca M.
      • Borgstahl G.E.O.
      • Boissinot M.
      • Burke P.M.
      • Williams W.R.
      • Slater K.A.
      • Getzoff E.D.
      ,
      • Oesterhelt D.
      • Hess B.
      ) of PYP. The pH dependence of this presumed phenol/phenolate transition has a surprising feature. When the absorbance difference data at 416 nm are fitted with the Henderson-Hasselbalch equation, a pK > 10 is calculated, significantly higher than the pK of 8.8 measured for the phenolic group of 4-hydroxycinnamic acid and related model compounds (
      • Kroon A.R.
      • Hoff W.D.
      • Fennema H.
      • Gijzen J.
      • Koomen G.-J.
      • Verhoeven J.W.
      • Crielaard W.
      • Hellingwerf K.J.
      ). We did obtain this latter value upon titration of the chromophore of PYP when the protein was dissolved in 8 mguanidinium-HCl (data not shown).
      Figure thumbnail gr4
      Figure 4PH dependence of the spectral characteristics of the long-lived (blue-shifted) photocycle intermediate of PYP .Absorbance difference spectra (“after-minus-before” actinic illumination) of wild type PYP at pH 7.47 (●) and 10.91(▪). From the absolute spectrum of the illuminated PYP sample, a scaled spectrum of the ground state (i.e. pG) of PYP was subtracted, to remove the contribution of this state from the difference spectrum.

      Analysis of Site-directed Mutants of PYP

      To obtain more insight into the nature of the specific groups of PYP that contribute to the light-induced reversible (de)protonation, variants were constructed through site-directed mutagenesis. The pH dependence of the pH changes caused by illumination of PYP suggests that (a) histidine(s) is involved in the light-induced proton uptake at acidic pH. PYP contains two histidines, His-3 and His-108. As His-3 is positioned close to the relatively exposed N terminus of the protein (see also under “Discussion”), we selected His-108 and changed it into phenylalanine. The H108F protein indeed shows a similar pH dependence of the reversible protonation at alkaline pH and adecreased reversible proton uptake at acidic pH (Fig.5).
      In the E46Q mutant of PYP (
      • Genick U.K.
      • Devanathan S.
      • Meyer T.E.
      • Canestrelli I.L.
      • Williams E.
      • Cusanovich M.A.
      • Tollin G.
      • Getzoff E.D.
      ,
      • Imamoto Y.
      • Mihara K.
      • Hisatomi O.
      • Kataoka M.
      • Tokunaga F.
      • Bojkova N.
      • Yoshihara K.
      ), direct protonation of the chromophore by its hydrogen-bonding partner in the chromophore-binding pocket cannot take place. It may therefore be anticipated that in this derivative, upon formation of pB a proton has to be taken up from the bulk aqueous phase. The results presented in Fig. 5 confirm that the extent of proton uptake per molecule of pB has indeed increased in E46Q (Fig. 5). Unfortunately, however, the reduced stability and altered photocycle kinetics of this protein preclude analyses over the same pH range as with wild type PYP, because with the E46Q protein significant amounts of a pB-like intermediate are formed already at pH values below 6. A pK of 4.2 was measured for the transition between pG and this pB-like intermediate of the E46Q protein. This latter protein also shows very little pB formation at alkaline pH because of the accelerated rate of recovery of pG (data not shown).

      DISCUSSION

      The results obtained fully confirm the reported protonation of PYP at slightly acidic pH (
      • Genick U.K.
      • Devanathan S.
      • Meyer T.E.
      • Canestrelli I.L.
      • Williams E.
      • Cusanovich M.A.
      • Tollin G.
      • Getzoff E.D.
      ,
      • Meyer T.E.
      • Cusanovich M.A.
      • Tollin G.
      ). Binding of the pH indicator dye bromcresol purple to the hydrophobic contact surface that is exposed by PYP in its pB state (
      • Van Brederode M.E.
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Groot M.-L.
      • Hellingwerf K.J.
      ,
      • Hoff W.D.
      • Xie A.
      • Van Stokkum I.H.M.
      • Tang X.
      • Gural J.
      • Kroon A.R.
      • Hellingwerf K.J.
      ,
      • Rubinstenn G.
      • Vuister G.W.
      • Mulder F.A.A.
      • Düx P.E.
      • Boelens R.
      • Hellingwerf K.J.
      • Kaptein R.
      ) can not explain the results obtained. This conclusion can be reached from Fig. 1 B, which shows that the reversible pH changes disappear upon buffering of the PYP solution. The extent of protonation (∼1 H+/pB, measured at 100 μm bromcresol purple) also agrees with the value reported by Meyer et al. (
      • Genick U.K.
      • Devanathan S.
      • Meyer T.E.
      • Canestrelli I.L.
      • Williams E.
      • Cusanovich M.A.
      • Tollin G.
      • Getzoff E.D.
      ).
      The sensitivity of the pH electrode and the stability of PYP against extreme pH values allowed us to record light-induced pH changes in a wide pH range. Parallel measurement of absorbance transitions then allow one to calculate the extent of this (de)protonation in terms of the H+/pB ratio, if a simplified photocycle scheme is used (
      • Miller A.
      • Leigeber H.
      • Hoff W.D.
      • Hellingwerf K.J.
      ,
      • Oesterhelt D.
      • Hess B.
      ), in which only two intermediates (i.e. pG and pB) are considered. This simplification is supported by steady state “light-minus-dark” absorbance difference spectra of these samples. Significant amounts of intermediates other than pG and pB were not detected (note, however, that at very high pH the spectrum of the pB intermediate changes (see below)). The amount of pB intermediate formed is calculated from the extent of pG bleaching, as the latter intermediate can be detected more accurately. The pG intermediate has a 2–3-fold higher extinction coefficient (
      • Meyer T.E.
      • Yakali E.
      • Cusanovich M.A.
      • Tollin G.
      ,
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Van Ramesdonk J.
      • Van Brederode M.E.
      • Brouwer A.M.
      • Fitch J.C.
      • Meyer T.E.
      • Van Grondelle R.
      • Hellingwerf K.J.
      ) and absorbs in a more favorable wavelength region with respect to the signal to noise ratio of our detection systems (see e.g. Fig. 1).
      Analysis of the steady state pH changes revealed that PYP in the pB state can show reversible net proton uptake from as well as net proton release to the solvent. At slightly alkaline pH (i.e. 7.5–8.5), however, neither significant proton uptake nor proton release occurs in the pB state of PYP. This absence of photocycle associated pH changes is in line with simultaneous proton uptake by the chromophore and proton release by Glu-46. Because these counteracting processes are also expected to occur at and below pH 6 (e.g. see Refs.
      • Genick U.K.
      • Devanathan S.
      • Meyer T.E.
      • Canestrelli I.L.
      • Williams E.
      • Cusanovich M.A.
      • Tollin G.
      • Getzoff E.D.
      ,
      • Xie A.
      • Hoff W.D.
      • Kroon A.R.
      • Hellingwerf K.J.
      , and
      • Imamoto Y.
      • Mihara K.
      • Hisatomi O.
      • Kataoka M.
      • Tokunaga F.
      • Bojkova N.
      • Yoshihara K.
      ), most likely an additional group(s) is involved in this net proton uptake at low pH. A fit of the data for wild type PYP revealed that the pK of the transition from “absence of proton uptake/release” at pH 8 to “proton uptake” at pH < 5 has an apparent pK of 6.6 (assuming n = 1 in the fit). Because of the numerical value of this apparent pK, the two histidines of PYP (His-3 and His-108) are candidates to provide the side chain to mediate this proton uptake. His-3 is located close to the protein surface, and in the crystal structure it appears not to be involved in the hydrogen bonding responsible for the closure of the N-terminal helical lariat (
      • Borgstahl G.E.O.
      • Williams D.R.
      • Getzoff E.D.
      ). Furthermore, in solution the five residues at the N terminus of PYP are in a disordered state (
      • Düx P.
      • Rubinstenn G.
      • Vuister G.W.
      • Boelens R.
      • Mulder F.A.A.
      • Hård K.
      • Hoff W.D.
      • Kroon A.R.
      • Crielaard W.
      • Hellingwerf K.J.
      • Kaptein R.
      ). Therefore, in the pG state of PYP, His-3 is already exposed to solvent and thus is not likely to contribute to the observed proton uptake at acidic pH. His-108 is buried inside the hydrophobic core of PYP at a considerable distance from the chromophore (>15 Å). Analysis of the H108F derivative (Fig. 5) showed that His-108 does indeed contribute to the light-induced pH changes at acidic pH. Analysis of the titration curve of the H108F mutant with the Henderson-Hasselbalch equation gives an apparent pK of 5.5. However, because of the uncertainty about the end point of this titration, we can not unambiguously interpret this latter value.
      Multiple groups with a high pK (>10) must be involved in the reversible deprotonation of PYP. For these Tyr-42, Tyr-118, and Arg-110 are candidates, as they are buried inside the hydrophobic core of PYP in the pG state and are expected to have a pK of ∼10 (
      • Stryer L.
      ). However, because the protein environment may significantly modulate the pK of functional groups within a protein, other groups may be involved as well.
      In this study we have used steady state accumulation of the pB intermediate by continuous illumination to measure the net change in protonation level of this intermediate rather than selecting a range of pH indicators that could be used for transient absorption measurements of pH changes from pH 4 to 11. The results obtained with the E46Q protein, however, show the limitations in this approach. Because of the altered recovery kinetics that are displayed by this protein (see also Ref.
      • Genick U.K.
      • Devanathan S.
      • Meyer T.E.
      • Canestrelli I.L.
      • Williams E.
      • Cusanovich M.A.
      • Tollin G.
      • Getzoff E.D.
      ), no significant amount of pB intermediate accumulates during actinic illumination of this protein at alkaline pH values. The use of monochromatic actinic light of 450 nm to prevent interference by the light-induced branching reaction from pB back to pG (
      • Miller A.
      • Leigeber H.
      • Hoff W.D.
      • Hellingwerf K.J.
      ) did not significantly increase the amount of pB of the E46Q protein in the steady state in the light.
      J. Hendriks and K. J. Hellingwerf, unpublished observations.
      Kinetic analyses of the simultaneously measured pH and absorbance signals are possible only in a limited time domain. These analyses, however, have not revealed discrepancies in the rate of dark recovery of the two signals. The latter would have provided evidence for heterogeneity of the pB state. Such heterogeneity may be anticipated because in the recovery reaction of the ground state of PYP, several partial reactions have to take place, like re-isomerization of the chromophore, refolding of the protein to the pG conformation, and re-formation of the hydrogen-bonding network between the chromophore and Glu-46 and Tyr-42. The kinetics of the recovery reaction of wild type PYP and the E46Q protein were in general agreement with previously obtained results (e.g. Refs.
      • Meyer T.E.
      • Yakali E.
      • Cusanovich M.A.
      • Tollin G.
      ,
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Van Ramesdonk J.
      • Van Brederode M.E.
      • Brouwer A.M.
      • Fitch J.C.
      • Meyer T.E.
      • Van Grondelle R.
      • Hellingwerf K.J.
      ,
      • Meyer T.E.
      • Tollin G.
      • Hazzard J.H.
      • Cusanovich M.A.
      ,
      • Genick U.K.
      • Devanathan S.
      • Meyer T.E.
      • Canestrelli I.L.
      • Williams E.
      • Cusanovich M.A.
      • Tollin G.
      • Getzoff E.D.
      , and
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Gural J.
      • Hellingwerf K.J.
      ).
      Whereas the pK of the chromophore of PYP is tuned to a very low value in its pG state (i.e. pK 2.7 (
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Gural J.
      • Hellingwerf K.J.
      )), in the pB state it is tuned to a very high value (i.e.pK > 10). In agreement with this, it was observed that the position of the absorbance maximum of this presumed anionic form of the chromophore in pB, is tuned measurably from its position in aqueous solvents (i.e. 400 nm (
      • Hoff W.D.
      • Devreese B.
      • Fokkens R.
      • Nugteren-Roodzant I.M.
      • Van Beeumen J.
      • Nibbering N.
      • Hellingwerf K.J.
      )) to ∼425 nm. Hydrogen bonding between the phenolate moiety and Arg-52 (
      • Genick U.K.
      • Borgstahl G.E.
      • Ng K.
      • Ren Z.
      • Pradervand C.
      • Burke P.M.
      • Srajer V.
      • Teng T.Y.
      • Schildkamp W.
      • McRee D.E.
      • Moffat K.
      • Getzoff E.D.
      ) may contribute to this tuning of pB. Furthermore, the results of this study are in line with kinetic, mass spectrometric, and NMR analyses on PYP, which also suggests that this protein shows a partial unfolding upon formation of its presumed signaling state pB (
      • Van Brederode M.E.
      • Hoff W.D.
      • Van Stokkum I.H.M.
      • Groot M.-L.
      • Hellingwerf K.J.
      ,
      • Hoff W.D.
      • Xie A.
      • Van Stokkum I.H.M.
      • Tang X.
      • Gural J.
      • Kroon A.R.
      • Hellingwerf K.J.
      ,
      • Rubinstenn G.
      • Vuister G.W.
      • Mulder F.A.A.
      • Düx P.E.
      • Boelens R.
      • Hellingwerf K.J.
      • Kaptein R.
      ).
      Assuming that the (de)protonation reactions in PYP in a crystalline matrix are the same as in solution, we must conclude that Glu-46 in the pB state at neutral pH will be ionized and present in a relatively hydrophobic pocket. In view of the differences noted between the structure of the pB intermediate as measured with NMR and with Laue diffraction (
      • Genick U.K.
      • Borgstahl G.E.
      • Ng K.
      • Ren Z.
      • Pradervand C.
      • Burke P.M.
      • Srajer V.
      • Teng T.Y.
      • Schildkamp W.
      • McRee D.E.
      • Moffat K.
      • Getzoff E.D.
      ,
      • Rubinstenn G.
      • Vuister G.W.
      • Mulder F.A.A.
      • Düx P.E.
      • Boelens R.
      • Hellingwerf K.J.
      • Kaptein R.
      ), this assumption is not necessarily true; but if it is true, it may be a significant factor in the free energy that drives pB back to the ground state pG.
      The current results, recent data on PYP (e.g. Refs.
      • Genick U.K.
      • Soltis S.M.
      • Kuhn P.
      • Canestrelli I.L.
      • Getzoff E.D.
      ,
      • Perman B.
      • Srajer V.
      • Ren Z.
      • Teng T.
      • Pradervand C.
      • Ursby T.
      • Bourgeois D.
      • Schotte F.
      • Wulff M.
      • Kort R.
      • Hellingwerf K.J.
      • Moffat K.
      , and
      • Rubinstenn G.
      • Vuister G.W.
      • Mulder F.A.A.
      • Düx P.E.
      • Boelens R.
      • Hellingwerf K.J.
      • Kaptein R.
      ), and the effects of high pressure on protein structure (
      • Hummer H.
      • Garde S.
      • Carcia A.E.
      • Paulaitis M.E.
      • Pratt L.R.
      ) lead to the following view of the photocycle of PYP. Light initiates a two-bond isomerization of the p-coumaryl chromophore in PYP, which then leads to proton transfer from Glu-46 to its phenolate moiety. This process in turn leads to significant unfolding of the protein and exposure of titratable groups to the solvent, all of which is reversed in the recovery reaction from pB to pG. Tuning of the pK of the functional groups involved, possibly via reversible (de)hydration of the chromophore in the binding pocket, may be at the heart of this mechanism.

      CONCLUSION

      The results presented in this study demonstrate that transient (de)protonation of PYP in its pB state is not observed at neutral to slightly alkaline pH. This conclusion provides further support for the assumption that the chromophore in the pB state is most likely protonated by the proton from Glu46.
      Extending this interpretation to the data obtained at acidic and alkaline pH then leads to the conclusion that the formation of pB is paralleled by a significant solvent exposure of functional groups that are buried inside the protein in the pG state.

      Acknowledgments

      The authors acknowledge the expert technical assistance of Robert Cordfunke and Martial Cijs and the advice of Dr. Ivo van Stokkum in the mathematical analyses.

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