The influence of the halide ions on the photochemical reaction cycle of pharaonis halorhodopsin

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1 Journl of Photochemistry nd Photoiology B: Biology 82 (26) The influence of the hlide ions on the photochemicl rection cycle of phronis hlorhodopsin Klár Mgyri, Vioric Simon, György Váró, * Deprtment of Physics, University Bes-Bolyi Cluj-Npoc, Romni Institute of Biophysics, Biologicl Reserch Center of the Hungrin Acdemy of Sciences, P.O. Box 521, Szeged H-671, Hungry Received 2 June 25; received in revised form 4 August 25; ccepted 8 August 25 Aville online 1 Octoer 25 Astrct Sodium slt of chloride, romide nd iodide were used to elucidte the effect of the size of the nion on the inding to phronis hlorhodopsin nd its trnsport during the photocycle of this retinl protein. Spectroscopic titrtion reveled n pprent strong inding constnt of 2 mm for chloride, 3 mm for romide nd 5 mm for iodide. In the cse of iodide second, week inding constnt of out 1 M could e estimted. This second inding constnt ws similr to tht oserved erlier for nitrte. By chnging the hlide ions, only the trnsitions in the second hlf of the photocycle were ffected, which contined intermedites N, O, nd HR. The O to HR trnsition ecomes fster with incresing ion volume, mening tht the ion uptke is ccelerted. This effect shows direct correltion with the ion rdius. With incresing ion concentrtion the N O HR equilirium chnged in such wy tht the ccumulted O tended to decrese. This tendency ws overruled in iodide, y the ppernce of the second inding constnt. The incresing iodide concentrtion, up to 1 mm decreses the ccumultion of the intermedite O, due to kinetic resons, ut t higher ion concentrtion the mount of O increses, lthough its decy ecomes fster. This effect correltes with the ppernce of the second iodide ound to the protein. Ó 25 Elsevier B.V. All rights reserved. Keywords: Retinl protein; Chloride trnsport; Photocycle; Asorption kinetics 1. Introduction Phronis hlorhodopsin (phr) is light-driven chloride ion pumping retinl protein, found in the cell memrne of Ntronocterium phronis [1,2]. Its mino cid sequence ws estlished to e very similr to the slinrum hlorhodopsin nd cteriorhodopsin found in Hlocterium slinrum [3], showing 66% nd 25% homology, respectively. The photoctive molecule in the phr is retinl ound to lysine 256 in helix G vi protonted Schiff-se. Under ll conditions, 85% of the retinl in the protein is in ll-trns, 15-nti nd 15% in 13-cis, 15-syn configurtion [4,5]. Although the retinl * Corresponding uthor. Tel.: ; fx: E-mil ddress: vro@nucleus.szk.u-szeged.hu (G. Váró). composition of the smple is not homogeneous, there is no evidence tht the 13-cis contining phr is photoctive or tkes prt in the trnsport process, resulting homogeneous photocycle [4]. The structure of slinrum hlorhodopsin is known with 1.8 Å resolution [6]. Bsed on the high homology etween the two chloride ion pumps nd their similr photocycle, it cn e ssumed tht the phr hs similr structure. In the structure, the inding site of the ion ws determined nd it shows tht cluster of three wter molecule is in the inding pocket [6], suggesting tht ions of different size could e ccommodted. Spectrl titrtion in function of different sodium slts show tht the phr inds different nions, inducing lue shift in the spectrum [7]. If the titrtion ws effectuted t constnt sodium concentrtion y exchnging the nononding sulfte to the titrted nion, the inding constnt /$ - see front mtter Ó 25 Elsevier B.V. All rights reserved. doi:1.116/j.jphotoiol

2 K. Mgyri et l. / Journl of Photochemistry nd Photoiology B: Biology 82 (26) of the phr t ph 6 ws found to e 2 mm with.75 inding order for chloride [4], 1 mm for zide [8], nd 11 mm with.9 order for nitrte [9]. In the cse of nitrte second, low-ffinity inding constnt of out 7 M ws oserved [9]. In sodium sulfte the phr hs photocycle with only one red shifted, K like intermedite. Electric signl mesurements revel tht during the photocycle there re internl chrge motions in the protein, ut there is no net chrge trnsfer cross the memrne [4]. In 1 mm chloride the photocycle contins succession of intermedites (noted y K, L, N, O nd HR ). Ech intermedite hs welldetermined sorption spectrum in visile. Their kinetics could e descried y model contining sequentil, first order equilirium rections [4]. At 1 M chloride concentrtion the intermedite O cn not e oserved, for kinetic resons. It ws shown tht the ion relese nd uptke steps re tht of N O nd O HR trnsitions, respectively [1]. In nitrte the photocycle is very similr to tht oserved in chloride, ut the trnsported ion is much lrger [7]. The electric signl mesurements show tht chrge is trnsported cross the memrne [9]. At severl molr nitrte concentrtion the O intermedite does not dispper form the photocycle, which ws concluded to e the effect of the second, low-ffinity inding of the ion [9]. Beside of these chnges in the photocycle nother effect ws lso oserved, the pprent relese nd reinding of smll frction of the excited retinl, inside the retinl pocked, during the photocycle [9]. By using cell envelope vesicles of phr, it ws shown tht oth chloride nd nitrte re trnsported with the sme efficiency [2]. When the thing solution of the smple contins zide, insted of chloride, the trnsported ion ws estimted to e proton [8]. The photocycle looks similr to tht of the proton trnsporting cteriorhodopsin [7,11] nd y the use of cell envelope vesicles ws shown tht not the zide ion is trnslocted through the memrne [8]. Bsed on kinetic isotope effect mesurement, recently ws proposed tht the trnsported ion, insted of proton could e negtive hydroxyl ion [12]. In proton trnsporter cteriorhodopsin nd proteorhodopsin the wter to deuterium oxide exchnge cused mrked chnge in the photocycle. The deuteron exchnge in phr did not ffect the chloride nd nitrte photocycle of it, nd surprisingly the zide photocycle lso remined unffected [12]. In cteriorhodopsin t low ph, y using the method of exchnging HCl to HBr it ws possile to show tht the trnsported ion ws the hlide [13]. There re only preliminry informtion out the effect of different nions on the photocycle of phr [7]. Bsed on these the effect of the hlide ions (chloride, romide, nd iodide) ws investigted, to etter understnd the process of ion trnsport y phronis hlorhodopsin cross the memrne. 2. Mterils nd methods phr ws produced in Hlocterium slinrum strin L33, in which the Ntronocterium phronis hop gene ws introduced [4]. The mesurements were performed on memrnes encsed in polycrylmide gel [14]. All slt solutions were mixed y using 1 M N 2 SO 4 nd 2 M of NCl, NBr or NI in such wy tht lthough the hlide concentrtion vried, the totl N concentrtion ws constnt t 2 M. The solutions contined 5 mm MES (2-[Nmorpholino]ethnesulfonic cid) t ph 6. During the experiment the smple ws in 4 1 mm cuvette incuted in temperture controlled smple holder. The photocycle ws initited y lser flsh from frequency-douled Nd-YAG lser (Surelite I-1, Continuum, Snt Clr, C). The mesuring light ws provided y 55 W hlogen lmp with het filter. Mesurements were effectuted t four wvelengths (5, 59, 62 nd 64 nm) selected y monochromtor plced etween the smple nd the photomultiplier. By these the chnge of the mesuring wvelength did not ffect the light intensity going through the smple. The up to one second long signls were recorded with trnsient recorder crd (NI- DAQ PCI-512, Ntionl Instruments, Austin, TX) with 5 ns resolution. Ech mesurement ws the verge of 1 signls. At the end of the mesurement the liner time se ws converted to logrithmic y verging in the time intervl etween logrithmiclly equidistnt points. The signls were fitted to photocycle model y using RATE progrm [15]. 3. Results nd discussion Although there were preliminry informtion out the inding of different hlide ions to phr [7] the exct inding constnts were determined y spectrl titrtion with NCl, NBr nd NI. In ll three cses, the spectr shifted towrd lue with incresing nion concentrtion s shown for romide titrtion (Fig. 1()). The reltive chnges clculted t two wvelengths 56 nd 64 nm were plotted nd titrtion curves were fitted to the points (Fig. 1()). For chloride the inding constnt nd order were s determined erlier (not shown) [4]. Similr to tht oserved in nitrte [9], in the cse of iodide second, low-ffinity inding constnt could e estimted, lthough its exct vlue ws not determined, due to limited numer of points t the high hlide concentrtions. The inding constnts nd rection orders re listed in Tle 1. The strength of the second inding constnt seems to correlte to the rdius of the ions. Compring the sorption kinetic signls mesured in 1 mm hlide concentrtion (Fig. 2) revels tht the fst prt of the photocycle does not depend on the nion. The signls mesured t 5 nd 59 nm look unchnged in the whole time domin. In the ms time domin, the positive mplitude of the sorption signls mesured over 6 nm decreses y chnging chloride to romide or iodide. To emphsize the chnges the signls mesured t 64 nm were normed t the 1 ls time point (Fig. 3). With the increse of the size of the nion the positive mplitude, locted t round 1 ms, decreses.

3 18 K. Mgyri et l. / Journl of Photochemistry nd Photoiology B: Biology 82 (26) 16 2 s. relt. mpl wvelength (nm) NBr NI log cc (M) Fig. 1. Hlide dependent chnges in the spectrum of phronis hlorhodopsin. () The sorption spectr increses nd shifts towrd lue y incresing romide concentrtion. () The reltive chnge clculted t 56 nm (r, m) nd 64 nm (j, d) yields titrtion curves for ech hlide ion, from which the inding constnts nd rection order, were clculted (see Tle 1). s. chnge s. chnge s. chnge c 5 nm 64 nm 62 nm 59 nm 5 nm 64 nm 62 nm 59 nm 5 nm 64 nm 62 nm 59 nm Fig. 2. Asorption kinetic signls mesured t four wvelengths on phronis hlorhodopsin in 1 mm sodium chloride (), sodium romide () nd sodium iodide (c). The solution contined 95 mm sodium sulfte, 5 mm MES t ph 6. Tle 1 The moleculr mss, ion rdius nd the inding prmeters of nions, ound to phr Anion m w R (nm) Binding constnt Binding order Cl mm.75 Br mm 1 I mm.75 NO c 3 15 M mm.9 7M 1 Ion rdii from [16]. Binding prmeters from [4]. c Binding prmeters from [9]. The set of signls shown in Fig. 2 could e fitted with the previously pulished sequentil equilirium rection model [4] K () L () N () O () HR ) HR The RATE progrm chrcterized the goodness of the fit with clculting v 2 (the sum of the squre of the residuum of the fit). In ll three cses, this vlue ws very low nd vried in nrrow rnge, etween.18 nd.22, showing tht the fits were very good nd they cn e compred. The result of the fit to the signls contining chloride (Fig. 4) ws very similr, to tht pulished erlier [4]. The sorption chnges in the ls time domin correspond to the ppernce of the equilirium etween the K nd L intermedites. The following prt up to.1 ms is dominted y this equilirium, which is nion independent. The decy prt of the K L equilirium, gives rise to the N, O nd HR intermedites, which steps re lredy nion dependent. By chnging the chloride to romide nd iodide the mximum concentrtion of intermedite N decreses, the intermedite O disppers totlly, while the concentrtion of intermedite HR increses (Fig. 5). The oserved concentrtion chnges cn e explined y the ccelertion of the rise of HR with lmost one order of mgnitude, ccompnied y slight ccelertion of its decy (Fig. 5(c)), wht cn e oserved lso on the decy of the sorption kinetic signls (Fig. 2). In the sme time the rise of N nd the N O trnsition does not chnge considerly, resulting in decresing mount of N nd O without oservle chnge in their kinetics (Fig. 5() nd ()). The chnges in the finl decy of the equilirted K nd L intermedites re lso minor (not shown).

4 K. Mgyri et l. / Journl of Photochemistry nd Photoiology B: Biology 82 (26) NCl.6 NCl s. chnge.4 NBr NI.4 NBr NI Fig. 3. The mplitude chnge of the sorption kinetic signl mesured t 64 nm. The signls were normed t 1 ls. Mesuring conditions were the sme s in Fig c NCl NBr NI.4 NBr NCl K L Fig. 4. The time dependent concentrtion chnges of the photocycle intermedites, clculted from the sorption kinetic signls shown in Fig. 2(). From the literture is known tht the chloride relese step is the N O trnsition, while the uptke hppens t the O HR trnsition [1]. From the ccelertion of the rise of HR intermedite n unexpected oservtion cn e concluded. By incresing the size of the ion the uptke of it is fcilitted. This could e due to some steric enhncement, which is supported y the ppernce of the second inding site, s the size of the ion increses. One possile explntion could e tht s the size of the nion increses the negtive chrge is more deloclized. On the phr contining negtively chrged memrne the chrges re loclized, the interction of chrge close to the entrnce to the inding site with the pproching ion could diminish, incresing the proility of the ion to enter in the interior of the protein, where the inding site is locted [6]. In the sme time y penetrting in the protein the lrger ion, chnges the conformtion of the protein giving spce for second inding site. O N HR' HR NCl -4-2 Fig. 5. Chnge of the concentrtion of the photocycle intermedites, y chnging the hlide ion: () intermedite N; () intermedite O; (c) intermedite HR. To understnd the effect of the second inding site on the photocycle, the sorption kinetic signl t 64 nm ws mesured t different nion concentrtions. In chloride (not shown) nd romide (Fig. 6()) the concentrtion dependence ws similr to tht mesured erlier [1]. By incresing ion concentrtion the positive mplitude t round 1 ms decresed nd disppered t round 1 M. This led to the conclusion tht t high ion concentrtion, due to kinetic reson, the intermedite O does not ccumulte [1]. In the cse of iodide the positive mplitude decresed up to 1 mm concentrtion nd strts to increse, ut with fster decy kinetics, s the concentrtion ws further incresed. In the sme time smll negtive component ppered t the end of the trce (Fig. 6()). The similr, ut lrger effect oserved erlier in nitrte, ws explined s the temporl rek of the Schiff-se in some of the photoexcited phr [9]. The ccumultion of intermedite O t high iodide concentrtion seems to correlte with the inding of the second iodide to the protein. The nomly in spectrl titrtion, the increse of the positive mplitude t round 1 ms nd the ppernce of the very slow negtive component oserved in the cse of iodide nd even etter ccentuted in nitrte, seems to correlte to the rdius of the trnsported ion (see Tle 1). These

5 2 K. Mgyri et l. / Journl of Photochemistry nd Photoiology B: Biology 82 (26) 16 2 s. chnge s. chnge suggest tht the size of the trnsported ion does not influence the efficiency of the trnsport [2], ut introduces chnges in the inding nd the trnsport steps of the photocycle. Acknowledgment mm 1 mm 1 M 2 M 1 mm 1 mm The Ntionl Science Reserch Fund OTKA T4876 supported this work. 2 M 1 M Fig. 6. Chnge of the mplitude of the sorption kinetic signl mesured t 64 nm in function of incresing concentrtion of sodium romide () nd sodium iodide (). The signls were normed t 1 ls. Mesuring conditions were the sme s in Fig. 2. of Hungry References [1] D.B. Bivin, W. Stoeckenius, Photoctive retinl pigments in hlolklophilic cteri, J. Gen. Microiol. 132 (1986) [2] A. Duschl, J.K. Lnyi, L. Zimányi, Properties nd photochemistry of hlorhodopsin from the hlolklophile, Ntronocterium phronis, J. Biol. Chem. 265 (199) [3] J.K. Lnyi, A. Duschl, G.W. Htfield, K.M. My, D. Oesterhelt, The primry structure of hlorhodopsin from Ntronocterium phronis: structurl, functionl nd evolutionry implictions for cteril rhodopsins nd hlorhodopsins, J. Biol. Chem. 265 (199) [4] G. Váró, L.S. Brown, N. Sski, H. Kndori, A. Med, R. Needlemn, J.K. Lnyi, Light-driven chloride ion trnsport y hlorhodopsin from Ntronocterium phronis 1. The photochemicl cycle, Biochemistry 34 (1995) [5] L. Zimányi, J.K. Lnyi, Fourier trnsform Rmn study of retinl isomeric composition nd equilirtion in hlorhodopsin, J. Phys. Chem. B 11 (1997) [6] M. Kole, H. Besir, L.O. Essen, D. Oesterhelt, Structure of the lightdriven chloride pump hlorhodopsin t 1.8 Ĺ resolution, Science 288 (2) [7] B. Schrf, M. Engelhrd, Blue hlorhodopsin from Ntronocterium phronis: wvelength regultion y nions, Biochemistry 33 (1994) [8] G. Váró, L.S. Brown, R. Needlemn, J.K. Lnyi, Proton trnsport y hlorhodopsin, Biochemistry 35 (1996) [9] Z. Bálint, M. Lktos, C. Gne, J.K. Lnyi, G. Váró, The nitrte trnsporting photochemicl rection cycle of the Phronis hlorhodopsin, Biophys. J. 86 (24) [1] G. Váró, R. Needlemn, J.K. Lnyi, Light-driven chloride ion trnsport y Hlorhodopsin from Ntronocterium phronis. 2. Chloride relese nd uptke, protein conformtion chnge, nd thermodynmics, Biochemistry 34 (1995) [11] A. Kulcsár, G.I. Grom, J.K. Lnyi, G. Váró, Chrcteriztion of the proton trnsporting photocycle of phronis hlorhodopsin, Biophys. J. 79 (2) [12] J. Szkács, M. Lktos, C. Gne, G. Váró, Kinetic isotope effect in the photochemicl rection cycle of ion trnsporting retinl proteins, J. Photochem. Photoiol. B 79 (25) [13] A. Dér, S. Szárz, R. Tóth-Boconádi, Z. Tokji, L. Keszthelyi, W. Stoeckenius, Alterntive trnsloction of protons nd hlide ions y cteriorhodopsin, Proc. Ntl. Acd. Sci. USA 88 (1991) [14] P.C. Mowery, R.H. Lozier, Q. Che, Y.W. Tseng, M. Tylor, W. Stoeckenius, Effect of cid ph on the sorption spectr nd photorections of cteriorhodopsin, Biochemistry 18 (1979) [15] K. Ludmnn, C. Gergely, G. Váró, Kinetic nd thermodynmic study of the cteriorhodopsin photocycle over wide ph rnge, Biophys. J. 75 (1998) [16] J. Isrelchvili, Intermoleculr nd Surfce Forces, Acdemic Press Ltd., New York, 1992.