The early events in halorhodopsin after light excitation are studied with picosecond time resolution. Absorption and fluorescence measurements show that the electronically excited state of the incorporated retinal has a lifetime of 5 ps. Within that time a red-shifted photoproduct is formed that remains stable for at least 2 ns

Franz, M.A.

Hegemann, P.

Kaiser, W.

Oesterhelt, D.

Polland, H.J.

Zinth, W.

Elsevier - Publisher Connector 

Picosecond events in the photochemical cycle of the light-driven chloride-pump halorhodopsin 

Polland, Hans-Joachim

Franz, M. A.

Zinth, Wolfgang

Kaiser, Wolfgang

Oesterhelt, Dieter

Open Access LMU ( Ludwig-Maximilians-Univ. München)

PICOSECOND EVENTS IN THE PHOTOCHEMICAL CYCLEOF THE LIGHT-DRIVEN CHLORIDE-PUMPHALORHODOPSINH.-J. POLLAND, M. A. FRANZ, W. ZINTH, AND W. KAISERPhysik Department der Technischen Universitat Miunchen, Muinchen, Federal Republic ofGermanyP. HEGEMANN AND D. OESTERHELTMax-Planck-Institutfur Biochemie, Martinsried/Munchen, Federal Republic ofGermanyABSTRACT The early events in halorhodopsin after light excitation are studied with picosecond time resolution.Absorption and fluorescence measurements show that the electronically excited state of the incorporated retinal has alifetime of 5 ps. Within that time a red-shifted photoproduct is formed that remains stable for at least 2 ns.INTRODUCTIONHalobacterium halobium contains three different retinalproteins (1): bacteriorhodopsin (BR) (2), halorhodopsin(HR) (3, 4), and slow cycling rhodopsin (5). The mostextensively studied protein is BR (6-8). It acts as a lightenergy transducing pigment by the generation of a protongradient across the cell membrane (9). Less information isavailable on HR, which is identified as an inwardlydirected chloride pump (10, 1 1). Electrical measurementson black lipid membranes demonstrated that the chromo-protein alone exhibits the transport activity and, therefore,the chloride pump is identical with HR (12). Little isknown about the intermediates that are formed after lightexcitation. Two different photocycles occur for high chlo-ride and low chloride concentrations, respectively (13, 14).Only the high chloride photocycle is associated with trans-port activity (10) and a hypsochromically shifted long-lived intermediate has been reported (13). It has anabsorption maximum at X = 520 nm and decays to theinitial state of HR within several milliseconds (14). Attemperatures of liquid nitrogen, a red-shifted intermediatecould be trapped (13), but the time scale of its occurrenceunder physiological conditions is unknown. Here we reporton spectroscopic experiments with purified HR on a pico-second time scale that identify an early intermediate in theHR photocyle.GLOSSARYn quantum yieldTr intrinsic radiative lifetimeTr fluorescence lifetimetD time delay between exciting and probing pulse.BIOPHYS. J. e Biophysical Society * 0006-3495/85/01/55/06Volume 47 January 1985 55-59MATERIALS AND METHODSHR was isolated from OD2 cells by the method described in reference 15with reported modifications (12). After detergent exchange on the secondphenylsepharose column, the sample was concentrated in an Amicon cell(YM-10 filter; Amicon Corp., Scientific Sys. Div., Danvers, MA) up to40 umol/l, dialyzed against 1% octylglucoside, 1 M NaCl, 10mM MOPS(pH 7) for 16 h at 40C, and concentrated further to 150 ,umol/l by thesame procedure.Picosecond experiments were performed using single, frequency-doubled picosecond pulses (X = 526.5 nm) of a mode-locked Nd:glass-laser system. The green input pulse was divided by a beam splitter.One part was frequency shifted by transient stimulated Raman scatteringto X = 540 nm (pulse energy 100 nJ, pulse duration 2.5 ps, peak intensity102 MW/cm2). The second part generated a picosecond light continuumin water. The level of excitation was kept small, -10% of the moleculesabsorbed one photon per pulse at 540 nm, to prevent nonlinear effects orheating of the sample. The low irradiance led to a linear response of thesample, i.e., the observed fluorescence signal or the absorption changeswere proportional to the laser energy. The light path through the samplewas I mm and the absorbance A(578 nm) = -lgl/10 - 0.75. Themonitoring pulse was produced by selecting a wavelength interval (AX '10 nm) from the light continuum. The intensity ratio of probe pulse toexcitation pulse was small: Ip/IeI < 0.1. Parallel polarization of excita-tion and probe pulses was chosen. The improved electrooptical detectorsystem (16) made it possible to measure changes in the energy of theprobe pulses with high accuracy of AE/Eo < 5 x 10-4.After each run, the cross-correlation curve of the excitation and probepulses was recorded. This experiment yielded the time resolution (<1 ps)and the zero point (tD = 0), which is determined by the temporalcoincidence of the two pulses. The dashed line in Fig. 3 gives an exampleof such a cross-correlation curve between the exciting pulse at 540 nm andthe probe pulse at 675 nm. From the half-width of the cross-correlationcurve of 4.5 ps, a pulse duration of the two experimental pulses of 3 ps wasdeduced.The fluorescence spectrum (unpolarized, corrected for spectral sensi-tivity) and the streak data were measured with excitation pulses at 526.5nm (only 5% of the molecules were excited). The spectral investigationswere made with the help of a 25-cm monochromator-photomultipliercombination (spectral resolution 3.5 nm). The time dependence of thefluorescence emission (X > 570 nm) was measured by a streak camera$1.00 55with a time resolution of 3 ps. The results are corrected for nonlinearinstrumental response of the camera. We ascertained that stray light orfluorescence from impurities did not falsify our results (i.e., by measuringcontrol samples without HR). The absorption spectrum of the sampleswas controlled before and after each experiment. No irreversible bleach-ing of the specimen was observed.RESULTSAbsorption and FluorescenceThe absorption and fluorescence spectra of HR are pre-sented in Fig. 1. The broad unstructured absorption bandwith Xmax = 578 nm (light adapted) resembles that of BR.The extinction coefficient of 50,000 M -'cm-' at 578 nm issomewhat smaller than that of BR (63,000 M - 'cm -'). Thefluorescence spectrum is approximately the mirror imageof the absorption spectrum (Fig. 1). A large Stokes shift of-4,000 cm-' is found when the absorption and fluores-cence are plotted in the form of e(v)/v and F(v)/v3,respectively.The fluorescence quantum yield is determined to be(5 ± 2) x 10-4 by comparing the emission ofHR with thatof oxaine 1 in ethanol (q = 0.1 1) (17). From the absorptionspectrum we calculate (18) an intrinsic radiative lifetimeof - rr = 8 ns and predict a fluorescence lifetime of r, - 4 psfor the first excited singlet state of retinal in HR (;s = q xTr) .Fluorescence Lifetime of HalorhodopsinThe time dependence of the fluorescence (X> 570 nm) wasmeasured with a streak camera using an excitation pulse at526.5 nm with a pulse duration of 4 ps. The fluorescenceintensity rises rapidly to a peak value and decays exponen-tially (Fig. 2). Deconvolution with the time dependence ofthe excitation pulse gives a fluorescence lifetime of TS = 5 +2 ps, in agreement with the estimate mentioned above.n'EI0IIWavelength X Cnm]FIGURE 1 Absorption (-) and fluorescence spectrum (O-O) of HR.We find a fluorescence quantum yield of 5 x 10-4 and a Stokes shift of4,000 cm-'. The wavelengths of the probe pulses in Figs. 3 and 4 aremarked by arrows, the fluorescence intensity is given in arbitrary units(a.u.).00~~~~~~~~0~~~~0 10 20 30 40Time t 0ps.FIGURE 2 Fluorescence intensity as a function of time (X > 570 nm)measured with a streak camera. A relative time scale is used. We deduce afluorescence lifetime of 5 ± 2 ps.Transient Absorption and AmplificationThe transmission of probe pulses at various frequencieswas carefully measured during and after excitation at 540nm. Four interesting probing frequencies at 675, 646, 592,and 573 nm were selected and are marked by arrows inFig. 1. Strong fluorescence with negligible absorptionoccurs at 675 nm but strong absorption with small fluores-cence occurs at 573 nm. We observed three differentphysical processes when the sample was excited by a lightpulse: (a) an additional signal at the probing frequency,which is induced by the monitoring pulse and emitted fromthe populated excited state (amplification), (b) a reducedabsorption (bleaching of the initial state), and (c) anincrease of absorption due to a photoproduct.First, the data obtained with a probe pulse at 675 nmare discussed in Fig. 3. A transient amplification isfollowed by an increase in absorption. The amplificationrises quickly during the excitation of the sample andreaches a maximum value of 2% at tD = 1.5 ps. Later whenthe exciting pulse has left the specimen, the amplificationdecays with a time constant of 5 ps and, finally, for tD > 2ps, the probe pulse indicates absorption. The amplificationis caused by the fast population of the rirst excited singletstate. The decay time of amplification of 5 ps agrees withthe fluorescence lifetime discussed above. The generatedabsorption for tD > 20 ps belongs to an intermediatephotoproduct that persists during the observation time of300 ps.The results obtained with the three other monitoringwavelengths at 573, 592, and 646 nm are presented in Fig.4. In all three cases, the probe pulse experiences changes ofabsorption of different sign and magnitude during andafter the excitation process. At 573 nm (Fig. 4 a), a strongand rapid decrease of absorption is seen as a result of thepassage of the exciting pulse. This observation is due to thebuild-up of a population in the first excited singlet state, SI,BIOPHYSICAL JOURNAL VOLUME 47 198556*A. , ....-1i,; .j,.~~~~O T. -, :;2j-\~hf$675nm0s^ lt* .~~~~~,.:..~, '|~ *t~~~4S 941 W.e. -; X4: ^* IO 3. ; :FIGURE 3 Change of the probe intensity at X,, = 675 nm. The probepulse is amplified (-2%) during the population of the excited singlet state.The small absorption increase for tD > 15 ps is caused by the excessabsorption of a photoproduct. The dashed line gives the cross-correlationcurve between the exciting and probing pulse at 540 and 675 nm,respectively.and the depopulation of the ground state (see Fig. 1). Thebleaching disappears with a time constant of 5 ps, which isthe same value as found for the lifetime of the S, state (seeabove). For times 300 ps> tD> 20 ps, the absorption doesnot return to its original value at all four monitoringwavelengths (Figs. 3 and 4). We therefore suspect that a_*|t XviiXj F"u -l_t*.....* _ _..:-..4o 4U-aI...%'! r.... 4c01.11-ICf,., ". ::... '21it.111I.; *j5p~~~~~~~hLo GA S.!.'IkmS-''-- = -.* st - - - Sa -_9J>rn*aaBaN..a'aS.-N p Al'1i0J0 31;; RFIGURE 4 Absorption changes of the probe pulse as a function of timedelay between exciting (Xe. = 540 nm) and probing (AX, = 573, 592, 646nm) pulse. The rise of the excess absorption at 646 nm reflects the growthof the first photoproduct. It remains constant during the experimentaldelay time of 300 ps. Absorption (absorbance) changes are normalized.red-shifted photoproduct, which has a smaller absorptioncross section at 573 nm than the initial nonirradiated HR,exists.At 592 nm (Fig. 4 b), the same early bleaching is foundas was found at 573 nm. A small excess absorption fortimes tD> 20 ps appears, i.e., the generated photoproducthas a larger absorption at 592 nm than the original HR,and the isosbestic point of the two species must occurbetween 573 and 592 nm.At 646 nm (Fig. 4 c), the early decrease of absorption issmall, but for tD> 20 ps the transient absorption of thephotoproduct is large. The absorption change after initialbleaching has a time constant of 5 ps at all four wave-lengths, i.e., the photoproduct seems to rise with a timeconstant of 5 ps. We recall that the lifetime of the S1 statemeasured by fluorescence (Fig. 2) and by decay of amplifi-cation (Fig. 3) is 5 ps. The photoproduct is found to persistfor at leat 2 ns (within the 300 ps of observation <10O%variation was measured.Our finding may be summarized as follows. (a) Thefluorescence spectrum of HR is quite broad with a largeStokes shift of 4,000 cm-'. (b) The fluorescence quantumefficiency is small with v = 5 x 10-4. (c) The fluorescencelifetime is found to be 5 ps, which is in good agreementwith the value calculated from the radiative lifetime andthe quantum efficiency. (d) At 3000K, a red-shifted photo-product is observed. (e) The build-up of the intermediateproceeds within a time of 5 ps, the lifetime of the SI state.It is interesting to compare the present data ofHR withour recent investigations on BR (19). We found evidencefor a very short-lived (0.5 ps) excited singlet state in BRwith a smaller fluorescence quantum efficiency (1 x 10-4).Furthermore, the first red-shifted intermediate(19, 20, 21) in BR appears very rapidly within 1 ps(19, 21, 22), whereas it seems to be 5 ps in HR (Fig. 4).The slow formation of the first photoproduct in HR maylead to a lower efficiency of the photoreaction in HR thanin BR.DISCUSSIONOur results clearly demonstrate the conversion of theexcited state ofHR into a product with red-shifted absorp-tion. The maximum is between 590 and 670 nm and mightwell be similar to the absorption maximum of the interme-diate K in the photocycle of BR. Furthermore, the photo-product could be identical with HR632 found at low temper-ature (13). A detailed description of the photoproduct thatwe refer to as HRK will be possible if transient differencespectra and the quantum yield of the primary processbecome available. Preliminary experiments indicate asmaller quantum yield in HR than in BR.The lifetime of the excited state obtained from fluores-cence and amplification measurements is 5 ps. A fluores-cence lifetime of 5 ps was also calculated from the radiativelifetime and the quantum efficiency. The large Stokes shiftPOLLAND ET AL. Picosecond Events in Halorhodopsin* ; e ' s 1' - '- ' ;t - Z - -}1. - .-T 5--'k t/57is similar to that in BR and indicates a considerabledifference in the geometric arrangement of retinal in the*ground and the excited state.The time of formation of the first photoproduct, HRK,appears to be 5 ps. At present it is not possible to decidewhether the time of formation is even faster. Nevertheless,we try to explain our results with the most simple model.Both processes, the repopulation of the ground state SO(HR) and the formation of the intermediate HRK, occur inparallel from the excited singlet state SI with a timeconstant of 5 ps.So (HRK)SO (HR) *SISO (HR).A faster build-up of the intermediate HRK may beexplained by a more complicated model. After excitationimmediate branching forms the photoproduct HRK and alonger-living excited state SI' (lifetime r = 5 ps) thatdiffers from the optically excited state SI in its electronic orconformational structure.So (HRK)SO (HR) SI (IS; K SO (HR).Thus, the transition of SI' to SO (HR) would determinethe measured dynamics (r = 5 ps) of absorption andfluorescence. The present experimental data do not decidebetween the two models.The red-shifted absorption of the intermediate K in thephotocycle of BR may be interpreted as an increaseddistance of the positively charged nitrogen of the Schiff'sbase and the negative counterion in the protein (23). In BRthis is very likely due to a rotation of the retinal moleculearound the 13th double bond and the 14th single bond(24). It seems unlikely that within a few picoseconds theprotein structure could respond to the excited state ofretinal with molecular movements increasing the distancebetween the charges. We, therefore, prefer the interpreta-tion that the red-shift in HR is also due to a rotation ofbonds in retinal molecule (isomerization). Presently,whether this rotation includes the 13th double bond is notknown. However, an increased 13-cis retinal content wasfound upon extraction of illuminated HR-containing enve-lope vesicles, which indicates the possibility of a trans to cisisomerization during the photocycle in HR.We wish to thank Hamamatsu-Television Europe GmbH. Seefeld, Fed-eral Republic of Germany, for placing the streak camera model C 1 370-01at our disposal.This work was supported by the Deutsche Forschungsgemeinschaft (SFB143).Receivedfor publication 17 April 1984 and infinalform 3 July 1984.REFERENCES1. Spudich, J. L., and R. A. Bogomolni. 1983. Spectroscopic discrimi-nation of the three rhodopsinlike pigments in Halobacteriumhalobium membrane. Biophys. J. 43:243-246.2. Oesterhelt, D., and W. Stoeckenius. 1971. Rhodopsinlike proteinfrom the purple membrane of Halobacterium halobium. NatureNew Biol. 233:149-152.3. Matsuno-Yagi, A., and Y. Mukohata. 1977. Two possible roles ofbacteriorhodopsin. A comparative study of strains of Halobacter-ium halobium differing in pigmentation. Biochem. Biophys. Res.Comm. 78:237-243.4. Wagner, G., D. Oesterhelt, G. Krippahl, and J. K. Lanyi. 1981.Bioenergetic role of halorhodopsin in Halobacterium halobiumcells. FEBS (Fed. Eur. Biochem. Soc. ) Lett. 131:341-345.5. Bogomolni, R. A., and J. L. Spudich. 1982. Identification of a thirdrhodopsinlike pigment in phototactic Halobacterium halobium.Proc. Natl. Acad. Sci. USA. 79:6250-6254.6. Stoeckenius, W., and R. A. Bogomolni. 1982. Bacteriorhodopsin andrelated pigments of Halobacteria. Annu. Rev. Biochem. 52:587-616.7. Lozier, R. H., R. A. Bogomolni, and W. Stoeckenius. 1975. Bacteri-orhodopsin: A light-driven proton pump in Halobacterium halo-bium. Biophys. J. 15:955-962.8. Oesterhelt, D., and B. Hess. 1973. Reversible photolysis of the purplecomplex in the purple membrane of Halobacterium halobium.Eur. J. Biochem. 37:316-326.9. Oesterhelt, D., and W. Stoeckenius. 1973. Functions of a newphotoreceptor membrane. Proc. Natl. Acad. Sci. USA. 70:2853-2857.10. Schobert, B., and J. K. Lanyi. 1982. Halorhodopsin is a light-drivenchloride pump. J. Biol. Chem. 257:10306-10313.11. Bamberg, E., P. Hegemann, and D. Oesterhelt. 1984. Reconstitutionof the light-driven electrogenic ion pump halorhodopsin in blacklipid membranes. Biochem. Biophys. Acta. 773:53-60.12. Bamberg, E., P. Hegemann, and D. Oesterhelt. 1984. The chromo-protein of halorhodopsin is the light-driven electrogenic chloridepump in Halobacterium halobium. Biochemistry. In press.13. Weber, H. J., and R. A. Bogomolni. 1981. P588, a second retinal-containing pigment in Halobacterium halobium. Photochem.Photobiol. 33:601-608.14. Schobert, B., J. K. Lanyi, and Cragoe. 1983. Evidence for a halidebinding site in halorhodopsin. J. Biol. Chem. 258:15158-15164.15. Steiner, M., and D. Oesterhelt. 1983. Isolation and properties of thenative chromoprotein halorhodopsin. EMBO (Eur. Mol. Biol.Organ. ) J. 2:1379-1385.16. Polland, H.-J., T. Elsaesser, A. Seilmeier, W. Kaiser, M. Kussler, N.J. Marx, B. Sens, and K. H. Drexhage. 1983. Picosecond dye laseremission in the infrared between 1.4 and 1.8 ,um. Appl. Phys.B32:53.17. Sens, R., and K. H. Drexhage. 1981. Fluorescence quantum yield ofoxazine and carbazine laser dyes. J. Lumin. 24/25:709.18. Strickler, S. J., and R. A. Berg. 1962. Relationship betweenabsorption intensity and fluorescence lifetime of molecules. J.Chem. Phys. 37:814.19. Polland, H. J., W. Zinth, and W. Kaiser. 1984. Ultrafast PhenomenaIV. Springer Ser. Chem. Phys. 38:456-458.20. Applebury, M. L., K. S. Peters, and P. M. Rentzepis. 1978. Primaryintermediates in the photochemical cycle of bacteriorhodopsin.Biophys. J. 23:375-382.21. Shichida, Y., S. Matuoka, Y. Hidaka, and T. Yoshizawa. 1983.Absorption spectra of intermediates of bacteriorhodopsin mea-sured by laser photolysis at room temperatures. Biochem. Bio-phys. Acta. 723:240.58 BIOPHYSICAL JOURNAL VOLUME 47 198522. Ippen, E. P., C. V. Shank, A. Lewis, and M. A. Marcus. 1978.Subpicosecond spectroscopy of bacteriorhodopsin. Science (Wash.DC). 200:1279.23. Honig, B., T. Ebrey, R. H. Callender, U. Dinur, and M. Ottolenghio.1979. Photoisomerization, energy storage, and charge separation.A model for light energy transduction in visual pigments andbacteriorhodopsin. Proc. Natl. Acad. Sci. USA. 76:2503.24. Schulten, K., and P. Tavan. 1978. A mechanism for the light-drivenproton pump of Halobacterium halobium. Nature (Lond. ).272:85.POLLAND ET AL. Picosecond Events in Halorhodopsin 59

English

A mechanism for the light-driven proton pump of Halobacterium halobium.

Absorption spectra of intermediates of bacteriorhodopsin measured by laser photolysis at room temperatures.

and charge separation. A model for light energy transduction in visual pigments and bacteriorhodopsin.

Bacteriorhodopsin and related pigments ofHalobacteria.

Bacteriorhodopsin: A light-driven proton pump in Halobacterium halobium.

Bioenergetic role of halorhodopsin in Halobacterium halobium cells.

Evidence for a halide binding site in halorhodopsin.

Fluorescence quantum yield of oxazine and carbazine laser dyes.

Functions of a new photoreceptor membrane.

Halorhodopsin is a light-driven chloride pump.

Identification ofa third rhodopsinlike pigment in phototactic Halobacterium halobium.

Isolation and properties ofthe native chromoprotein halorhodopsin.

P588, a second retinalcontaining pigment in Halobacterium halobium.

Primary intermediates in the photochemical cycle of bacteriorhodopsin.

Reconstitution of the light-driven electrogenic ion pump halorhodopsin in black lipid membranes.

Relationship between absorption intensity and fluorescence lifetime of molecules.

Reversible photolysisofthepurple complex in the purple membrane of Halobacterium halobium.

Rhodopsinlike protein from the purple membrane of Halobacterium halobium.

Spectroscopic discrimination of the three rhodopsinlike pigments in Halobacterium halobium membrane.

The chromoprotein of halorhodopsin is the light-driven electrogenic chloride pump in Halobacterium halobium.

Two possible roles of bacteriorhodopsin. A comparative study ofstrains ofHalobacterium halobium differing in pigmentation.

Ultrafast Phenomena IV.

Picosecond events in the photochemical cycle of the light-driven chloride-pump halorhodopsin

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Picosecond events in the photochemical cycle of the light-driven chloride-pump halorhodopsin

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