J. Phys. Chem. 1991,95, 2351-2356 appear to be. a consensus regarding the value of the rate constant. Our data at room temperature disagree with the value a t 292 K of Li et al.13 of (2.3 f 0.4) X cm3/(molecule s). For temperatures below 900 K, our observations of the absence of CN C02reaction is consistent with those of Jacobs et al.” However, we observed CN decays beginning at temperatures of 914 K, and Jacobs et al. saw no evidence of reaction for temperatures up to 1000 K. The complex behavior we observed suggests that additional processes are present which are affecting the CN decays,
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and thereby precludes the determination of reliable reaction rate constants. These processes will have to be addressed in more detail in order to properly resolve these uncertainties in the data.
Acknowledgment. We thank Drs. J. Durant and R. Oldenborg for providing copies of data prior to publication. R.J.B. acknowledges support from the Oak Ridge Associated Universities Postgraduate Research Training program and the U S . Department of Energy.
Vibrationally Excited Retinal in the Bacteriorhodopsin Photocycle: Picosecond Time-Resolved Anti-Stokes Resonance Raman Scattering T. L. Brack and G. H. Atkinson*st Department of Chemistry and Optical Science Center, University of Arizona, Tucson, Arizona 85721 (Received: February 9, 1990; In Final Form: October 4, 1990)
Time-resolved (6 ps) spontaneous resonance Raman (RR) scattering from the retinal chromophore in the purple membrane of Halobacterium halobium is recorded as Stokes and anti-Stokes spectra. Anti-Stokes RR scattering is used to identify and characterize vibrationally excited, ground-state populations in retinal formed during the optical initiation of the bacteriorhodopsin photocycle. The frequencies and intensities of anti-Stokes RR bands, relative both to the corresponding Stokes RR features and to each other, are shown to be functions of the energy used in single and pumpprobe, pulsed laser experiments. Time-dependent intensities of anti-Stokes RR bands demonstrate that relaxation of vibrationally excited BR-570 occurs with a rate of ==7ps. The vibrational relaxation properties characterizing retinal as a chromophore embedded within a protein environment are discussed relative to those found in room temperature solutions of polyatomic molecules.
Introduction The changes that occur in the electronic and vibrational states of the retinal chromophore during the early stages of the bacteriorhodopsin (BR) photocycle have been examined extensively in order to elucidate the molecular mechanisms(s) by which optical excitation is utilized to store the -15 kcal/mol needed to drive proton translocation across the Halobacterium halobium memb r a ~ ~ e . ’ - These ~ initial photocycle events include the optical population and subpicosecond relaxation of the lowest energy, excited electronic state of retinal (SI)as well as the formation within the initial 40 ps of photochemical intermediates having distinct configurational and conformational forms of retinal.4,s These transient photophysical and photochemical intermediates have been identified primarily through the time-resolved electronic@ and/or vibrational’*’* spectroscopies of the retinal chromophore and, therefore, the photocycle is described almost exclusively in terms of the retinal transformations. It remains clear, nonetheless, that at least some of these retinal intermediates interact directly with the surrounding protein environment which, analogously, undergoes its own changes during the photocycle. Of the BR photocycle intermediates that have been identified, none have involved vibrationally excited populations. Specifically, vibrationally excited, ground-state retinal, So(v*),has not been previously reported, although a t least two important photocycle processes could reasonably result in such vibrational excitation: (i) nonradiative, vibronic relaxation from SIduring optical excitation to form vibrationally excited BR-570 (Le., BR’) and (ii) the ground-state transformation of 5-625 to form vibrationally excited K-590(Le., K’). Both processes are described schematically in Figure 1. Vibrationally excited species merit attention since (i) they may contribute significantly to the pumping process and/or the energy storage mechanism and (ii) the decay of So@*) *Author to whom correspondence should be addressed. ‘Senior Alexander von Humboldt Awardee, Technical University of Munich, Federal Republic of Germany.
0022-3654/91/2095-235 1$02.50/0
provides a new opportunity to characterize the relaxation processes by which retinal interacts with its surrounding in vivo protein environment. Recently, quantitative modeling of both picosecond transient absorption and fluorescence data has supported the inclusion of the 5-625 K’ K-590 processes into the photocycle and has discussed the evidence supporting the presence of BR’ and K’.I3 Independent experimental evidence demonstrating the presence of vibrationally excited populations either during optical excitation (e.g., BR’) or as part of the photocycle (e.g., K’),however, has not been reported previously. In this paper, the formation of BR’ during the 4-6-ps optical initiation of the BR photocycle is measured directly by picosecond time-resolved anti-Stokes RR scattering. The molecular processes which produce BR’ during the optical excitation of BR-570 and their relevance to the BR photocycle are examined through the
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(1) Stoeckenius,W.; Bogomolni, R. A. Annu. Reo. Biochem. 1982, S I , 587. (2) Birge, R. R. Annu. Rev. Biophys. Biwng. 1981, IO, 315. (3) Birge, R. R.; Cooper, T. M. Biophys. J . 1983,42, 61. (4) Athnson, G. H.; Brack, T. L.; Blanchard, D.; Rumbles, G. Chem. Phys. 1989, 131, 1 . (5) Brack, T. L.; Atkinson, G. H. J . Mol. Srrucr. 1989, 214, 289. (6) Sharkov, A. V.; Pakuvev, A. V.; Chekalin, S. V.; Matveetz, Y. A. Biochim. Biophys. Acra 1985, 808, 94. (7) Polland, H.-J.; Franz, A.; Zinth, W.; Kolling, E.; Oestrerhelt, D. Biophys. J . 1986, 49, 651. (8) Mathies, R. A.; BritoCruz, C. H.; Polland, W. T.: Shank, C. V. Science 1988, 240, 711.
(9) Atkinson, G. H.; Blanchard, D.; Lemaire. H.; Brack, T. L.; Hayashi, H. Biophys. J . 1989, 55, 263. (10) Hsieh, C.-L.; El-Sayed, M. A.; Nicol, M.; Nagumo, M.; Lee, J.-H. Photochem. Photobiol. 1983, 38, 83. ( 1 1) Smith, S. 0.;Braiman, M.; Mathits, R. In Time-resolved Vibrational Spectroscopy; Atkinson, G. H., Ed.;Academic Press: New York, 1983; p 219. (12) Atkinson, G.H.; Brack, T. L.; Blanchard, D.; Rumbles, G.;Siemankowski, L. In UlfrafartPhenomena V; Fleming, G.R.; Siegman, A. E., Eds.; Springer Verlag: Berlin, 1986; p 409. (1 3) Blanchard, D.; Gilmore, D.; Brack, T. L.; Lemaire, H.; Hughes, D.; Atkinson, G. H. Chem. Phys., in press.
0 1991 American Chemical Society
2352 The Journal of Physical Chemistry, Vol. 95, No. 6, 199'1
J*
\
Brack and Atkinson
I n
Antistokes
Stokes
x50 ,
76MHz
x 50
Reaction Coordinate Figure 1. Schematic representation of the potential energy surfaces
together with the photophysical and photochemical processes occurring during the initial part of the BR photocycle. Vertical arrows refer to absorption and wavy and curved arrows refer to nonradiative (vibrational and chemical) relaxation. CES refers to the common excited state to which BR*, BR'*, and J* relax while FC refers to the Franck-Condon states initially populated by absorption. time and power dependencies of the intensities and frequency displacements observed for the anti-Stokes R R bands. Intermolecular vibrational relaxation from retinal, presumedly to the protein environment, is measured from changing R R band intensities to occur in =7 ps, thereby providing the first direct view of the vibrational relaxation of retinal within its protein environment. In addition, differences in the band maxima positions observed in both Stokes and anti-Stokes spectra are assigned to R R scattering originating in the excited vibrational levels comprising So(u*).
Experimental Section The instrumentation and procedures utilized have been described in detail previous195 and, therefore, only a brief description is given here. The BR samples are prepared by standard techniques from cultured Halobacterium halobium strain R1 l4 and are held at 1 1 OC by ice water baths throughout each laser experiment. Picosecond laser excitation is obtained from two independently tunable dye lasers (Coherent model 702) that are synchronously pumped by the second-harmonic output (532 nm) of a modelocked Nd:YAG laser (Quantronix Model 416). A cavity dumper is used with each dye laser to obtain single 4-6-ps (fwhm) pulses a t variable repetition rates of 1-76 MHz and average output powers of 0.5-45 mW. An optical delay line is used with the probe laser beam to provide timing delays needed for pump-probe, timeresolved RR measurments. The delayed probe beam is made collinear with the pump laser beam before both are directed to the flowing (20 m/s) BR sample which is formed by a jet stream (380 Mm diameter) of the purple membrane suspension (OD565 = 4). The focused laser spot (
