J. Phys. Chem. B 1998, 102, 8109-8112
8109
Quantitative Determination of the Protein Catalytic Efficiency for the Retinal Excited-State Decay in Bacteriorhodopsin S. L. Logunov, T. M. Masciangioli, and M. A. El-Sayed* Laser Dynamics Laboratory School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400 ReceiVed: March 3, 1998; In Final Form: July 13, 1998
It was previously found that by removing the negative charge of Asp85 in bacteriorhodopsin (bR), either by protonating it (as in deionized bR) or by mutation to Asn, the decay time of the retinal excited state increases from 0.5 ps to either 1.5 or 10 ps. The two decay components result from the presence of all-trans and 13-cis,15-syn (13-cis) retinal isomers in the modified retinal protein. To quantitatively determine the protein catalysis for the primary process in native bR, we need to determine which decay component results from the excited state of the all-trans isomer (present in the native bR). It is known that the all-trans isomer absorbs at longer wavelength than the 13-cis isomer in blue bR. In this communication, we report the results of pump-probe experiments using 100 fs laser pulses. Probing is carried out at 490 nm, where the excited state in both isomers absorbs. It is found that the ratio of the amplitudes of the two decay components in blue bR changes with variation of the excitation wavelength. The shorter-lived component is found to increase in amplitude as the excitation wavelength increases, i.e., as we excite more of the all-trans isomer. This leads to the conclusion that the short-lived component (1.5 ps) is for the decay of the all-trans excited state while the long-lived component (10 ps) is for the 13-cis retinal excited-state decay. Thus, the presence of the negative charge of Asp85 in native bR catalyzes the rate of the excited-state decay of the all-trans retinal by 300% and that of the 13-cis isomer by >2000%.
Introduction Bacteriorhodopsin (bR) is the only protein in the purple membrane of Halobacterium salinarium.1-3 After photoexcitation, light-adapted bR goes through a photocycle with at least seven intermediates with different absorption spectra. In the past decade, primary events in bR have been studied extensively by using ultrafast laser pulses.4-11 Recent time-resolved Raman spectroscopy suggests that retinal isomerization takes place on the subpicosecond time scale.12 It is known that within 500 fs of photoexcitation of bR, the first intermediate (J625) appears. This intermediate is considered to be the vibrational excited state of the K590 intermediate (13cis,15-anti isomer). After the J625 intermediate thermally relaxes to give K590, the next intermediate in the cycle (L550) is formed. Upon formation of L550, retinal becomes planar and the protein around the retinal is adjusted. The retinal protonated Schiff base (PSB) is then deprotonated to give M412. Light-adapted bR has almost 100% all-trans retinal chromophore, while dark-adapted bR contains 2/3 of the retinal in the 13-cis,15-syn (13-cis) configuration and 1/3 in the all-trans configuration.13 Only bR with the all-trans retinal configuration, however, goes through the proton pump photocycle. The 13cis chromophore in the mixture of isomers in dark-adapted bR goes through a photocycle, but does not pump protons.14,15 It was found that after excitation the 13-cis retinal formed an intermediate similar to the K intermediate observed in bR with the all-trans retinal photocycle. This intermediate, however, has a long lifetime and decays predominantly back to its initial state.16 * Author to whom correspondence should be addressed.
Removal of metal cations from bR by cation exchange chromatography17 or by lowering the pH of the solution changes the purple membrane into blue membrane (blue bR). Studies using site-directed mutagenesis to study the D85N mutant of bR lead to the conclusion that in the blue bR, Asp85 is protonated.18,19 From this result it was concluded that the unprotonated state of the charged Asp85 amino acid residue is essential for proton pumping. In the D85N mutant, in which the negatively charged COO- group is replaced with the neutral residue Asn, the proton pumping is inhibited.18,20 Similarly, deionized or acid blue bR does not produce a large percentage of the M intermediate21,22 in its photocycle and thus does not pump protons across the membrane.21 From the fingerprint region of the time-resolved Raman spectrum22 and transient absorption data it was shown, however, that the K and L type intermediates are formed22,23 in both deionized and acid blue bR. In deionized blue bR, there are at least two major isomers (13-cis,15-syn and all-trans) under light-adapted conditions21,24 as a result of a rapid dark adaptation process. This is also the case for a number of bR mutants.25 Transient excited-state decay kinetics of these bR variants always show two decay components, ∼1.5 ps (fast component) and ∼10 ps (slow component),26,27 which are attributed to the presence of the two isomers. When Asp85 is neutralized,26 at low pH, or by replacing it by the neutral residue Asn,27,28 the observed retinal excited-state lifetimes are found to be 4 and 20-30 times longer than that observed for the all-trans retinal in the wild-type bR.26-28 Thus the negative charge of the Asp85 catalyzes the rate of the excited-state decay of retinal by a factor of either
S1089-5647(98)01360-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/19/1998
8110 J. Phys. Chem. B, Vol. 102, No. 41, 1998 300% or >2000%, depending on which of the two components observed in the modified protein is due to the retinal excitedstate decay of the all-trans isomer. To determine the exact efficiency of the protein catalysis of the primary process in wild-type bR, we need to determine the decay component corresponding to the excited state of the alltrans retinal isomer in deionized blue bR (which is the same for the D85N mutant). To do this we need to answer two questions. First of all, is the biexponential decay due to the presence of two isomers? If yes, which one is due to the alltrans isomer, and which is due to the 13-cis isomer? In the present study we examine the relationship between the amplitudes of the fast and slow components of the excitedstate decay in deionized blue bR as a function of the excitation wavelength. It is known23 that the broad absorption spectrum of the all-trans isomer in blue bR is red shifted from that of the 13-cis isomer. If the decay components result from the excitation of the two isomers, changing the excitation wavelength is expected to lead to changes in both the decay amplitude ratio and the position of the bleach (optical hole) spectra produced. The decay component with the amplitude that increases as the excitation wavelength increases must belong to the all-trans isomer. This is found to be the case for the short-lived component. We are thus able to assign the shortlived component to the excited-state decay lifetime of the alltrans isomer. This leads to the conclusion that the negative charge on the Asp85 of the protein catalyzes excited-state decay of the all-trans retinal in bR by 300%. Material and Methods Bacteriorhodopsin containing cells were grown from the master slants of H. salinarium ET1-001 strain kindly provided by Professor Bogomolni at UC Santa Cruz. The purple membrane was isolated and purified as described previously in refs 29, 30. The blue (deionized) bR was prepared by filtration of wild-type bR through a cation exchange column (BioRad AG 50W-X8, hydrogen form).31 All measurements were completed at room temperature (∼23 °C). All samples were light adapted for at least 30 min prior to taking measurements. The transient absorption spectroscopy setup with femtosecond time-resolution was constructed as follows. Pulses of 100 fs duration, 1 mJ, and a 1 kHz repetition rate were generated by a Ti-Sapphire laser pumped with a 4 W Innova argon ion laser (Coherent), amplified by a regenerative amplifier (Clark MXR, Inc.). The output of the laser was split into two equal parts and was used to pump two identical OPO (“TOPAS” Light Conversion Ltd.). A tunable excitation beam in a wide spectral window with energy up to a few 100 mJ was generated in each of the OPO. Also, one of the harmonics from the Ti-Sapphire laser was used as an excitation source. The excitation beam went through two computer-controlled optical delay lines with a resolution of 3 mm (22 fs). A small portion of the fundamental frequency (about 40 mJ) was used to generate a femtosecond continuum in a 1 mm sapphire plate. The spectral range of the femtosecond continuum was between 400 and 1000 nm. In some cases when the probe wavelength was outside this window, output of another OPO was used as a probe beam. The probe beam was split into two, one a signal beam and the other a reference. Pump and signal beams were overlapped on the sample in such a way that the focus of both pump beams were slightly behind the sample. The signal and reference beams were focused into optical fibers coupled into a monochromator, and were then detected by a photodetector.
Logunov et al.
Figure 1. Transient absorption spectra recorded 200 fs after excitation with 100 fs pulses at 660 nm (thin line) and 530 nm (thick line).
The excitation beam was modulated by an optical chopper at a frequency of 500 Hz. Two photodiodes were employed for the kinetic measurements at the exit slit of the monochromator. The excitation energies were measured with another photodiode. The output from the signal and reference photodiodes were amplified and passed through the sample and hold circuitry, and then coupled to a lock-in amplifier locked at 500 Hz. Each point in the kinetic measurements at a single wavelength required 200 shots at a fixed delay, and there were about 100-300 points in one delay line scan. The delay line repeatedly scanned until a reasonable signal-to-noise ratio was achieved. The typical OD change measured was in the range of 5-50 mOD. The sample was measured in a spinning cuvette to exclude thermal effects and photodegradation. For the spectral measurements the exit slit of the monochromator was removed and a CCD camera (Princeton Instruments, EUV-1024, controller ST-130) was attached. The spectra of the signal and reference beam were detected and stored in the computer. The measurements of the absorbance changes for each point were detected by comparison of the intensity ratio of the signal to the reference beam, with and without excitation. This was achieved by using a slow shutter, blocking and unblocking the excitation beam. The data were collected until a reasonable signal-to-noise ratio was achieved. Time-resolved difference absorbance spectra were obtained by averaging about 3000 pairs of unexcited and excited spectra. The group velocity dispersion of the white light continuum in the optics of the probe channel and in a cell with water was compensated by using cross-correlation data between the pump and probe pulses. Results The effect of varying the excitation wavelength on the transient spectrum of blue bR, measured 200 fs after excitation with 530 and 660 nm irradiation, is shown in Figure 1. It is apparent that the two spectra are different from one another. In the case of 530 nm excitation, the maximum of the bleach is found to be at 580 nm and the width of the hole is about 200 nm. Upon excitation at 660 nm, the maximum of the bleach is red shifted to 605 nm and the width of the hole increases to 300 nm. A similar effect is also observed in the excited-state absorption spectrum. Upon excitation at 530 nm the excitedstate absorption maximum is observed around 490 nm, while in the case of 660 nm excitation the maximum is found to be 515 nm. Furthermore, the excited-state absorption intensity induced by the 660 nm irradiation is higher by ∼20% than that induced with irradiation at 530 nm. These results suggest that
Protein Catalytic Efficiency in Retinal Decay
J. Phys. Chem. B, Vol. 102, No. 41, 1998 8111 belongs to isomer b, one can derive the following expression:
[
] ()
IL IS / IS + IL IS + IL
Figure 2. Excited-state decay of deionized blue bR measured at 490 nm, after excitation with 660 nm (thin line) and 530 nm (thick line) laser pulses.
TABLE 1: Effect of Excitation Wavelength on the Lifetimes and the Intensity Amplitudes of the Two Decay Components of the Excited States (monitored at 490 nm) of the Two Retinal Isomers wavelength (nm) AS AL τS, ps τL, ps
530
580
600
645
660
0.4 0.6 1.5 11.0
0.5 0.5 1.4 9.0
0.5 0.5 1.6 12.0
0.66 0.33 1.4 10.0
0.65 0.35 1.5 10.0
the absorption is a result of the excitation of a mixture of species and is consistent with the fact that the retinal in blue bR has two isomers in almost equal proportions.25 The two excited-state decay curves, measured at 490 nm after excitation with 530 and 660 nm 100-fs pulses, are shown in Figure 2. The two excitation pulses are located at opposite sides of the ground-state absorption maximum of retinal in blue bR (600 nm). The difference in the two kinetic profiles can be attributed to the effect of different amounts of 13-cis and alltrans isomers excited at these two wavelengths. To examine the effect of excitation wavelength on the dynamics of the retinal excited state in bR, decay curves are obtained and analyzed when the blue bR sample is excited at different wavelengths in the range between 400 and 660 nm. In this range both isomers absorb, with the all-trans isomer being stronger in the longer wavelength region.23 As shown in Table 1, as the excitation wavelength increases, the amplitude of the short excited-state lifetime increases. The different decay curves are each fitted to the biexponential function:
∆A(t)490 ) AS exp(-t/τS) + AL exp(-t/τL), where AS + AL ) 1 (1) and S and L refer to the short and long components, respectively, keeping the lifetime of both the fast and slow kinetic components at ∼1.5 and ∼10 ps, respectively. The amplitudes of both components are then calculated as a function of the excitation wavelength and are given in Table 1. Discussion Below, we study the changes in the relative absorption intensity (at 490 nm) of the short and long decay components at zero time in the decay curves obtained at different excitation wavelengths. If the short component results from the excitedstate decay of the absorption of isomer a and the long component
λexc
)
490
AS AL
λexc
) 490
N*a I490σa490 N*bI490σb490
)
( )
Na σa490 σaλexc (2) Nb σ 490 σ λexc b b
where N is the ground-state concentration, N* is the excitedstate concentration, σ490 is the absorption cross section of the excited state at λ ) 490 nm, and σλ is the ground-state absorption cross section at the variable excitation wavelength (λ). From eq 2, the change in the amplitude ratio monitors the changes in the ratio of the absorption cross section of the two isomers. Based on the assignment made32 in previous studies of the absorption of the two isomers, and on our previously33 observed correlation between the ratio of the amplitudes of the two decay components and the isomer composition of different mutants (determined chromatographically25), the ratio of the ground-state absorption cros section (σa/σb)λ in eq 2 increases with λ if a is the all-trans and b is the 13-cis isomer. From Table 1, the ratio AS/AL increases as λexc increases. This leads to the assignment that the short decay component is that of the excited state of the all-trans isomer while the long component is that of the excited state of the 13-cis isomer. From the above assignments, we conclude that the protein catalytic efficiency for the primary step in native bR is 300% by decreasing the all-trans excited-state lifetime from 1.5 to 0.5 ps. To determine the efficiency of catalysis for 13-cis retinal, we need to know the rate of the excited-state decay of the 13cis retinal in bR. Early femtosecond pump-probe measurements show that the lifetime of the excited state of 13-cis retinal is very similar to that of all-trans retinal9 in wild-type bR. Thus, removing the negative charge of Asp85, by deionization, reducing pH, or mutagenetic replacement with the neutral residue Asn, increases the excited-state lifetime of 13-cis retinal from 0.5 ps to >10 ps. Thus the protein catalytic efficiency for the primary step of the13-cis retinal in the bR cavity is >2000%! The valence bond theory predicts27 that the positive charge on the protonated Schiff base (PSB) is transferred to all the odd numbered carbon atoms (C13, C11, C9,...) along the retinal chain upon photoexcitation of retinal. If this is the case, then the negative charge of Asp85 should stabilize the positive charge on C13 and lower the potential energy of the excited state. Due to the orthogonality of the excited state and the ground state, stabilization of C13+ in the excited state will lower the barrier for isomerization of the C13-C14 bond in the excited state, and increase the barrier of the ground state.27 Because the quantum yield of the K-like intermediate is similar for the all-trans and 13-cis isomers,34-36 it follows then that the symmetry of the excited state and the ground state is the same for both isomers around the crossing point. The different effect of removing the negative charge of Asp85 on the excited-state decay of the 13-cis isomer, as compared to the all-trans, could reflect a large change in the cavity-retinal interaction for the 13-cis isomer. As a result, the charge distribution of the positive charge on the different carbon atoms allows for a smaller positive charge localization on C13 in the 13-cis than in the all-trans isomer. Acknowledgment. We gratefully acknowledge the Department of Energy (Office of Basic Energy Sciences) for funding this work under grant FG02-97ER14799.
8112 J. Phys. Chem. B, Vol. 102, No. 41, 1998 References and Notes (1) Stoeckenius, W.; Bogomolni, R. A. Annu. ReV. Biochem. 1982, 51, 587-616. (2) Oesterhelt, D.; Stoeckenius, W. Nature (London), New Biol. 1971, 233, 149-152. (3) Lozier, R. H.; Bogomolni, R. A.; Stoeckenius, W. Biophys. J. 1975, 15, 955-962. (4) Applebury, M. L.; Peters, K. S.; Rentzepis, P. M. Biophys. J. 1978, 23, 375. (5) Dobler, J.; Zinth, W.; Kaiser, W.; Oesterhelt, D. Chem. Phys. Lett. 1988, 144, 215-220. (6) Doig, S. J.; Reid, P. J.; Mathies, R. A. Proc. SPIEsInt. Soc. Opt. Eng. 1991, 1432, 184-196. (7) Mathies, R. A.; Cruz, C. H. B.; Pollard, W. T.; Shank, C. V. Science 1988, 240, 777-779. (8) Nuss, M. C.; Zinth, W.; Kaiser, W.; Koelling, E.; Oesterhelt, D. Chem. Phys. Lett. 1985, 117, 1-7. (9) Petrich, J. W.; Breton, J.; Martin, J. L.; Antonetti, A. Chem. Phys. Lett. 1987, 137, 369-375. (10) Polland, H. J.; Franz, M. A.; Zinth, W.; Kaiser, W.; Koelling, E.; Oesterhelt, D. Biophys. J. 1986, 49, 651-662. (11) Sharkov, A. V.; Pakulev, A. V.; Chekalin, S. V.; Matveets, Y. A. Biochim. Biophys. Acta 1985, 808, 94-102. (12) van den Berg, R.; Jang, D. J.; Bitting, H. C.; El-Sayed, M. A. Biophys. J. 1990, 58, 135-141. (13) Scherrer, P.; Mathew, M. K.; Sperling, W.; Stoeckenius, W. Biochemistry 1989, 28, 829-834. (14) Fahr, A.; Bamberg, E. FEBS Lett. 1982, 140, 251-253. (15) Drachev, L. A.; Kaulen, A. D.; Skulachev, V. P.; Zorina, V. V. FEBS Lett. 1988, 239, 1-4. (16) Sperling, W.; Carl, P.; Rafferty, C. N.; Dencher, N. A. Biophys. Struct. Mech. 1977, 3, 79. (17) Chang, C. H.; Chen, J. G.; Govindjee, R.; Ebrey, T. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 396-400. (18) Subramaniam, S.; Marti, T.; Khorana, H. G. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1013-1017.
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