10322
J. Phys. Chem. 1994, 98, 10322-10334
Femtosecond Spectroscopy of Chlorosome Antennas from the Green Photosynthetic Bacterium Chlorojlexus aurantiucus Sergei SavikhinJ Yinwen Zhu,* Su Lin: Robert E. Blankenship: and Walter S. Struve**t Ames Laboratory and Department of Chemistry, Iowa State University, Ames, Iowa 50011, and Department of Chemistry and Biochemistry and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-1604 Received: April 8, 1994; In Final Form: July 8, 1994@
The antenna kinetics of bacteriochlorophyll (BChl) c- and a-containing chlorosomes from the thermophilic filamentous green photosynthetic bacterium Chlorojlexus aurantiacus were investigated using two independent pump-probe techniques with subpicosecond resolution. Isotropic one- and two-color absorption difference experiments using probe wavelengths between 710 and 770 nm reveal BChl c photobleaching (PB) and stimulated emission (SE) decay kinetics with major lifetime components of 50-100 fs, 1-2 ps, and 7-10 ps. Two-color PB/SE profiles pumped at 770 nm and probed at 800 nm (where BChl a pigments absorb) exhibit no detectable rise time. However, two-color experiments using 790 and 820 nm pump and probe wavelengths, respectively, yield PB/SE rise components of 100 fs, 2 ps, and 10 ps. Upon excitation at 720 nm, the BChl c PB/SE spectrum observed using a broad-band probe continuum displays surprisingly little spectral evolution during the first 2 ps. Upon 760 nm excitation, the BChl c PB/SE spectrum experiences a small blue shift (from -750 to -744 nm) during the first picosecond. The one-color anisotropies r(t) in the BChl c spectrum initialize very close to 0.4 and subsequently exhibit little decay (r(w) 0.36). Singleexponential analyses of one- and two-color anisotropies probed at 800 nm yield a component with a lifetime -10 ps; the final anisotropies are generally small, indicating that the energy transfers in this region are accompanied by a large reorientation of Qy transition moments. The absorption difference profiles in the BChl c (but not in the BChl a ) region of the chlorosome spectrum contain oscillating components at early times, which are damped within -1 ps. They likely arise from vibrational coherences in the BChl c aggregate. Our results do not appear to be consistent with subpicosecond spectral equilibration among inequivalent BChl c pigments within the main BChl c absorption band. The essentially prompt PB/SE absorbance changes at wavelengths up to 800 nm suggest that some pigments absorbing near the latter wavelength are kinetically strongly coupled to the main BChl c pigment aggregates. This is accompanied by apparent energy transfer and depolarization (with kinetics that exhibit a 10 ps component) to longer-wavelength BChl a pigments that exhibit PB/SE in the 820 nm region. The latter BChl a pigments have orientations that differ considerably from those of the 800 nm pigments. Whether the 800 nm absorbing pigments are BChl a or long wavelength forms of BChl c is not yet clear.
-
Introduction Chlorosomes are the principal light-harvesting assemblies in green photosynthetic bacteria. Their ellipsoidal bodies (100 x 30 x 12 nm3)are attached to the inner cytoplasmic membrane,’-3 and contain rodlike elements composed of bacteriochlorophyll (BChl) c aggregates. In addition to several thousand BChl c pigments, each chlorosome contains proteins, lipids, carotenoids, and BChl a. In the green thermophilic bacterium Chloroflexus aurantiacus, electronic excitation in the carotenoids and BChl c pigments is transferred to reaction centers through at least two types of BChl a complexes: B808-866 complexes that are located with the reaction centers in the cytoplasmic membrane and B795 complexes that occupy a baseplate connecting the chlorosome to the membrane.4 While C. aurantiacus contains chlorosomes similar to those found in green sulfur bacteria, it is not otherwise closely related to green sulfur ba~teria.~ A striking property of the BChl c pigment organization in chlorosomes is that its Q, absorption, linear dichroism (LD), and circular dichroism (CD) spectra closely resemble those of BChl c aggregates that assemble spontaneously in solution. +
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Iowa State University. Arizona State University. Abstract published in Advance ACS Abstracts, September 1, 1994.
0022-365419412098- 10322$04.5010
Since the pigment organization in all other known antennas depends critically on details of protein conformation,6 this unusual capacity for “self-aggregation” has been extensively ~ t u d i e d . ~ -BChl ’ ~ c aggregates in vitro, like BChl c assemblies in chlorosomes, exhibit Q, absorption bands that are shifted by -1400 cm-’ from the monomer band and show similar CD spectra.9,11,19-23 Both the in vivo and in vitro BChl c assemblies display ultrashort excited state lifetime^.^^-^^ In certain ways they may be likened to linear J-aggregate~?~,~~ where the radiative lifetime of the lowest ( k = 1) exciton component is shorter than the monomer radiative lifetime by a factor of approximately N , the number of coupled pigments.36 However, the fluorescence is strongly quenched in all aggregated BChl c systems. The radiative decay pathway is therefore not dominant, indicating that either energy transfers or nonradiative processes are the principal decay pathways.37 The kinetics of energy transfers between the BChl c and BChl a antennas have been extensively in~estigated?~-~~ Chlorosome fluorescence decays in whole cells are multiexponential, with BChl c decay components exhibiting lifetimes of -15 ps?7-29 The latter two research groups reported that these BChl c decay times are mirrored by similar BChl a fluorescence rise times, although Mimuro et al.27did not observe such a correlation.
0 1994 American Chemical Society
Femtosecond Spectroscopy of Chlorosome Antennas
J. Phys. Chem., Vol. 98, No. 40, 1994 10323
components with nonparallel transition moments are coherently The extent of protein influence on the BChl c pigment it is of interest to determine whether experiments architecture in chlorosomes has remained unclear. Chlorosomes performed under higher time resolution show evidence for shortcontain very little protein compared to other antennas4 The lifetime anisotropy decay components that may have escaped spectroscopic properties of BChl c are reportedly unchanged detection in the earlier pump-probe study.32 Initial anisotropies when all proteins have been extracted from Chlorojlexus larger than 0.4 have already been reported in photobleaching/ chlorosomes using dodecyl s ~ l f a t e . ~ *However, -~ formation stimulated emission decays of BChl a-protein antenna comof chlorosome-like structures in water solution from proteinfree lipid-pigment chlorosome extracts has been o b ~ e r v e d . ' ~ . ~ ~ plexes (FMOtrimers, after Fenna, Matthews, and Olson) from the green bacterium Chlorobium t e p i d ~ m .Another ~~ issue is Brune et al.'O showed that addition of a 1% n-hexanol solution the kinetics of equilibration between BChl c spectral forms, e.g., to chlorosomes yields a BChl c species spectroscopically similar BChl c727 and BChl c744. Femtosecond pump-probe studies to BChl c monomers. This effect is reversible; dilution with have recently shown that a major portion of spectral equilibration fresh medium recovers the original absorption and LD spectra.10,16,42Such reversibility suggests that pigment-protein in FMO trimers occurs on a time scale of 350-450 fs.49 We have used two complementary femtosecond pump-probe interactions are not a major determinant of the BChl c pigment. organization in chlorosomes. techniques to study energy transfers involving the BChl c antenna in BChl a-containing chlorosomes from Chlorojlexus Several groups have studied the linear dichroism of the BChl c Qyabsorption band of uniaxially oriented chlorosomes.19~zz~43~M aurantiacus. The f i s t technique, based on a self-mode-locked Ti:sapphire laser, provides one- and two-color pump-probe Van Amerongen et al.43344established that these pigments are profiles with exceptional signal to noise ratios (S/N) at selected aligned with their transition moments at an average angle of wavelength combinations. The second technique, which uses 17" with respect to the chlorosome's long axis. Time-resolved a femtosecond spectrometer driven by amplified pulses from a fluorescence and absorption anisotropy data also support the dye laser, yields broad-band absorption picture that the pigments in chlorosomes are highly ~ r d e r e d . ~ ~ , Nd:YAG-pumped ~~ We find that under present resolution, the difference spectra. However, the BChl a fluorescence polarization resulting from initial BChl c anisotropies r(0) are not markedly larger than excitation of the BChl c antenna is low,2oindicating that BChl 0.4. The BChl c antenna PB/SE decay kinetics encompass at c and BChl a Qy transition moments are typically separated by least three time scales (-100 fs, 1-2 ps, and -10 ps at most large angles. A recent LD study42suggested that the transition probe wavelengths). Lifetimes very similar to these appear as moments of the BChl a species absorbing at 794 nm are PB/SE rise components in a two-color experiment using the essentially perpendicular to the BChl c moments. pump and probe wavelengths 790 and 820 nm, respectively. Whether or not chlorosomes contain forms of BChl c with The Tksapphire laser pump-probe profiles exhibit reproducible distinct absorption spectral properties has been a somewhat damped oscillations during the first several hundred femtoseccontroversial question. Holzwarth and co-workers have sugonds. Their occurrence in molecules and aggregates was gested that sequential energy transfer takes place from shorter predicted several years ago by Mukamel and c o - w ~ r k e r s , ~ ~ * ~ ~ ~ ~ ~ to longer wavelength absorbing forms of BChl c, on the basis and can arise from vibrational or exciton coherences in the BChl of fluorescencekinetic studies of chlorosomes treated to remove c antenna. the B795 BChl a baseplate that serves as the energy acceptor. The linear dichroism of the BChl c antenna Q, band varies Materials and Methods somewhat with wavelength;z2more recent LD and CD studies of three-dimensionally aligned chlorosomes42have suggested The Tksapphire laser system was constructed according to the presence of at least two major BChl c spectral forms within the design of Huang et al.,52and is described in refs 49 and 53. the main 740 nm absorption band. Gaussian simulations of the In one-color experiments, laser output was tuned with a singlepolarized absorption spectra yielded the optimized peak absorpplate intracavity birefringent filter, producing -80 fs pulse width tion wavelengths 727 and 744 nm. The transition moments of and 8-10 nm spectral bandwidth at wavelengths from 700 to the BChl c744 species are aligned nearly parallel to the 800 nm. The average output power (76 MHz repetition rate) chlorosome long axis, while the BChl c727 species apparently varied with wavelength but was typically 600 mW at 740 nm. exhibit significant transition moment components perpendicular In two-color experiments, the birefringent filter was omitted, to the long axis. Spectral decompositions of the polarized producing 740 nm in the earlier one-color experiment^.^^ The optimized time shifts between the experimental instrument function and the fitting model function are 20-30 fs. A 5-10 fs rise time feature does appear when this time shift is arbitrarily fixed at 0 fs (Table 2), but such short “lifetimes” can arise from systematic 1.5-3.0 pn optical path length differences between the autocorrelator and pump-probe apparatus. Contrasting early-time behavior is observed in the 790 820 nm two-color experiment (Figure 4 and Table 2). Profiles were accumulated using time windows of 8, 24, and 594 ps in order to study the rather complicated rise kinetics that appear at this combination of wavelengths. The optimized lifetimes from triple-exponential fits depend markedly on the time window used (Table 2 ) . Consequently, global analyses were performed on a set of 790 820 nm profiles from all of these windows simultaneously. A quintuple-exponential global analysis yielded 48% prompt rise plus three PB/SE rise components (103 fs, 2.08 ps, and 9.93 ps with amplitudes of 25%, 11%, and 16%) and two PB/SE decay components (145 ps and 378 ps with amplitudes of 67% and 33%). The three rise components correspond approximately to the PBISE decay components found in most one- and two-color experiments with a probe wavelength 5800 nm (Tables 1 and 2). The two decay component lifetimes here are considerably longer than any major PBISE decay components observed at shorter probe wavelengths. Intermediate behavior is exhibited in the 790 810 nm two-color experiment (Table 2), where the PBISE rise kinetics are empirically well described by a small-amplitude (9%) single-exponential term with a lifetime of 800 fs. Behavior very similar to that in Figure 4 is also found in broad-band absorption difference experiments probed at 800-820 nm under 760 nm excitation (not shown). Figure 5 shows broad-band absorption difference spectra of chlorosomes excited at 720 and 760 nm and probed at several
-
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Figure 4. Two-color isotropic absorption difference signals for pump-
probe wavelengths 790 820 nm, obtained using time windows of 8, 24, and 594 ps. These three perspectives reveal details of rise behavior, which exhibits components with lifetimes -100 fs, -2 ps, and -10 ps in addition to a prompt component. time delays from 0 to 10 ps. It clearly shows that the BChl c PBISE spectrum undergoes only subtle time evolution during the fist several hundred femtoseconds. With 720 nm excitation, the initial BChl c PB/SE maximum (-744 nm) exhibits no visible red-shifting that would signal downhill transfers to lower-energy BChl spectral forms. Under 760 nm excitation, the BChl c peak position experiences a small blue shift (from -750 to -744 nm) during the f i s t 500 fs, as shown in Figure 6. The latter figure also shows that 720 nm excitation yields a prompt BChl c PBISE band at -744 nm, which subsequently displays dynamic spectral shifts of -2 nm at most. Hence, during the first 2 ps there is no evidence of the large-scale spectral changes that would accompany sequential energy transfers among distinct BChl c species such as c727, c744, and c766. Holzwarth et aL30 found a fluorescence decay associated spectral (DAS) component with a 5 ps lifetime, arising from spectral equilibration within the BChl c antenna, in chlorosomes lacking the BChl a antenna. They found no similar DAS
10326 J. Phys. Chem., Vol. 98, No. 40, 1994
Savikhin et al.
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TABLE 3: Optimized Parameters for Single- and Double-Exponential Fits to Chlorosome Anisotropy Decays 2, nm window, ps tl, fs (AI) ZZ, PS (Az) r(m) r(O) 710 8 393 (0.023) 11.4 (0.059) 0.295 0.377 740 8 1450 (0.029) 0.362 0.391 750 8 352 (0.034) 0.366 0.400 760 8 1040 (0.030) 0.357 0.387 800 8 9.4 0.118 0.380
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Figure 5. Absorption difference spectra of chlorosomes excited at 720
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at 750 nm, in the main BChl absorption region of the chlorosome. Transform-limited laser pulses were used. Final parameters for singleexponential fits to this and other anisotropy decays are given in Table 3. Quantum oscillations at early times atise from vibrational coherences (see Discussion).
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wavelengths). The anisotropy function
appears to initialize very close to 0.4, and its limiting value r(-) at long times is large (-0.36). A single-exponential fit to r(t) yields the anisotropic lifetime = 352 fs. However, the BChl c anisotropy decay times yielded in our one-color experiments vary considerably with wavelength (Table 3). The optimized lifetimes from single-exponential fits to r(t) at 740 and 760 nm are 1.45 and 1.04 ps, respectively. A double-exponential model (with lifetimes of 393 fs and 11.4 ps) is required to describe the anisotropy at 710 nm. The apparent BChl c anisotropy lifetimes found in experiments under lower time resolution similarly varied with wavelength (-7 and -4 ps at 720 and 740 nm, respecti~ely).~~ The present femtosecond probe pulses are spectrally broad (10-12 nm), and so these measurements average the anisotropic decay kinetics over broader pump and probe bandwidths than the earlier experiments. If the anisotropy decays (like the isotropic decays) contain lifetime components of -100 fs, 1-2 ps, and -10 ps, the first component would have become pulse-limited in the picosecond pump-probe experiment; the outcome of a single-exponential fit to the 1-2 and 10 ps components would have yielded a result intermediate between these lifetimes. In summary, comparisons between the present and earlier results3* are clouded by differences in time resolution and bandwidth and by the fact that the total anisotropy changes are numerically small ( 5 : 0.1). The extraction of r(0) in Figure 7 is complicated by the presence of coherent coupling artifacts at early times. Figure
J. Phys. Chem., Vol. 98, No. 40, 1994 10327
Femtosecond Spectroscopy of Chlorosome Antennas 1
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Figure 8. One-color anisotropic profiles and anisotropy functions at 740 nm, obtained using transform-limited pulses (top) and with overcompensated pulses (bottom). In both cases, dashed curves give the laser cross-correlation (instrument) function. The oscillations observed in A&) and A&) at early times using transform-limited pulses are vibrational coherences (see Discussion); they do not appear in the anisotropy functions r(t). Sigmoid behavior in r(t) near t = 0 for overcompensated pulses is an artifact of intentional laser chirp. The marked, low-frequency “oscillation” in the anisotropic signals for overcompensated laser pulses is not a vibrational coherence but an artifact of clurp sweeping.
8 demonstrates how non-transform-limited pulses can be deliberately used to separate the early molecular response signal from the coherent spike. In the upper portion of Figure 8, the polarized absorption signals were obtained using group velocity dispersion (GVD) compensated pulses centered at 740 nm, yielding a 115 fs fwhm instrument function. The limiting value of r(0) at early times here is apparently 0.4. In the lower portion of Figure 8, the same profile was measured with overcompensated pulses, yielding a 200 fs fwhm instrument function. In the latter case, the width of the coherent spike is essentially unchanged (1 15 fs fwhm),since the Tixapphire cavity alignment is fixed. Hence, the anisotropy function at negative times, where the total responses are statistically significant but where the coherent spike has not set in (-150 fs It 5 - 60 fs in the lower portion of Figure 8), are dominated by the true molecular response signal. At such times, r(t) is seen to be very close to 0.4 (-0.42 at most). The sigmoid behavior of r(t) near t = 0 in the lower portion of Figure 8 is an artifact of laser chirp and does not appear when the laser pulses are transform-limited. This situation arises because the blue parts of overcompensated (anomalously chirped) laser pulses are biased toward their leading edges. The pump-probe signals at negative times in the lower portion of Figure 8 thus arise from temporal overlap between the (red) trailing edge and (blue) leading edge of the probe and pump pulses, respectively. These roles become reversed at early positive times, producing the sigmoid signal. While the effects of laser chirp are comparatively subtle in Figure 8, they become pronounced at wavelengths near the zerocrossing point (at -730 nm)32 in the BChl c Qy absorption difference spectrum (data not shown). Consequently, earlytime dynamics observed using non-transform-limited pulses can be dominated by chirp sweeping rather than antenna kinetics.
3-; --2l /
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anisotropy functions. Final parameters for these fits are listed in Table
Figure 9. Two-color anisotropy decays and anisotropy functions r(t) for pump-probe wavelengths 770 800 nm, 780 800 nm, and 790 800 nm. Smooth curves show single-exponential fits to 3.
As summarized in Table 3, the initial one-color anisotropies r(0) observed in the BChl c Qy spectrum from 710 to 760 nm are close to 0.4 (0.36 r(0) 0.42). This situation differs from that in FMO trimers, where initial anisotropies considerably larger than 0.4 have been observed.49 The residual anisotropies found for these wavelengths at long times (typically r(=) -0.36) are much larger than those found in FMO trimer^.^^,^^ Very different anisotropy behavior is found in two-color experiments using a probe wavelength of 800 nm in the BChl a antenna region, as shown in Figure 9. Single-exponential fits to these two-color anisotropies yield appreciably longer lifetimes (7.8-9.4 ps, Table 3) than the one-color anisotropies at most BChl c absorption wavelengths. These lifetimes are similar to that of a rise component found in the 790 820 nm two-color magic-angle profiles (Figure 4), and they resemble the time scale previously assigned to BChl c BChl a energy transfers on the basis of fluorescence e~periments.~~-~O The residual anisotropies are far smaller (-0.1 5 r(m) I+O.l) than those found in the BChl c spectrum, and the initial anisotropies r(0) extrapolate to values somewhat lower than 0.4. Similarly, a single-exponential fit to the one-color anisotropy at 800 nm (Table 3) yields a lifetime of 9.4 ps and a residual anisotropy of 0.118. The one-color anisotropy decays at 780 and 790 nm also display major components with a lifetime of -10 ps (not shown), while the 770 nm decay resembles those observed at 710-760 nm. Damped Oscillations. The early-time absorption difference signals All(t) and A l ( t ) contain reproducible oscillations when the probe spectrum overlaps the BChl c absorption bands (Figure 10). These oscillations, which are damped within -1 ps, are especially marked in one-color experiments using wellcompensated (i.e., transform-limited)laser pulses. They are also discemible in the residuals of multiexponential fits to two-color profiles (1-2% of the total absorption difference signal) if the pump and probe wavelengths overlap the 740 nm BChl c
-
-.
10328 J. Phys. Chem., Vol. 98, No. 40, 1994
Savikhin et al. spectra, with a relatively large number of frequency components in the neighborhood of 50 and 100-250 cm-'. In view of the -1 ps oscillation damping time, the frequency broadening for a single mode with a well-defined frequency should be on the order of 15 cm-'. Hence, while the Fourier spectra vary considerably with wavelength, there generally appear to be at least two frequencies in the neighborhood of 50 cm-', and several frequencies between 100 and 250 cm-'. No similar oscillations were observed using probe wavelengths (790-800 nm) which do not overlap the main BChl c absorption spectrum. In this regard, the femtosecond absorption difference spectroscopy of the BChl a antenna in chlorosomes resembles that of FMO trimers, in which no oscillations were
Discussion
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Frequency, cm-I Figure 11. Oscillating parts of one-color BChl c antenna absorption difference signals at several wavelengths from 710 to 760 nm (left) and their Fourier transform spectra (right). absorption band. The ratio of the oscillating components in All and A 1 is similar to the 3: 1 ratio of the monotonic components in All and A l ; hence, the oscillations are essentially absent in the anisotropy functions r(t). In Figure 11, the oscillating parts of the one-color profiles are shown together with their Fourier transform spectra. Unlike the vibrational coherences observed by Vos et al. in bacterial reaction center mutants57and by Wise et al. in organic dyes,58these oscillations exhibit complicated Time, ps
Isotropic Antenna Kinetics. The principal features in our isotropic absorption difference profiles are as follows: (a) Aside from a major femtosecond decay that overlaps the first coherent oscillation, the one-color profiles exhibit decay components of 1-2 ps and 7-10 ps at most BChl c wavelengths from 710 to 760 nm. (b) In two-color profiles accumulated using probe wavelengths shifted 20 nm to the red from the pump wavelength, no PBISE rise kinetics are detected when the probe wavelength is 5800 nm. Triple-exponential fits to these profiles for probe wavelengths '790 nm typically yield, in addition to 1-2 ps and 7- 13 ps PBISE decay components, a subpicosecond decay component with a lifetime of 48-109 fs. Markedly different long-component lifetimes are found in two-color profiles probed at 790 nm (20-30 ps) and 800 nm (120-130 ps), indicating that some of the pigments probed at these wavelengths belong to a different kinetic pool than the ones probed at 740-780 nm. (c) A global analysis of the 790 820 nm two-color kinetics (Figure 4) yields PB/SE rise components of 103 fs, 2.08 ps, and 9.93 ps and decay components of 145 and 378 ps. In addition, the rise kinetics contain a substantial (48%) prompt component. All three rise components mirror PBISE decay components from the two-color experiments obtained using probe wavelengths
