J. Phys. Chem. 1996, 100, 9537-9544
9537
Annihilation Processes in the Isolated D1-D2-cyt-b559 Reaction Center Complex of Photosystem II. An Intensity-Dependence Study of Femtosecond Transient Absorption†,‡ M. G. Mu1 ller, M. Hucke, M. Reus, and A. R. Holzwarth* Max-Planck-Institut fu¨ r Strahlenchemie, Stiftstr. 34-36; D-45470 Mu¨ lheim a.d. Ruhr, Germany ReceiVed: December 11, 1995; In Final Form: February 29, 1996X
The excitation intensity dependence of the kinetics of the primary processes and of the yield of radical pair formation in the isolated D1-D2-cyt-b559 reaction center of photosystem II has been studied by femtosecond transient absorption spectroscopy. It is shown that the kinetics is strongly dependent on the excitation intensity. The radical pair yield as a function of excitation intensity is compared with the theoretical annihilation curve and a good agreement between theory and experiment is observed, indicating that the intensity effects on the kinetics and radical pair yield arise primarily from annihilation processes. Sufficiently annihilation-free measurements require excitation intensities that give rise to e0.06 absorbed photons/RC while maintaining a high signal/noise ratio of g100:1 at most detection wavelengths in order to resolve the complex kinetics. It is shown that such low excitation intensities give rise to absorption changes that are at the edge of the capabilities of present femtosecond absorption equipment. We also compare the excitation conditions that have been used so far by other research groups for published transient absorption data on the isolated D1D2-cyt-b559 complex. This comparison shows that essentially all published data have been obtained under conditions where annihilation or quenching effects are expected to significantly distort the kinetics and timeresolved spectra and to reduce the radical pair yield.
Introduction Photosystem (PS) II is the antenna/reaction center complex in higher plants and green algae that performs the photoinduced splitting of water into molecular oxygen and protons and which is thus of prime importance for the maintenance of the biosphere.1-3 The so-called D1-D2-cyt-b559 complex was isolated for the first time in 1987 from PS II particles and has been shown to be the unit active in primary charge separation,4,5 and it is therefore called the reaction center (RC) complex of PS II. Since its isolation, there is vivid interest in understanding the mechanism and the kinetics of the primary processes in this complex. Quite a variety of spectroscopic techniques have been employed to elucidate these processes.6 Among those that potentially can provide direct information on the kinetics and the mechanisms of the charge separation and other fast processes, femtosecond transient absorption spectroscopy ranks high on the list. For this reason a number of femtosecond studies has been performed on the D1-D2 complex as a function of temperature, excitation intensity, redox state, etc. (for a review see e.g. ref 6). Unfortunately the outcome of these experiments carried out by various groups has led to highly controversial and fundamentally differing interpretations. Thus the group of Wasielewski and co-workers assigned a 3 ps component to the primary charge separation process,7,8 while the group of Klug and co-workers criticized that assignment and instead attributed a ∼21 ps component to that process.9-11 Both the groups of Holzwarth et al.12-16 and van Gorkom et al.17 favored the 3 ps interpretation for the primary charge separation based on fluorescence and transient absorption data, respectively. Up to now no agreement has been reached in this matter. Furthermore there was an additional disagreement on the lifetimes and * Author to whom all correspondence should be addressed. † This work has been presented in part at the ESF-Workshop on “Structure and Function of the isolated D1-D2 reaction center”, Wye College, England, April, 1995. ‡ Abbreviations: Chl, chlorophyll; Pheo, pheophytin; RC, reaction center; D1, D2, polypeptides of the reaction center of photosystem II. X Abstract published in AdVance ACS Abstracts, May 1, 1996.
S0022-3654(95)03715-4 CCC: $12.00
spectral range of slow energy transfer components present in the D1-D2 complex between the groups of Klug and coworkers9,18,19 and Holzwarth and co-workers12-16,20 which still persists in part. A further topic of controversy was the effect of using parallel vs magic-angle detection conditions.12,21 Despite these controversies a common conclusion prevailing from all of the studies was that the primary kinetics is highly complex, and unravelling it represents a challenging task. As it stands up to now no valid kinetic scheme has been presented that would be suitable to explain satisfactorily the observed transient absorption and fluorescence kinetics and would at the same time provide reasonable species-associated (fluorescence and/or transient absorption) spectra of the intermediates. This ongoing controversy is to a large part caused by the fact that (a) the absorption and absorption difference spectra of all the involved states are highly congested in the Qy region of the spectrum and (b) that no detailed kinetic models have been tested on the data. In addition, intensity-dependent effects, underestimated largely so far, might also be causing at least part of the discrepancies between different groups. This is the subject of the present study. If one sets out to unravel the ultrafast processes in the D1D2 complex it is important to determine exactly the upper limit of excitation pulse intensity that would allow one to observe the kinetics in the linear range, sufficiently undistorted from annihilation and other nonlinear effects that might influence the kinetics. Surprisingly such a study has not been performed in detail to our knowledge. Despite some claims in the literature from most groups that they actually performed their transient absorption measurements under annihilation free conditions, in fact rough estimates of the excitation intensities used already suggest that excitation intensities applied may have been quite high in many cases. Thus in some published data even the signs of transient absorption signals differed, let alone the shape of the kinetics.7,9 Furthermore most groups reported prominent transient absorption components with lifetimes comparable to the pulse widths used in their measurements.7,10,19,22 Such © 1996 American Chemical Society
9538 J. Phys. Chem., Vol. 100, No. 22, 1996
Figure 1. Absorption spectrum of D1-D2 complex at room temperature (full line) and spectrum of 680 nm femtosecond excitation pulse (dashed line).
ultrafast components could to a significant part be caused by annihilation effects since the majority of fast annihilation is expected to occur in the RC core on a time scale comparable to energy transfer.19 In view of this situation the aim of our present study is severalfold: First, to measure the kinetics of D1-D2 particles as a function of the excitation intensity over a large range. Second, to measure the annihilation curve for radical pair formation and to compare it with exact theoretical calculations. Third, to present a detailed comparison of the kinetics with published data from other groups and to compare the excitation conditions used by various groups. As a main result of this study we conclude that femtosecond transient absorption experiments published so far have not been performed under sufficiently annihilation-free conditions and that in some cases actually quite high pulse intensities or alternatively high average powers have been employed, giving rise to severe annihilation effects or substantial concentration of quenching intermediates, respectively, and thus to concomitant modifications in the kinetics. This has profound consequences on the interpretation and conclusions drawn from these experiments. Materials and Methods The D1-D2 cyt-b559 RCs used in this work have been isolated according to van Leeuwen et al.23 with slight modifications.24 All measurements reported here have been carried out on fractions of a sample pool which had a Chl/2 Pheo ratio of 6.1 ( 0.2 as determined by HPLC.24 The room temperature spectrum of the RCs are shown in Figure 1 along with the spectrum of the femtosecond excitation pulse peaking at 680 nm. For some measurements samples with a slightly higher Chl/2 Pheo ratio, i.e. 6.3 ( 0.2, have been used but the results did not differ significantly with respect to the results discussed here. For measurements, carried out at 4 °C, the sample was contained in buffer Tris-HCl 50 mM, pH ) 7.2, detergent dodecyl maltoside 0.1%. The sample was filled into a rotating cuvette (d ) 1 mm, volume 3 mL) under a nitrogen atmosphere. The cuvette is air tight so that with the additional use of the enzymatic oxygen-scrubbing system glucose/glucose oxidase and catalase25 the sample was kept under anaerobic conditions. The absorption of the sample at the excitation wavelength (680 nm) was adjusted to about 0.8-0.9/mm (i.e. OD ) 1/mm at 675 nm). A single excitation pulse hits the same sample volume only every ∼50 s on average. Femtosecond Absorption and Data Analysis. Excitation pulses for femtosecond absorption measurements were generated by chirped pulse amplification of 40 fs pulses from a Tisapphire laser oscillator (Tsunami, Spectra Physics) in a regenerative Ti-sapphire amplifier and stretcher/compressor unit (Quantronix model 4810). The output pulses from the amplifier (FWHM ) 80 fs) were frequency shifted using an
Mu¨ller et al. optical parametric generator (Topas, Light Conversion). The spectral width of the excitation pulses (∼120 fs FWHM; ca. 180 fs autocorrelation width) was limited to e6 nm spectral width at a repetition rate of 3 kHz. The pulses were close to being transform limited. Part of the 800 nm light from the amplifier was used to generate a white light continuum of 80 fs width (FWHM). The pump and probe pulses were polarized at magic angle relative to each other in order to exclude any kinetic depolarization effects. A narrow wavelength range from the white light continuum, after transmitting the sample, was selected by a double monochromator (DH10 Vis, Jobin Yvon, spectral width ∼2 nm) and was detected by a photomultiplier. Lock-in detection was used for recording the transient absorption kinetics. Full details of the apparatus will be published in a separate report. The rms noise of the detection system was as low as (5 × 10-7 OD units under actual measurement conditions in the best cases. This allowed data with a very high signal/noise (S/N) ratio to be obtained at very low excitation intensities which was actually a prerequisite for the measurements presented here. The exciting and detecting light were focused to a spot with a diameter (FWHM) of 0.13 mm. Determination of the Molar Absorption Coefficient per RC. The molar absorption coefficient (or absorption cross section) of a D1-D2 particle at room temperature is a key parameter for the theoretical calculation of the multiple hit probability as a function of excitation intensity. Our preliminary data indicated that this extinction coefficient is much higher than what was obtained on an estimate based on the absorption coefficients of Chl a and Pheo a in solution of organic solvents.26,27 We therefore determined the absorption coefficient of D1-D2 RCs experimentally in the following way: The absorption spectrum of a D1-D2 RC solution was measured on an absolute scale. An aliquot of this sample was taken, and the pigments were extracted using standard techniques,24 and the absorption spectrum of the extract was measured. The Chl/ Pheo ratio was determined by HPLC.24 The absorption spectra of solutions of known concentration of pure Chl a and Pheo a were measured in the same solvent (Note: It is important to use exactly the same water content in the solvent as used for measuring the extract, since in particular the Pheo a spectrum undergoes shifts and spectral changes in response to changing water content.) The spectrum of the extract was then composed as a linear superposition of the pure Chl a and Pheo a spectra by a fit over most of the Qy band region.24 The extinction coefficient of the intact D1-D2 particle was then determined from these data assuming that the integrated absorption coefficient of Chl a and Pheo a in the Qy region remains unchanged when going from the organic solvent to the protein. In this way the value of the molar absorption coefficient of the entire D1-D2 complex (6.1 Chl + 2 Pheo) was determined to be max ) 720.000 ( 10% (675.5 nm). This value is by roughly a factor of 2 higher than what would be obtained based on adding up the solution spectra of the chromophores in organic solvents (even after accounting properly for spectral shifts according to the spectral decomposition24). A detailed analysis of the absorption line-shape function is given in ref 24. The reason for the much higher absorption coefficient is the substantially smaller (by a factor of about 2) inhomogeneous width of the spectra of Chl a and Pheo a in the protein as compared to organic solvent. Thus in the protein the spectra are narrower and have a higher extinction coefficient. Data Analysis. Kinetic transient absorption data were analyzed in a global fashion by combining the data sets recorded at different detection wavelengths and excitation intensities.28,29 The global analysis included a deconvolution of the kinetics
Annihilation Processes in Photosystem II
a
J. Phys. Chem., Vol. 100, No. 22, 1996 9539
a
b
b
Figure 3. Plot of amplitude ratios of lifetime components for a fourexponential fit including all excitation intensities: (a) for 545 nm detection and (b) for 680 nm detection. Components are as follows: A1, 46 ps; A2, 1.1 ps; A3, long-lived (nondecaying); A4, 100 fs.
Figure 2. Intensity dependence of the transient absorption signal at a detection wavelength of 680 nm (a) and at 545 nm (b), λexc ) 680 nm. The signals were normalized to the maximum at time zero in order to enable a better comparison.
with the autocorrelation of the excitation pulse. The quality of the fits was judged in each case on the basis of χ2 values and plots of the weighted residuals.29 Results and Discussion Using nearly transform-limited 120 fs (FWHM) pulses with a spectral maximum at 680 nm (Figure 1) the intensity dependence of the transient absorption signals was measured for detection wavelengths of 545 nm (Pheo Qx band) and 680 nm (i.e. near the P680 absorption maximum) over a time range of up to 300 ps. The excitation energy/pulse was varied over nearly 3 orders of magnitude. The kinetic traces are compared in Figure 2a for 680 nm and in Figure 2b for 545 nm detection wavelength. As can be seen from these figures the kinetics is dependent on excitation energy down to about 1.7 nJ/pulse. Below this intensity the kinetics remains essentially constant. In addition to complex changes in the kinetics at short delay times (see below) a strong decrease of the intensity-normalized maximal bleaching at long delay times (300 ps) is observed with increasing excitation pulse intensity. The long-lived bleaching is taken as a direct measure of the amount of longlived radical pair formed. Thus these data point to a drastic decrease in the (relative) yield for radical pair formation at increasing excitation intensity (cf. Figure 2).
The data from all the kinetic traces for the two detection wavelengths and all excitation intensities were subjected to global analysis. The minimal number of exponential components required to achieve a reasonable fit to the kinetics was four with lifetimes of ∼100 fs, 1.1 ps, 46 ps, and g5 ns (nondecaying). Figure 3, parts a and b, shows the plots of the amplitude ratios for the components of the four-exponential fit as a function of excitation intensity. We chose to plot the amplitude ratios, rather than the absolute values, because it is thus easier to recognize any changes in the kinetics, since for intensity independent kinetics the amplitude ratios should remain constant. Since the kinetics depends drastically on the excitation intensity, the lifetimes from the global analysis are dominated by the (distorted) values from the high excitation intensities if all kinetic traces (at all excitation intensities) are included. This procedure thus leads to large distortions in the amplitudes for the low excitation intensities, since there the lifetimes are in fact quite different. For this reason we have performed an additional global analysis leaving out the decays for high excitation intensities (only for 680 nm detection, cf. Figure 4). These new lifetimes for the low intensity range are 250 fs, 48 ps, 2.7 ps, and long-lived (nondecaying). From comparison of Figures 3 and 4 it can be seen that the amplitude ratios change substantially even at relatively low excitation intensity and for some of the components (e.g. the ultrafast 100 fs component) even the sign changes in that intensity region. It is also clear that not only the amount of nondecaying component (A3) is reduced, but the entire kinetics is strongly dependent on the excitation intensity. This demonstrates that kinetics undistorted by annihilation and other artifacts can be measured only at very low excitation intensities, i.e. at energies e1.7 nJ/pulse under our focusing conditions (corresponding to e0.06 absorbed photons/RC). Intensity Effects on the Relative Radical Pair Yield. One of the most easily recognizable effects of high intensity should
9540 J. Phys. Chem., Vol. 100, No. 22, 1996
Figure 4. Plot of amplitude ratios of lifetime components from a global analysis leaving out the high intensities. Components are as follows: A1, 48 ps; A2, 2.7 ps; A3, long-lived (nondecaying); A4, 250 fs.
Figure 5. Plot of the relative yield of formation of radical pair (absorbance change after 300 ps at 680 nm normalized to the excitation intensity) as a function of excitation density at 680 nm (x axis 1) and as a function of the average number of absorbed photons/RC/pulse (x axis 2). Also shown is the annihilation curve (dashed) as calculated based on the multiple hit probability (See Appendix and text). The arrows indicate the excitation intensities used for various measurements: (1) lowest intensity measurement at 545 nm detection (cf. Figure 6b); (2) intensity used for recording a complete low intensity data set (7 nJ at 680 nm with 130 µm spot diameter, see text); (3) estimated intensity used in the measurements of ref 11; (4) 70 nJ/pulse as used for measuring the kinetics shown in Figure 6a; (5) estimated intensity used in the measurements of ref 22. For excitation wavelengths other than 680 nm the relevant numbers have been recalculated to the equivalent value for hypothetical 680 nm excitation taking into account the absorption spectrum.
be a reduction in radical pair yield which can be measured in transient absorption at long delay time. Thus in order to put the observed intensity effects on a more quantitative basis and to be able to compare it to the theory for annihilation and also to the data from other groups we have plotted in Figure 5 the intensity-normalized yield of radical pair formation (measured from the bleaching at 680 nm for a delay of 300 ps normalized to the excitation intensity). The experimental data are compared with the theoretical curve using the formalism and the assumptions given in the Appendix. Essentially the underlying theory is the same as that given by Paillotin30,31 except that we use here the exact solutions which apply to any optical density of the sample and also take into account the details of the excitation and detection conditions relevant for transient absorption detection. For the sake of simplicity, and since no better information was available for most studies reported in the literature, we assumed a square excitation profile (i.e. one filament only according to the formulae in the Appendix). It
Mu¨ller et al. follows from the plot in Figure 5 that the theory describes the experimental situation quite well up to the highest excitation intensities used. Thus it seems that at least the intensity effect on the relative radical pair yield can be described by a straightforward annihilation process. A 100% probability for annihilation upon multiple excitation within a single RC particle was used in the theoretical calculations. In order to be able to solve the relevant equations (see Appendix) the molar absorption coefficient for the D1-D2 particles was required. Using a first estimate for this value on the basis of solution spectra for Chl a and Pheo a (maxestimated ≈ 310.000 L mol-1 cm-1) we could not get a reasonable agreement between theory and experiment. Experimental determination of the absorption coefficient indeed led to an about two times higher absorption coefficient (maxexp ≈ 720.000 L mol-1 cm-1; see Materials and Methods). Using this value then resulted in a very good agreement with the experimental curve (see Figure 5). From the absorption change at low intensity excitation and long delay time we can calculate (see Appendix) a lower limit of 70.000 L mol-1 cm-1 ((10%) for the difference absorption coefficient ∆ of the radical pair (at 680 nm), i.e. the difference (P680-Pheo) - (P680+ - Pheo-).42 Even considering the substantial absorption increase overlapping with the bleaching, this number is surprisingly low, taking into account the fact that at least one Chl (from P680) and one Pheo should contribute to the bleaching. This number will presumably increase somewhat once a valid kinetic scheme is available. Comparison of Excitation Intensities Used by Various Groups. In order to be able to judge the conclusions drawn by various groups from their transient absorption experiments it is important to know the exact excitation conditions and the resulting annihilation probabilities. We have thus compiled in Table 1 the excitation and annihilation conditions at which the transient absorption experiments published in the literature on D1-D2 have been performed. Where the authors have provided all the necessary information to calculate the impinging photon density per pulse and unit area the values given in the respective references have been used as the basis for our calculations (performed according to the formalism given in the Appendix). In those cases where the exact excitation conditions have not been provided by the authors, we used their published experimental absorption changes at 680 nm and long delay times (radical pair formation) as the basis for the calculation. In that case normalization has been performed with our measured kinetics and absorbance change at low excitation intensity (see Figure 2). The group of Wasielewski and co-workers in their first measurements7 used excitation in the nonselective region around 610 nm. These experiments have been performed under extremely high excitation conditions leading to annihilation (on average) in more than 88% of the RCs (relative radical pair yield only 12%!). It should be noted that the initial suggestion of a ∼3 ps charge separation kinetics has been made on the basis of these experiments. By taking the annihilation data in Table 1 into account, it is clear that in case that this interpretation (i.e. ∼3 ps charge separation) should finally turn out to be correct indeed, the initial conclusions would have to be considered to be highly fortuitous. The Wasielewski group has recently reported new measurements (mainly at 545 nm detection) at substantially lower excitation intensity.32 Nevertheless in their new data set the annihilation probability is still ∼22%. The most extensive (in terms of both spectra and kinetics) transient absorption data sets on D1-D2 complexes have been reported by the group of Klug and co-workers.9-11,18,43 Our estimate of their excitation conditions for measurements at magic
Annihilation Processes in Photosystem II
J. Phys. Chem., Vol. 100, No. 22, 1996 9541
TABLE 1: Comparison of Excitation and Annihilation Conditions Used by Various Groups When Measuring Ultrafast Absorbance Changes on the D1-D2-cyt-b559 Reaction Center Complex research group
ref
Φrel
annihilation (%)
average absorbed photons/RC/pulse
approximate S/N ratio (680 nm)
8.7 0.53 0.4
∼5 ∼50-60 ∼20
0.023
∼20
11
0.23
g200
0.97
3
0.06
∼50
this work
0.994
0.6
0.012
∼40
22
0.61
1.15
- (only spectra)
Wasielewski et al. Wasielewski et al. Klug et al.
7 32 11
0.12 0.78 0.82
van Gorkom et al.
17
0.988
Holzwarth et al.
13 and this work
0.89
Holzwarth et al.
this work
Holzwarth et al. van Grondelle et al.
88 22 18 1.2
39
comment very high intensity data for lowest intensity (60 nJ) used estimated based on absorbance change at 680 nm for radical pair valid for 680 nm excitation; ≈0.9 photons/RC total over 40 pulses due to high repetition rate (average pump intensity 400 W/cm2); Qy detection range; from all the RCs in the irradiated/detected volume under these conditions 10% are in the radical pair state and 20% are in the triplet state measurement series using 7 nJ/pulse at 680 nm (average pump intensity 0.18 W/cm2) measurements at 1.7 nJ/pulse (545 nm detection); average pump intensity 0.04 W/cm2 measurements at 0.34 nJ/pulse, (680 nm detection); average pump intensity 0.008 W/cm2 77 K measurement for 680 nm excitation; based on absorption change at 680 nm for radical pair; the favorable assumption that ∆ at 77 K is about 30% higher than at room temperature was made
a,b The absorption coefficient for the RC particle at 675 nm (room temperature) has been determined to be 720 000 ( 10% (see text). The lower limit for the ∆ difference absorption coefficient (P680Pheo-P680+ Pheo-) at 680 nm has been estimated to be 70 000 ( 15% at 680 nm (room temperature). Φrel is the relative yield of radical pairs calculated as number of radical pairs/number of photons absorbed. b The data presented here have been calculated based on the data for excitation energy, wavelength, absorbance, and beam diameter given in the indicated references. Where these parameters were not given, the calculation has been made on the observed absorption change at 680 nm for the radical pair (long delay time). Good agreement between calculated and measured absorption changes was obtained in all cases where a comparison could be made.
angle11 results in a probability of ∼18% annihilation (relative radical pair yield 0.82), i.e. similar to the excitation conditions used for the new data from the Wasielewski group. Clearly the intensities used by either the Wasielewski or the Klug group are still too high for a reliable answer to the problem of the exact charge separation kinetics (see detailed discussion below). In this connection one may in particular also question the interpretation of the ultrafast (100 fs) component which has been assigned by the Klug group to equilibration among the strongly coupled RC core excited states. Undoubtedly a substantial contribution to this ultrafast component will arise due to annihilation under the excitation conditions used. Furthermore the exact kinetics for the equilibration process might be significantly slower if undistorted by annihilation. This is shown by our data at lower intensity. While higher excitation intensities in our intensity-dependent measurements led to an ultrafast component of about 100 fs (i.e. approximately the excitation pulse width), measurements at lower intensity gave a longer-lived ultrafast component of about 250-350 fs. Using picosecond excitation pulses of very high repetition rate, the group of van Gorkom and co-workers has reported a series of transient absorption data at very low excitation intensity per pulse.17 Our calculations indeed show that their measurements were essentially annihilation-free (∼1.2% annihilation) if judged on a per pulse basis. Thus these measurements are the only ones performed so far under sufficiently low excitation intensity to exclude singlet-singlet annihilation. Nevertheless the measurements from the van Gorkom group suffer from a severe problem: Due to the high repetition rate of 80 MHz every RC particle absorbed on average 0.9 photons since it “sees” about 40 excitation pulses on average before the sample could be exchanged. Consequently the multiple excitation probability is very high (according to multiple hit theory 12% double hits, ∼4% triple hits, i.e. altogether g36% multiple hits). These
multiple excitations, due to the fact that they occur from consecutive pulses with a pulse-pulse distance of multiples of ∼12 ns, will occur mostly in RCs that have already undergone charge separation and are thus either in the radical pair state or in the triplet state. The very efficient quenching effect of the radical pair on the newly created excited state is unpredictable in detail but is expected to lead to severe distortion in the observed kinetics.33-36 Similarly, singlet-triplet annihilation could be quite substantial as well (cf. Table 1). Thus despite the low excitation intensity per pulse any conclusions drawn from these measurements should be considered with caution. We have recently measured an extensive multi-wavelength femtosecond data set.37,44 The aim was to avoid as much as possible annihilation but nevertheless keep a high S/N ratio that would also allow to measure the 545 nm range under the same excitation conditions with a sufficiently high S/N ratio. The series was performed using 7 nJ/pulse in the excitation, focused to a spot of 130 µm diameter. Under these low intensity conditions (this corresponds to about two times lower effective photon density than used by the Klug group, corrected for the different excitation wavelengths) nevertheless the probability for annihilation events is still ∼11% (see Table 1). For some selected detection wavelengths we have also measured the kinetics at excitation intensities that were 20 times lower than that (leading to negligible annihilation). Low-temperature transient absorption measurements have been performed recently by the group of van Grondelle and co-workers.22 On the basis of the observed transient absorption changes of the radical pair at long delays and by making the favorable assumption of assuming a some 30% higher difference absorption coefficient than at room temperature, our calculation yields a relative radical pair yield of 0.61, i.e. about 40% annihilation probability, at their excitation conditions. This must be
9542 J. Phys. Chem., Vol. 100, No. 22, 1996 considered very high annihilation and is expected to lead to severe interference of annihilation and primary charge separation kinetics. In Figure 5 we have indicated by arrows on the annihilation curve the intensities at which transient absorption experiments on the D1-D2 complex have been performed so far by different groups. Arrow 3 indicates the excitation intensity at which most of the published measurements of the Klug group have been performed. This intensity is by a factor of ∼2 higher than the one used in the majority of our own measurements37 (arrow 2) and is in good agreement with the photon density and excitation probability that can be obtained from the parameters given in ref 11. However a direct comparison of our intensity-dependent transient absorption kinetics with the results published in refs 9 and 10 suggests, that for measuring the 545 nm range (Pheo Qx band) probably a g5 times higher excitation intensity has been used by these authors (indicated by arrow 4 in Figure 5) as compared for the measurements in the Qy range.45 This intensity is quite high in the annihilation region. Given the fact that a decisive part of the kinetic assignment of the Klug group is in fact based on this kinetics around 545 nm, one may question the validity of their assignment. In order to directly compare the signals we have plotted in Figure 6 our own measured kinetics at 545 nm together with that of the Klug group10 at a comparable excitation intensity (Figure 6a) and at a ∼40 times lower intensity (Figure 6b). Note in particular the substantial change in the zero-crossing time with excitation intensity and the differences in the ratio of absorption (at zero time) to bleaching amplitudes (at long time). Without any detailed analysis it is clear from this comparison that the published kinetics9,10 is distorted by nonlinear effects and that the radical pair yield (as judged by the signal at long times) is substantially decreased. Reasons for Excitation Intensity Effects on the Kinetics. The results presented above indicate that both the kinetics and the yield of radical pair are strongly dependent on the excitation intensity. The D1-D2 complex contains at least eight chromophores. Thus we expect at first glance the usual annihilation behavior of multiple chromophore assemblies, as has been studied intensively on various photosynthetic antenna systems (see e.g. refs 38 and 39). However, the situation is more complex in this case. First it is now well known that energy transfer among chromophores in the D1-D2 complex occurs on largely different time scales.12,13,19 Thus equilibration within the RC core pigments occurs most likely on a time scale of a few hundred femtoseconds.12,19 This should then also correspond approximately to the time scale of the major annihilation component since most of the absorption occurs initially by the chromophores in the core. Our data show that at high excitation intensity the lifetime of the fastest component drops from about 250 fs (at low excitation intensity) to about 100 fs at moderate and high excitation intensity. That is not the only effect, however, since the energy transfer of the external Chls with the core occurs on a 10-100 times slower time scale (∼6-30 ps).12,13 Thus also the annihilation kinetics should have secondary component(s) on a comparable picosecond time scale. This can explain the changes in the values and amplitudes of the intermediate lifetimes upon changing the excitation intensity. There is another effect however. This will come into play when a particle is hit by two photons and one of them gives rise to charge separation (on a ∼3 ps time scale as deduced from our time-resolved fluorescence experiments12,13). Thus after a few picseconds a Chl cation and most probably a Pheo anion are formed. Both of these species are known to be efficient quenchers of excited states.33-36 Thus we expect for particles with multiple excitation another component in the excited-state decay
Mu¨ller et al.
Figure 6. Comparison of the transient absorption traces at 545 nm for measurement carried out at (a) 70 nJ/pulse (dashed line) and (b) 1.7 nJ/pulse. In each case the absorption trace at 545 nm (full line) from ref 11 is overlaid, normalized to the absorption change of traces a and b at time zero. Note the difference in absorbance scales and also the ratio of absorbance maximum at time zero and at 55 ps. The measurement for b is averaged for a longer time in order to get a comparable S/N ratio as in a. The numbers in b represent the relative amount of radical pair formation, as deduced from the bleaching signal at about 55 ps delay.
due to the quenching by radical species. No exact data are available on the corresponding quenching rates, which will depend anyway strongly on the unknown distance of the different chromophores in the complex. Nevertheless the quenching is expected to be very efficient. Possible quenching mechanisms in that case are either Fo¨rster energy transfer and/or electron transfer. Finally the radical pair evolves into the triplet with high yield. Under such measuring conditions thus a relatively high steady state concentration of triplet may arise that gives rise to singlet-triplet quenching which may also be very efficient. These two latter effects may have caused substantial distortion of the kinetics reported by the van Gorkom group.17 Which Level of Annihilation Can Be Accepted? In conclusion, multiple excitation of an RC is expected to lead to very complex and not easily predictable changes in the overall kinetics and to a reduction in radical pair yield. It is clear from our data set presented here that even at low intensities the kinetics of primary processes in the D1-D2 RC is extremely complex (more components than have been deduced so far from transient absorption experiments are required for the full kinetic description37). If any meaningful analysis and interpretation of this complex kinetics is to be performed, we must be absolutely sure that we can exclude any nonlinear effects of the type(s) discussed above. Thus transient absorption kinetics have to be measured under conditions where the contribution of nonlinear effects is substantially less than the smallest amplitude component. It is important to note that most of the three or four relevant kinetic components indicative of energy transfer and/ or charge separation have small amplitude, generally e10% of the long-lived radical pair signal (for example at 680 nm and similarly at most other wavelengths).9,10,13 In this case our estimates indicate that a S/N ratio of better than 100:1 will be minimally required to unravel the five or even six exponential kinetics. In essence this amounts to the requirement that the
Annihilation Processes in Photosystem II probability of multiple excitation per particle and pulse must be reduced to a maximum of about 2%, which is still by about a factor of 4 lower than what has been used in our own extended low intensity measurement series (i.e. 1.8 × 1014 photons cm-2 pulse-1 corresponding to 7 nJ/pulse at 680 nm with a spot diameter of 130 µm). This figure is a tremendous challenge to the experiment since it is necessary to measure transient absorption changes in the range of 10-5 to a maximum of 10-3 OD units with a S/N ratio of g100, which for the lowest signals would require a noise level equivalent to e10-7 ∆OD units. Such low noise levels in a femtosecond transient absorption experiment are outside the reach of present transient absorption setups but may become possible with new developments on the experimental side. The apparatus used in our measurements allows under optimal conditions a noise level of e5 × 10-7 ∆OD units, as is demonstrated in Figure 6. This requires however rather long integration times on the order of 10 s per data point. Such long integration times are in partial conflict with the further necessity to measure the transient absorption changes at a large number of different detection wavelengths and the further requirement to keep the sample intact, i.e. in a photochemically undamaged state.40,41 Thus some compromise between sample stability, total sample volume required, S/N ratio, and acceptable annihilation effect has to be made. Conclusions In summary we conclude that recording sufficiently annihilation-free transient absorption signals on the D1-D2 complex is at present at the very edge of the technical feasibility even for the most sophisticated instruments. No such measurements have been performed so far. Even the best published measurements were obtained under excitation conditions that yield about 18% annihilation, which is clearly too high in the light of the above discussion, taking into account the high complexity of the kinetics. Any detailed conclusions drawn from the published transient absorption data must therefore at present be considered with great care. In contrast fluorescence kinetic measurements, in particular by single-photon-counting methods, are normally performed under substantially lower excitation conditions (typically e1 × 1013 photons cm-2 pulse-1) where annihilation can be excluded. If the repetition rate of the excitation is suitably reduced and if the sample is pumped through a flow cuvette all other possible kinetic artifacts can be excluded as well. In fluorescence measurements by single photon counting the main problem is to achieve a sufficiently high time resolution. It has been shown, however, that the time resolution that can be obtained in our single-photon-counting instrument is just sufficient to perform these measurements and resolve the ∼3 ps component.13 We conclude that the interpretation based on fluorescence kinetic experiments must at present be considered to be more reliable than those from most published ultrafast transient absorption measurements.
J. Phys. Chem., Vol. 100, No. 22, 1996 9543 ability. Near the front surface of the cuvette the annihilation probability is very high and it decreases to low values at the backside of the cuvette. Furthermore the excitation and detection may be inhomogeneous across the laser beam depending on the beam profiles. This means that the region with high annihilation (e.g. in the center part of the excitation spot) will contribute most to the ∆A signal due to the fact that most of the light is absorbed in that region. Taking average values for the various parameters in this situation leads to substantial errors in the estimation of the annihilation and detection probability and in the average number of absorbed photons. We have thus numerically integrated the equations for excitation and multiple hit probability in the excitation beam across the cuvette and the beam profile using a finite element approach. For simplicity we assume a constant beam waist across the cuvette. Thus the beam profile has been decomposed into nF concentric rings (filaments) and the cuvette has been splitted into nS layers where nF has been chosen to be in the order of 20 and nS about 10. We have taken the maximal number of absorbed photons/RC to be m ) 20. Likewise we have integrated the probability for detection of the annihilation (caused by the pump beam) by the probe beam across the beam profile. It is thus possible to also take into account different spot sizes of the pump and probe beams, as often used in experiments. Necessary input parameters are the total pulse energy Ep, the excitation wavelength λexc and the OD of the sample at the excitation wavelength, and the intensity profile or halfwidth HW of the excitation beam. Furthermore the particle concentration c is required which can be obtained from the absorption spectrum and the molar absorption coefficient per RC particle (see Materials and Methods). The relevant parameters are given by the following relationships:
Pj )
G(j,HW)*Aj nF
ptot with ptot )
∑G(j,HW)*Aj
Calculation of Multiple Excitation Probability. The general theory for annihilation has been developed by Paillotin and others for arrays of chromophores undergoing efficient energy transfer.30,31 For a proper description of the annihilation conditions we have to take into account the details of the specific experiment which are not considered in Paillotin’s equations, however. In a transient absorption experiment two beams, the excitation and the detection beam, are used. In order to increase the signal, the experiment is usually carried out at high optical density (usually near OD ) 1) at the excitation wavelength. This has the effect that the excitation probability across the cuvette is extremely unequal, and so is the annihilation prob-
hcL
j)1
(in) pi,j ) Pj10-(OD/nS)(i-1)
(out) pi,j ) Pj10-(OD/nS)(i)
tj ) cNA(d/nS)10-3Aj ui,j ) Φrel )
(in) (out) pi,j - pi,j tj
total number of radical pairs
)
total number of absorbed photons
[
]
(ui,j)k tj e-ui,j ∑ ∑ ∑ k! i)1 j)1 k)1 nL nF
nL nF
Appendix
Epλexc
m
m
[
(ui,j)k
tj e-u ∑ ∑ ∑ i)1 j)1 k)1 (k - 1)!
i,j
]
(1)
G(j,HW) is the intensity in filament j of the beam profile with halfwidth HW (full-width-half-maximum). This allows one to take into account nonuniform excitation profiles. The parameter Aj is the front surface of filament j, ptot is the total number of photons/pulse impinging on the surface of the cuvette as calculated from the total pulse energy and the excitation wavelength λexc, cL the speed of light, OD the optical density (out) are the number of at the excitation wavelength, p(in) i,j and pi,j photons entering and leaving segments i, j where i stands for the layer and j for the filament and NA the Avogadro number. The function tj gives the total number of RC particles in a section
9544 J. Phys. Chem., Vol. 100, No. 22, 1996
Mu¨ller et al.
of filament j. The function ui,j then gives the probability of photon absorption per RC particle in section i, j. Using the relationships for multiple hit probability the number of radical pairs created and the total number of absorbed photons can be calculated which then allows one to calculate the normalized yield of radical pair formation Q. The absorbance change ∆OD observed at a particular wavelength is given by the relationship:
[
∆OD ) -log
nF
]
G(j,HW)10∆(ca )dAj ∑ j)1 j
nF
G(j,HW)Aj ∑ j)1
(2)
where
caj )
[
]
(ui,j)k ∑ ∑tj k! e-ui,j i)1 k)1 nL
m
NAd10-3Aj
where caj is the average concentration of radical pairs in the filament j and ∆ is the difference absorption coefficient and d the length of the cuvette. The average number of absorbed photons/RC nA is given by nS
nA h)
nF
∑ ∑ui,j[pi,j(in) - pi,j(out)] i)1 j)1 nS
nF
(in) (out) [pi,j - pi,j ] ∑ ∑ i)1 j)1
We have used a 100% probability of annihilation in the case of multiple hits. Comparison with the experimental data shows that this is reasonable for the D1-D2 complex. Lower probabilities can be taken into account in a straightforward manner if desired, however. Acknowledgment. We thank Mrs. Iris Martin for developing the data analysis software used for the transient absorption data and Mrs, R. Kesslau and Mr. U. Pieper for able technical assistance. Partial financial support by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 189, Heinrich-Heine-Universita¨t Du¨sseldorf and Max-Planck-Institut fu¨r Strahlenchemie, Mu¨lheim a.d. Ruhr) is gratefully acknowledged. We thank Prof. K. Schaffner for supporting these investigations. References and Notes (1) Renger, G. Angew. Chem. 1987, 99, 660. (2) Sauer, K. Annu. ReV. Phys. Chem. 1979, 30, 155. (3) Renger, G. Photosynth. Res. 1993, 38, 229. (4) Danielius, R. V.; Satoh, K.; van Kan, P. J. M.; Plijter, J. J.; Nuijs, A. M.; van Gorkom, H. J. FEBS Lett. 1987, 213, 241. (5) Nanba, O.; Satoh, K. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 109. (6) Seibert, M. In The Photosynthetic Reaction Center; Anonymous, Ed.; Academic Press: New York, 1993; p 319. (7) Wasielewski, M. R.; Johnson, D. G.; Seibert, M.; Govindjee. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 524. (8) Seibert, M.; Toon, S.; Govindjee; O’Neil, M. P.; Wasielewski, M. R. In Research in Photosynthesis. II; Murata, N., Ed.; Kluwer Academic Publishers: Dordrecht, 1992; p 41.
(9) Hastings, G.; Durrant, J. R.; Barber, J.; Porter, G.; Klug, D. R. Biochemistry 1992, 31, 7638. (10) Durrant, J. R.; Hastings, G.; Joseph, D. M.; Barber, J.; Porter, G.; Klug, D. R. Biochemistry 1993, 32, 8259. (11) Klug, D. R.; Rech, T.; Joseph, D. M.; Barber, J.; Durrant, J. R.; Porter, G. Chem. Phys. 1995, 194, 433. (12) Holzwarth, A. R.; Mu¨ller, M. G.; Gatzen, G.; Hucke, M.; Griebenow, K. J. Lumin. 1994, 60/61, 497. (13) Gatzen, G.; Mu¨ller, M. G.; Griebenow, K.; Holzwarth, A. R. J. Phys. Chem. 1996, 100, 7269. (14) Gatzen, G.; Griebenow, K.; Mu¨ller, M. G.; Holzwarth, A. R. In Research in Photosynthesis. II; Murata, N., Ed.; Kluwer Academic Publishers: Dordrecht, 1992; p 69. (15) Roelofs, T. A.; Kwa, S. L. S.; van Grondelle, R.; Dekker, J. P.; Holzwarth, A. R. Biochim. Biophys. Acta 1993, 1143, 147. (16) Roelofs, T. A.; Gilbert, M.; Shuvalov, V. A.; Holzwarth, A. R. Biochim. Biophys. Acta 1991, 1060, 237. (17) Schelvis, J. P. M.; van Noort, P. I.; Aartsma, T. J.; van Gorkom, H. J. Biochim. Biophys. Acta 1994, 1184, 242. (18) Rech, T.; Durrant, J. R.; Joseph, D. M.; Barber, J.; Porter, G.; Klug, D. R. Biochemistry 1994, 33, 14768. (19) Durrant, J. R.; Hastings, G.; Hong, Q.; Barber, J.; Porter, G.; Klug, D. R. Chem. Phys. Lett. 1992, 188, 54. (20) Holzwarth, A. R.; Roelofs, T. A. J. Photochem. Photobiol. B 1992, 15, 45. (21) Wiederrecht, G. P.; Seibert, M.; Govindjee; Wasielewski, M. R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8999. (22) Visser, H. M.; Groot, M.-L.; van Mourik, F.; van Stokkum, I. H. M.; Dekker, J. P.; van Grondelle, R. J. Phys. Chem. 1995, 99, 15304. (23) van Leeuwen, P. J.; Nieveen, M. C.; van de Meent, E. J.; Dekker, J. P.; van Gorkom, H. J. Photosynth. Res. 1991, 28, 149. (24) Konermann, L.; Holzwarth, A. R. Biochemistry 1996, 35, 829. (25) McTavish, H.; Picorel, R.; Seibert, M. Plant Physiol. 1989, 89, 452. (26) Porra, R. J.; Thompson, W. A.; Kriedemann, P. E. Biochim. Biophys. Acta 1989, 975, 384. (27) Lichtenthaler, H. K. Methods Enzymol. 1987, 148, 350. (28) Holzwarth, A. R.; Schatz, G. H.; Brock, H.; Bittersmann, E. Biophys. J. 1993, 64, 1813. (29) Holzwarth, A. R. In Biophysical Techniques. AdVances in Photosynthesis Research; Amesz, J., Hoff, A., Eds.; in press. (30) Paillotin, G.; Swenberg, C. E. Dynamics of excitons created by a single picosecond pulse; Ciba Foundation Symposium 61; Excerpta Medica: Amsterdam, 1979. (31) Paillotin, G.; Swenberg, C. E.; Breton, J.; Geacintov, N. E. Biophys. J. 1979, 25, 513. (32) Greenfield, S. R.; Wasielewski, M. R.; Govindjee; Seibert, M. In Photosynthesis: from Light to Biosphere; Mathis, P., Ed.; Kluwer Academic Publishers: Dordrecht, 1995; p 663. (33) Dobek, A.; Deprez, J.; Geacintov, N. E.; Paillotin, G.; Breton, J. Biochim. Biophys. Acta 1985, 806, 81. (34) Geacintov, N. E.; Breton, J. Energy transfer and fluorescence mechanisms in photosynthetic membranes. 5; Chemical Rubber Company: Boca Raton, 1987. (35) France, L.; Geacintov, N. E.; Lin, S.; Wittmershaus, B. P.; Knox, R. S.; Breton, J. Photochem. Photobiol. 1988, 48, 333. (36) France, L. L.; Geacintov, N. E.; Breton, J.; Valkunas, L. Biochim. Biophys. Acta 1992, 1101, 105. (37) Mu¨ller, M. G.; Hucke, M.; Reus, M.; Holzwarth, A. R. J. Phys. Chem. 1996, 100, 9527. (38) van Grondelle, R.; Amesz, J. In Light Emission by Plants and Bacteria; Govindjee, Amesz, J., Fork, D. C., Eds.; Academic Press: New York, 1986; p 191. (39) van Grondelle, R. Biochim. Biophys. Acta 1985, 811, 147. (40) Chapman, D. J.; Gounaris, K.; Barber, J. Photosynthetica 1989, 23, 411. (41) Seibert, M.; Picorel, R.; Rubin, A. B.; Connolly, J. S. Plant Physiol. 1988, 87, 303. (42) A lower limit only is obtained for the difference absorption coefficient ∆ because the radical pair even at 300 ps is not populated to 100% due to efficient back reaction to the excited state. A more exact value can be obtained after proper kinetic modeling. (43) In the calculations and discussion of the data from the group of Klug et al. we refer to their most recent data set measured under magic angle conditions.11 (44) A preliminary account of these data has been given at the ESF Workshop on “Structure and Function of the D1-D2 reaction center complex”, Wye College, England, April 1995. (45) We should like to note that an increase in excitation intensity is not mentioned in the work of Hastings et al.9 and that the authors claim that the intensity has not been increased as compared to the measurements in the Qy range (J. Durrant, private communication).
JP953715A
