7712
J . Phys. Chem. 1994, 98, 7712-7716
Site-Selection Spectroscopy of the Reaction Center Complex of Photosystem 11. 2, Identification of the Fluorescing Species at 4 Kt Stefan L. S. Kwa,. Nina T. Tilly, Camiel Eijckelboff, Rienk van Grondelle, and Jan P. Dekker Department of Physics and Astronomy and Institute for Molecular Biological Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands Received: December 17, 1993; In Final Form: March 22, 1994’
Fluorescence emission spectra at 4 Kof the D l-D2-Cytb559 reaction center complex of photosystem 11, presumably containing six chlorophyll a (Chl a ) and two pheophytin a (Pheo a ) molecules, were obtained by selective excitation with a tunable, continuous laser (& = 680-691 nm). The bands in the vibrational region (700-800 nm) showed a characteristic structure, which could be used as a “fingerprint” to identify the emitting pigment at low temperature. By comparison of this “fingerprint” with those of monomeric Chl a and Pheo a in Triton X-100 detergent, it is unambiguously show that the steady-state emission arises from a Chl a species for &, > 683 nm. The nonselectively excited emission spectrum (A,, < 679 nm) reveals primarily Chl a emission bands, peaking at 683.7 f 0.3 and 744.0 f 0.5 nm. The shape of this spectrum is significantly different from that of earlier reported emission spectra of Dl-D2-Cytb559 complexes with presumably a lower Chl a content (four to five Ch1/2 Pheo). These show characteristic bands for Pheo a emission. We conclude that D1-D2Cytb559complexes with a higher Chl a content (6 Ch1/2 Pheo) contain an additional long-wavelength (680-683 nm) Chl a molecule, which is not related to P-680. The steady-state fluorescence at low temperatures mainly arises from emission of this Chl and not from charge-recombination fluorescence of P-680.
Introduction
The primary photosynthetic processes in the Dl-D2-Cytb559 reaction center complex of photosystem I1 are the absorption of light by the reaction center pigments, the excitation energy transfer to the primary donor P-680, and electron transfer from singletexcited P-680 to a pheophytin a (Pheo a ) molecule. At temperatures between 300 and 4 K the formation of the radical pair (P-680+ Pheo a)occurs within some tens of picoseconds after the e~citation.l-~Because the reaction center complex lacks the secondary electron acceptor QA, which is lost during the isolation procedure,s the charge-separated state will recombine in about 20-100 n ~ 2 7 ~ J to O form the singlet and triplet excited state of P-680 (P-680* and P-680T, respectively). At temperatures between 300 and 77 K thedecay of thechargerecombination fluorescence from P-680* corresponds to the lifetimes of the radical pair.z.9J0 At lower temperatures these relatively long fluorescence lifetimes werevirtually absent, which suggests that the activation energy for the charge recombination cannot be surpassed.13 A puzzling question is the origin of the major fluorescencecomponent at 13 K with a decay time of 4-8 ns.I3 Such a time has been ascribed to uncoupled pigments in damaged reaction centers, which fluoresce at 670-673 nm.12 However,the major fluorescencecomponent of carefully prepared particles at 13 K has the emission maximum at around 682 nm,13 meaning that it does not arise from uncoupled pigments. The 4-8-ns component may in principle be ascribed to a relatively fast recombination process, but this would also imply a very small energy difference between P-680* and the radical-pair state.l3 In addition, it does not explain the long lifetimes of the radical pair observed in absorption.2~~.~0 Alternatively the observed fluorescence may also originate from a coupled pigment but different from P-680. Considering the emission wavelength of 684 nm at 4 K14this fluorescing pigment should absorb at around 680-68 1 nm, the absorption region of P-680 and Pheo a.2J5J6 In this report we characterized the emitting pigment at 4 K by fluorescencesite-selectionspectroscopy. Previously we applied f Abbreviations: Chl, chlorophyll; BChl, bacteriochlorophyll; Phco, pheophytin; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethy~)propane1,3-diol; CP47, core antenna complex CP47 of Photosystem 11. *Abstract published in Advance ACS Abstracts, June 1, 1994.
0022-3654/94/2098-7712$04.50/0
this technique to monomeric Chl a in detergent at low temperature and found a very characteristic pattern of vibrational emission bands.’’ By comparing the site-selected fluorescence emission spectra of Chl a, Pheo a, and Dl-D2-Cytb559 we show that Chl a is the dominant emitting species in the reaction center complex of photosystem I1 at 4 K. Materials and Methods Sample Preparation. Dl-D2-Cytb559was isolated with a short Triton X-100 treatment as described elsewhere’s and was diluted for the measurements with a buffer containing 0.03% (w/v) n-dcdecyl 8-D-maltoside,20 mM BisTris (pH 6.5), 20 mM NaCl, and 70% (w/v) glycerol (final concentrations). The absorption spectrum at 4 K (see ref 19) is characterized by Qy(m) maxima at 67 1 and 679 nm and a weak shoulder at 684 nm and is virtually identical to the ones reported by Van Gorkom and co-workers on similar preparations.2J4 For the preparation of monomeric Chl a and Pheo a we used the core antenna complex CP47 as starting material. CP47, containing Chl a and &carotene as the only cofactors,20 was isolated as described elsewhere.2I Chl a in a protein-detergent environment was obtained by incubating CP47 at a Chl a concentration of 150 gg/mL with 2-3% (w/v) Triton X-100 as described in ref 17. For the measurements the Chi a solution was diluted with a buffer containing 20 mM BisTris (pH 6.5), 20 mM NaCl, and 70% (w/v) (finalconcentrations),resulting inan optical density of = 0.3/cm at 4 K. The Chl a was monomeric, which has been checked by circular dichroism, polarization, and site-selection spectroscopy (see ref 17). To obtain Pheo a, 2 mL of the diluted Chl a solution was pheophytinized by acidification with 1 or 2 drops of 10 M HC1. In Figure 1 the absorption spectra of monomeric Chl a (solid line) and Pheo a obtained from Chl a with 1 and 2 drops of HCl (dashed and dotted line, respectively) at room temperature are shown. The lower oscillator strength of the Qy(m) band and the presence of the Q, band at 538 nm and the Soret band at 414 nm are typical for Pheo a.22 The similarity between the two Pheo a spectra indicatesthat the pheophytinizationwas complete. Note that &carotene, not removed from the samples, is responsible for the absorbance between 450 and 520 nm. 0 1994 American Chemical Society
The Journal of Physical Chemistry, Vol. 98, No. 31, 1994 7713
Reaction Center Complex of Photosystem 11. 2
2
0 4
360
I
I
In
I
.
I
i!
I
I
I
I
430
500
570
640
I
.
I !
I
I
I
1
710
Wavelength (nm) Figure 1. Absorbance spectra of Chl u (-) and Phco u (- - - and .-) at room temperature. Monomeric Chl u was made by incubating CP47 with Triton X-100. Phco u was obtained by titrating 2 mL of the Chl u solution with one or two drops of 10 M HCl (- - - and respectively). Note that @-carotene,not removed from the samples, is responsible for the absorption bctwe.cn 450 and 520 nm. e-,
The sample was placed in a perspex cuvette (1 .O X 1.O X 4.0 cm) and cooled to 4 Kin a He-flow cryostat (Oxford). The width of spectral bands, whether symmetric or asymmetric, is defined here as the full width measured at half height. Fluorescence Measwmenb. Site-selectedfluorescence measurementswere performed with the experimentalsetup described in ref 17. In brief, excitation light (A = 610-710 nm, 5-30 mW/ cm2) was provided by a CW dye laser (Coherent 599, DCM dye, spectral bandwidth 2 cm-I) pumped by an Ar+ laser (Coherent Innova 310). Detection of the fluorescence was at right angle with respect to the excitation beam and was achieved with a double 1/8 m monochromator (Oriel) and a photomultiplier (S20 photocathode,Thorn EMI 9658). The spectral bandwidth in the detection was 1.0 nm and the wavelength calibration was checked at 632.8 nm with a HeNe laser. The emission spectra were corrected for the sensitivity of the detection system as described in ref 18. The excitation light was horizontally polarized to minimize scattering in the direction of the detection. No polarizer was used in the detection system.
-
Resulk3 Fluorescence emission spectra of Dl-D2-Cytb559 at 4 K are shown in Figure 2a. The excitation wavelengths were 620,630, 670,675,677, and 679 nm. The large peaks coinciding with the excitation wavelengths are due to direct scattering of laser light. The observed emission spectra are indistinguishable for these excitation wavelengths, implying that for LcI679 nm the emitting molecule(s) is mainly excited via energy transfer from other molecules. Since the absorbance of the sample at K was OD679= 0.3, the emission maximum located at 683.7 nm (width -8.5 nm) is not significantlyshifted due to self-absorption. The spectra in Figure 2a seem identical to the 4 K spectrum reported by Van Leeuwen et al. on similar preparations excited at 437 nm with lamp and monochromatorl4 and are slightly narrower than the spectra measured at 77 K.18 For measurements in the vibrational region and at longer excitation wavelengths we increased the absorbance to OD679 = 1.2 (for all Dl-D2-Cytb559 spectra described below) in order to achieve a higher signal-to-noise- and fluorescence-to-scattering ratio. A consequence is that the emission spectra are somewhat distorted due to self-absorptionin the wavelength region below 685 nm (in this case, the main emission band appears smaller with respect to the vibrational bands, and appears red-shifted to
Emission wavelength (nm) Figure 2. (a) Fluorescence emission spectra of Dl-DZ-Cytb559 at 4 K excited with a laser at 620,630,670,675,677, and 679 nm. OD679 was 0.3 at 4 K. The large peaks coinciding with the excitation wavelengths are due to direct scattering of laser light. Part b shows fluorescence emission spectra of Dl-DZ-Cytb559 at 4 K excited with a laser at 677, 680,685, and 689 nm. OD679 was 1.2 at 4 K due to which bands at I685 nm appear slightly red-shifted (seetext). All spectra in Figure 2 were normalized in the fluorwnce peak (not the scattering peak).
almost 685 nm). Above 685 nm the effect of self-absorption on the emission spectra can be neglected. Figure 2b shows fluorescence emission spectra of D1-D2Cytb559 at 4 K for haxc = 677,680,685, and 689 nm. It can be seen that the main emission band shifts with the excitation wavelength for long excitationwavelengths (Lxc I680 nm). This effect occurs in the absence of energy transfer and has well been explained in termsof site selection.23 The spectra were normalized to the maximum of the fluorescence peak (not the scattering peak). The relative height of the scattering peak is largest at longer excitation wavelengths, since there the absorbance and thus the fluorescence intensities are the lowest. For Lc= 685, 687, 689, and 691 nm the main emission band has its center of gravity at 3.5 f 1.0 nm (or 80 20 cm-1) to the red of the excitation wavelength (see Figure 2b for LC = 685 and 689 nm). This value is an upper limit for the peak position of this band, because due to the interference of the scattering laser light the real maximum could be obscured. Since the width (10-12 nm) is comparable to that found for Chl a in detergent (9 nm, see ref 17) this band must be largely attributed to phonon side bands of the Qy~m) transition. In site-selected triplet-minus-singlet( T S ) absorption difference spectra a nontrivial effect was 0b~erved.l~ Upon excitation at very long wavelength (Le = 691 nm) a bleaching of pigments at 684 nm on the blue side of the laser wavelength was observed.19
*
7714 The Journal of Physical Chemistry, Vol. 98, No. 31, 1994
Kwa et al.
- Dl-D2-Cytb559, I
I
:
hex,= 685 nm I
I
-
hex, = 691 nm
689
687
I
I
I
Pheo a
I
---I
I
1-
620,677,679
1
._.
\
m hex,= 685 nm
I
I
I
I
I
715 735 755 675 Emission wavelength (nm) Figure 3. The integralfluorescenceemission spectrumof Dl-D2-Cytb559 at 4 K excitedwith a laser at 685 nm (lowestspectrum)and thevibrational region of emission spectra excited at 620,677,679, 680, 685, 687, 689, and 691 nm (insets). OD679 was 1.2 at 4 K. The latter spectra were normalized to the vibrational emission band at around 744-755 nm. 695
The fact that a similar effect is not observed in fluorescence is reasonable, since anti-Stokes fluorescence cannot occur a t 4 K and only pigments with their zero-phonon line a t equal or longer wavelength than the laser wavelength contribute to the emission. Figure 3 shows the vibrational region for emission spectra of Dl-D2-Cytb559 at 4 K excited a t 620,677,679,680,685,687, 689, and 691 nm (insets). As a reference we also plotted the entire emission spectrum excited at 685 nm (lowest spectrum). All spectra show a characteristic pattern of vibrational Qybands. For A, 1 680 nm this pattern shifts with the excitation wavelength, similar to the main band in Figure 2b, and becomes better resolved. This is again due to site selection. The specific shape of the vibrational region can now be used as a "fingerprint" to identify the emitting species as Chl a or Pheo a. Figure 4 (all solid lines) shows the emission spectrum of D1D2-Cytb559 excited a t 685 nm taken from Figure 3. Also plotted in Figure 4 are the site-selected fluorescence emission spectra of Chl a (taken from ref 17) and Pheo a in Triton X-100a t 4 K (dashed lines in upper and lower panels, respectively). These spectra were excited a t 676 and 670 nm, respectively, but were afterward shifted to the red by computer (on energy scale) to align the vibrational bands with those of Dl-D2-Cytb559. Exact alignment was performed a t the only vibrational band which is clearly present in all emission spectra of Chl a, Pheo a, and D1D2-Cytb559 (see also later in Figure 5) and which is observed at 723.5 nm in the Dl-D2-Cytb559 spectrum excited at 685 nm. After the alignment procedure the main emission bands and the scattering peaks of the spectra in Figure 4 also coincide. It can immediately be seen that the positions of at least six vibrational bands of Chl a and D 1-D2-Cytb559perfectly coincide (see arrows, upper panel). For Pheo a the positions of only two bands are
II
I
615
I
695
715
735
755
775
Emission wavelength (nm) Figure 4. Fluorescence emission spectrum of Dl-D2-Cytb559 at 4 K excited at 685 nm (- in upper and lower panels) taken from Figure 3, compared with emission spectra at 4 K of Chl u excited at 676 nm (- - upper panel) and of Pheo n excited at 670 nm (- - - lower panel). The Chl a and the Pheo u spectrum were shifted by computer to the red (on energyscale)to align the vibrationalbands with those of D1-D2-Cytb559. Exact alignment was performed at the band at 723.5 nm. All spectra were normalized to the vibrational emission band in the region 735-755 nm. The vibrational regionsof the spectra were also enlarged by a factor of 6 (insets). Coincidingvibrationalbands aremarked witharrows, while bands which clearly differ are marked with crosses. similar (see arrows, lower panel), one of which was used for the alignment. Two prominent emission bands of Pheo a (marked with crosses, lower panel) are not at all present in the spectrum of Dl-D2-Cytb559. Therefore we conclude that Chl a is the only emitting species in Dl-D2-Cytb559 at 4 Kupon excitation at 685 nm. The relatively higher QY(m) emission band of Chl a in D1D2-Cytb559 as compared to that of monomeric Chl a (see Figure 4, upper panel) has also been observed for another Chl a-containing protein complex, the light-harvesting complex LHC-I1 (see ref 17). There seems to be a relation between excitonic coupling and the suppression of vibrational bands, but the physical origin is not well understood. Following the same procedure as in Figure 4 we compared in Figure 5 the emission spectrum of Dl-D2-Cytb559 excited nonselectively at 620 nm (taken from Figure 3) with the spectra of Chl a and Pheo a. Note that the emission maximum of D1D2-Cytb559 appears slightly red-shifted because of self-absorption. Since the vibrational bands are less resolved than when exciting at 685 nm, the interpretation is more ambiguous: only three band positions clearly correspond to those of Chl a (see arrows, upper panel), whereas only one is the same as in Pheo a. The spectra show that a Chl a contributes significantly to the emission spectrum. It can, however, not be ruled out that under these nonselective excitation conditions Pheo a is also contributing to the emission spectrum. We estimate that less than 30% of the
Reaction Center Complex of Photosystem 11. 2 Dl-D2-C@559, I
1 i
I
!
The Journal of Physical Chemistry, Vol. 98, No. 31, 1994 7715
hex,= 620 r
I
~ n
I
- - _ _ Chl a
740.5
,I I44
((I;
TABLE 1: Positions of Emission Bands in Two Kinds of DI-DZ-Cytb559 heparations at Low Temperatures’
largest QY(m) vibrational F ~ , & ~ U / F ~TU Chl content (46) (K) (Chlsper2Pheo) (nm) Qy(nm) 739 740 141 144 744
684 684
683 683.1 683.4 683.5
144
15 16 9
6 6 6
4 17 77 4 4 77
4-5 4-5
4 6 6 6
ref 20 21 28 this work 14 18
a F73&7~/Fmis the peak intensity ratio of the vibrational and 0-0 band (integrated intensities could not be obtained from the figuresin the literature). For all spectratheexcitation was nonselcctively in thevibronic or Soret absorption region.
-
Emission wavelength (nm) Figure 5. Fluorescence emission spectra as in Figure 4, except that the Dl-D2-CytbS59 spectrum was excited nonselectively at 620 nm (- in upper and lower paneis) and that the Chl a (-- - upper panel) and the Phea a spectrum (- lower panel) were aligned to this Dl-DZ-Cytb559 spectrum. Exact alignment was performed at the band at 720.5 nm. Indicated are the peak wavelengths of the largest vibrational emission bands of Chl a and Phea a. The maxima of the Qy(w) bands of Chl a and Phea a were here at 684 & 1 nm. Note that the Q y ( ~ )band of D1-D2-Cytb559 is distorted due to self-absorption in this region (see text).
--
total emission of Dl-D2-Cytb559 at 4 K arises from Pheo a upon nonselective excitation (bXc I679 nm).
Discussion AbsorptionWavelength of the Emitting Pigment. By using the vibrational region of the site-selected emission spectrum of D1D2-Cytb559 at 4 K as a “fingerprint”,we have conclusivelyshown that the fluorescing molecule at this temperature is Chl a for Lc > 683 nm, and mostly (for more than 70%) Chl a for b,,, < 681 nm. The absorption wavelength of the emitting pigment(s) can be estimated using the Stokes’ shift generally found for Chl a. At 4 K the Stokes’ shift has been measured to be 4 nm for Chl a in Triton X-100.17 Alternatively the Stokes’ shift can be estimated from parameters obtained by spectral hole-burning, since it is approximately equal to 2wmS,where wm is the mean phonon frequency and S the Huang-Rhys factor.25 Measured values for P-680 and other Chl a molecules in photosynthetic complexes at 1 4 K are typically om 25 cm-1 and S 0.3-2,2s which yield a Stokes’ shift of 1-4 nm. Using these Stokes’ shifts weconclude that theemissionat 683.7 nmof theDl-DZCytbS59 containing six Chls per two pheophytins (Figure 2a and ref 14) mainly arises from Chl absorbing at around 680-683 nm. A LongWavelength Antenna Chlorophyll? It is not likely that the steady-state fluorescence at 4 K contains a significant contribution from the primary donor P-680. The fluorescence decay times (4-8 ns) do not correspond with the radical pair lifetimes (- 100 ns). Furthermore, the activation energy for the
-
-
charge recombination should be assumed to be nearly zero (AE < keT 0.3 meV at 4 K). The alternative interpretation is that a Chl a molecule at about 680 nm, Chbm, not belonging to P-680 is the terminal emitter, i.e. serves as an energy trap. Even if Ch1680 would be in the vicinity of P-680, its zero-point energy needs only to be slightly lower than that of P-680 to introduce an activation energy for energy transfer which cannot be surpassed at 4 K (AE> 0.3 meV, or Ah > 0.1 nm). Since this required energy difference is much smaller than the spread in zero-point energies of molecules due to inhomogeneous broadening (typically several nanome t e r ~ ~the ~ ) energy , of P-680 could be sufficiently higher than that of C h h o in a significant fraction of the reaction centers in the sample to prevent energy transfer from Ch1680to P-680. The presence of a Chl at around 680 nm immediately raises the question how many Chl a molecules, including P-680, absorb at this wavelength? Our investigated Dl-D2-Cytb559 reaction center complex presumably contains six Chl a and two Pheo a and is characterized by two peaks at 672 and 679 nm in the steady-state absorption spectrum at low temperature (see refs 2 and 19). The bands have about equal oscillator strength, although the band at 679 nm is slightly higher but also narrower than the band at 672 nm. Therefore at least two Chls (including P-680) have to be assumed to absorb at 678-682 nm. The answer to this question can be found by (re)analyzing emission spectra which have been reported earlier20927 and very recently2* for chemically different D l-D2-Cytb559 reaction centers. The complexes used in those studies do contain the photoactive primary donor P-680, but presumably have a lower Chl content (four to five Chl). In Table 1 we have listed features from emission spectra of these preparations, which are emission wavelengths and intensity ratios. All spectra were excited nonselectively at short wavelength (Lxc < 630 nm) and have the main emission band at 683-684 nm. It can be seen that except for the position of the 0-0 emission band there are systematic differences in emission properties for preparations containing four to five, and six Chls. In particular, the D1-D2 Cytb559 complexes with a high Chl content (six Chls) have the largest vibrational emission band positioned at 744.0 k 0.5 nm (T = 4-77 K), while this is at 739 f 2 nm ( T = 4-77 K) for the preparations with a low Chl content (four to five Chls; see Table 1). The latter wavelength is characteristic for a Pheo a molecule emitting at 684 nm since this value is the (weighted) average of the positions of the Pheo a bands at 736 and 741 nm (see Figure 5, lower panel). Therefore we conclude that the steady-state emission at low temperature (4-77 K) of the Dl-D2-Cytb559 complexes with a low Chl content (four to five Chls) is dominated by Pheo a fluorescence and not by emission of P-680. This is further supported by the observation that charge-recombination fluorescence of P-680 is not the dominating component at low temperatures.”-13 With the use of the Stokes’ shift measured for Pheo a in Triton X-100 (4 nm, data not shown), the absorption wavelength of this Pheo a molecule can be found to be about 680
7716 The Journal of Physical Chemistry, Vol. 98, No. 31, 1994
nm. This is additional proof for the presence of at least one Pheo a molecule a t 680-681 nm as found earlier with spectral hole burning15 and ADMR measurements16 and which we will call Phe0680. The consequence of these results is that in Dl-D2-CytbS59 complexes with a high Chl content (six Chl/two Pheo) the lowenergy exciton band of P-680, C h b o and Pheo680absorb around 680 nm. This leaves the high-energyexciton band of P-680 (which absorbs at 667 nm with small oscillator strengthlg), three antenna Chl molecules and perhaps the second Pheo for the absorption region at around 672 nm, which is consistent with the notion that the 672- and 679-nm bands in the steady-stateabsorption spectrum have about equal amplitudes. A straightforward explanation for the significant differences in the low-temperature steady-state emission properties between the two kinds of preparations (see Table 1) is that the fluorescent Ch1680molecule does not seem to be present in Dl-D2-Cytb559 complexes with a low Chl content (four to five Chl/two Pheo). During the long incubation with Triton X-100 it could have been extracted from the complex, or it could have been pheophytinized (in which case only the Chl/Pheo ratio, but not the pigment content decreased upon prolonged Triton exposure). Spectral Inhomogeneity: Consequences. Inhomogeneous spectral broadening is a manifestation of the uncertainty in the absorption wavelength of a specific pigment in an individual D1D2-Cytb559 complex. This uncertainty is due to slightly different environmental interactions for each complex and is typically several nanometers.25 Only by averaging the wavelengths of many of such pigments in the sample a Gaussian distribution is formed, which gives rise to a more accurate wavelengths. However the width of this distribution is larger than the spectral separation between the different pigment pools, due to which only two Qy(w) bands are observed in the low-temperature absorption spectrum. In our opinion it is (almost) impossible to obtain accurate absorption wavelengths for all inhomogeneously broadened pools of pigments, because the spectral resolution is in principle limited by the widths of the absorption bands. The large uncertainty in the wavelength of an individual pigment as compared to thesmaller difference between the average wavelengths of different pigment pools has the consequence that at 4 K the photochemistry is different for every individual D1D2-Cytb559 complex. A relatively red-absorbing primary donor has a higher probability than a blue-absorbing P-680 to find a Chl68o or PheO68o molecule a t a higher energy in its complex. Consequently, at low temperature only a red-absorbing subpopulation of the P-680 pool in the sample will receive excitation energy from Chl6a0or Pheo68o. This means, for example, that in triplet-minus-singlet (T-S) absorption difference spectra, even under nonselective excitation conditions (see refs 2 and 19), a red-absorbing subpopulation of the P-680 pool is selected and excited to the triplet state with the radical pair mechanism.9 Therefore the spectral contribution of P-680 to this T S spectrum, if already obtained accurately from deconvolution, is principally different from the spectrum of the P-680 pool in the steady-state absorption spectrum. The consequences of large inhomogeneous broadening in the Dl-D2-CytbS59 complexes can be summarized as follows: (1) the photochemistry can differ completely for every individual complex, making accurate wavelength values for pigment pools irrelevant; the photochemistry is in that sense heterogeneous and this must be accounted for explicitly in any kinetic model, and (2) a spectral shape ofa pigment poolcontributing toan absorption
Kwa et al. difference spectrum is not necessarily the same as the shape in the steady-state absorption spectrum. Acknowledgment. The authors thank Mrs. M. L. Groot for critical discussions. Dr. H. J. van Gorkom and Dr. G. J. Small are acknowledged for kindly sending us their manuscripts prior to publication. The research was supported in part by the Dutch Foundations for Chemical Sciences (SON) and for Biophysics (SvB), with financial support of the Netherlands Organization for Scientific Research (NWO). J.P.D. was supported by a fellowship from the Royal Netherlands Academy of Arts and Sciences (KNAW). References and Notes (1) Wasielewski,M. R.; Johnson, D. G.; Govindjee;Preston, C.; Seibert, M. Photosynth. Res. 1989,22. 89-99. (2) VanKan,P. J.M.;Otte,S.C.M.;Kleinherenbrink,F.A.M.;Nicveen, M. C.; Aartsma, T. J.; Van Gorkom, H. J. Biochim. Biophys. Acta 1990, 1020. 146-152. (3) Jankowiak, R.; Tang, D.; Small, G. J.; Seibert, M. J . Phys. Chem. 1989. 93. 1649-1654. (4) Roelofs,T.A.;Gilbert,M.;Shuvalov,V.A.;Holnvarth,A.R.Biochim. Biophys. Acta 1991, 1060,237-244. (5) Hastings, G.; Durrant, J. R.; Barber, J.; Porter, G.; Klug, D. R. Biochemistry 1992,31, 7638-7647. (6) Durrant, J. R.; Hastings, G.; Joseph, M. D.; Barber, J.; Porter, G.; Klug, D. R. Biochemistry 1993,32, 8259-8267. (7) Schelvis, J. P. M.; Van Noort, P. I.; Aartsman, T. J.; Van Gorkom, H. J. Biochim. Biophys. Acta 1994,1184,242-250. (8) Nanba, 0.; Satoh, K. Proc. Natl. Acad. Sci. U.S.A. 1987.84, 109~
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