Energy Redistribution in Heterodimeric Light-Harvesting Complex

Excitation energy flow at 77 K in the photosynthetic apparatus of overwintering evergreens. A. M. GILMORE , S. MATSUBARA , M. C. BALL , D. H. BARKER ,...
0 downloads 0 Views 107KB Size
Download PDF
J. Phys. Chem. B 1998, 102, 8183-8189

8183

Energy Redistribution in Heterodimeric Light-Harvesting Complex LHCI-730 of Photosystem I Alexander N. Melkozernov,‡,§ Volkmar H. R. Schmid,||,† Gregory W. Schmidt,|| and Robert E. Blankenship*,‡,§ Department of Chemistry and Biochemistry, Arizona State UniVersity, Tempe, Arizona 85287-1604; Center for the Study of Early EVents in Photosynthesis, Arizona State UniVersity, Tempe, Arizona 85287-1604; and Department of Botany, UniVersity of Georgia, Athens, Georgia 30602-7271 ReceiVed: February 6, 1998; In Final Form: May 8, 1998

Time-resolved fluorescence of the LHC I-730 complex and its monomeric subunits of the light-harvesting complex (LHC) of photosystem I was studied in complexes reconstituted from Lhca1 and Lhca4 apoproteins and HPLC purified chlorophyll a, chlorophyll b, and carotenoids [Schmid, V. H. R.; Cammarata, K. V.; Bruns, B. U.; Schmidt, G. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 7667]. Fluorescence kinetics of the monomeric subunits that make up the LHC I-730 heterodimer are characterized at room temperature by three decay processes with lifetimes of 150-350 ps, 0.8-1.8 ns, and 2-3.5 ns. The 2-3.5 ns process represents an overall relaxation of light-harvesting complexes while the other decay processes possibly reflect kinetic heterogeneity due to different pigment-protein interactions. In LHC I-730 heterodimers, which are characterized by an assembly of more Chl b and a change in pigment-protein interactions, an additional 30-50 ps energy-transfer component was found. This component is absent in both Lhca1 and Lhca4 monomers. This energy-transfer component is due to intersubunit energy redistribution, from Lhca1 to Lhca4 in a heterodimer. The spectral overlap of fluorescence of Lhca1 and absorption of long wavelength spectral forms of Chl a in Lhca4 suggests the energy transfer is most possibly via the Fo¨rster inductive resonance mechanism.

Introduction Photosystem I (PS I) of algae and higher plants is associated with a supramolecular complex of chlorophyll a/b binding proteins that function as an accessory antenna absorbing light and delivering excitation energy to the PS I core complex. The light-harvesting complex (LHC) of PS I consists of four chlorophyll a/b-binding proteins, Lhca1, Lhca2, Lhca3, and Lhca4, with molecular masses of 21-24 kDa.1,2 LHC I contains 80-120 Chl a and Chl b molecules. The complex is characterized by a higher Chl a/Chl b ratio (≈3.5) than in LHC II complexes.3,4 In higher plants two major LHC I subfractions, LHC I-730 and LHC I-680, with different protein composition and fluorescence properties were isolated.5 The LHC I-730 complex is composed of Lhca1 (22 kDa) and Lhca4 (21 kDa) proteins that are assembled as heterodimers.6,7 Lhca2 and Lhca3 subunits are thought to assemble as homodimers forming the LHC I-680 subpopulations.6 The emission spectrum of the LHC I-680 complex is dominated at 77 K by a band with maximum at 680 nm while the broad band peaking at 730 nm dominates the spectrum of the LHC I-730 complex.7,8 All light-harvesting polypeptides of algae and higher plants seem to show similarity in specific Chl a/b-binding protein † Present address: Institut fuer Allgemeine Botanik, Johannes Gutenberg Universitaet, 55099 Mainz, Germany. ‡ Department of Chemistry and Biochemistry, Arizona State University. § Center for the Study of Early Events in Photosynthesis, Arizona State University. | University of Georgia. * To whom correspondence should be addressed at: Department of Chemistry and Biochemistry, Arizona State University, Arizona State University, Tempe, AZ 85287-1604, U.S.A. Phone: (602)965-1439; Fax: (602)965-2747; Email: [email protected]

sequences.2 The electron crystallography study of the most abundant Chl a/b protein of LHC II9 revealed with 3.4 Å resolution a structure of the Lhcb1 monomer that form in vivo the LHC II trimer. Three membrane spanning R-helices of the monomer bind about 12 molecules of Chl and two molecules of lutein. Two of three membrane spanning regions that are highly conserved in protein sequences of different Chl a/b proteins were reported to provide a close contact of pigments with interpigment distances that could enable relatively strong exciton couplings between pigments and a rapid subpicosecond energy transfer between molecules. The latter is consistent with observed 100-600 fs processes in transient absorption spectra of the LHC II complexes attributed to Chl b to Chl a energy transfer.10,11 The fast energy equilibration populates Chl a molecules with the overall relaxation times of 1-3 ns.12,13 The observed close contact of Chl a molecules and two carotenoids was suggested to prevent the formation of toxic singlet oxygen.9 The structural model of LHC II is broadly consistent with results of extensive spectroscopic study14-16 attributing the resolved spectral forms of inhomogeneously broadened absorption and fluorescence spectra of LHC II complexes to 12 interacting Chl molecules (possibly 7 Chl a and 5 Chl b). Unlike the LHC II complexes, the properties of LHC I complexes and their subfractions are less well-characterized spectroscopically. Despite the possible structural similarity to LHC II, LHC I complexes are characterized by several different spectral forms. The remarkable feature of the LHC I antenna is the presence of pigments with energy levels significantly lower in energy than the absorption maximum of the PS I reaction center.17 These long wavelength forms of Chl were reported to fluoresce at 735-740 nm both in vivo17 and in

S1089-5647(98)01046-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/20/1998

8184 J. Phys. Chem. B, Vol. 102, No. 42, 1998 vitro.18 The function of these spectral forms absorbing above 705 nm was proposed to be as a trap of excitation energy in the antenna chlorophyll system of PS I.17-19 Recently, the redshifted fluorescence of LHC I complexes was localized and ascribed to Lhca4 of the LHC I-730 subcomplex.7,20 It was shown earlier that the low-energy chromophore responsible for the 735 nm fluorescence preferentially becomes excited by a Chl b pool.18,19 In a time-resolved fluorescence study, Mukerji and Sauer19 reported for detergent-isolated spinach bulk LHC I complexes at 77 K and excitation at 650 nm two components with lifetimes of 30 and 200 ps, indicating energy transfer from Chl species emitting at 680-685 nm to Chl species emitting at 735 nm. In the subpopulation LHC I-730 Pålsson et al.8 found by femtosecond transient absorption spectroscopy a 15 ps phase that was assigned to a transfer of excitation energy to the pigment giving rise to far-red emission component F735 of the PS I holocomplex. Although all reports concluded that F735 fluorescence reflects the presence of an energy trap, the function of this trap is under debate.21 Mukerji and Sauer19 suggested that this trap concentrates excitons in the vicinity of the RC and focuses an excitation to the P700 interacting with long wavelength Chls of the PS I core complex. According to the other reports (see discussion in review by van Grondelle et al.21), the transfer of excitations to P700 via long wavelength Chls of LHC I and PS I core is a parallel and less favored energy path since P700 is still oxidized below 77 K with a relatively high quantum efficiency. Unlike LHC II polypeptides which form trimers, subfractions of LHC I complex preferentially form dimers. Trimerization in LHC II and dimerization in LHC I may be a result of specific protein differences. Recently, complexes of LHC I-730 subfractions were successfully reconstituted in vitro.7 Protein interactions between Lhca1 and Lhca4 subunits were found to be highly specific so that combinations with LHC II polypeptides did not result in dimerization. Furthermore, it was shown that at 77 K the emission of reconstituted Lhca1 and Lhca4 monomers is characterized by different spectral bands with maxima at 686 and 735 nm, respectively, which indicates the principal difference in emitting forms of Chl a. Heterodimerization in the LHC I-730 complex was shown to involve a preferential ligation of more chlorophyll b and a change in Chlprotein interactions as seen by circular dichroism spectra. In LHC I-730 heterodimers the low-temperature emission is dominated by the 735 nm band. The fluorescence yield of the individual complexes was estimated to be nearly equal to that of dimeric complex indicative of the proper energy transfer among Chls in monomeric as well as dimeric complexes. The authors suggested that Chl b and carotenoids most effectively promote the 735 nm fluorescence of the heterodimer. LHC I-730 is the only known heterodimeric antenna complex in higher plants. Because heterodimerization promotes changes in pigment organization and protein-protein interactions and spectral properties of the complex, it is interesting to study excitation relaxation and energy transfer in reconstituted LHC I-730 heterodimer and its constituents by time-resolved fluorescence. In this paper we report on SPC measurements of reconstituted heterodimers that closely resemble the native LHC I-730 complexes with regard to their spectroscopic properties as well as time-resolved fluorescence data on monomeric polypeptides that make up the LHC I-730 complexes. The data show that in both reconstituted and native LHC I-730 heterodimers an intersubunit 30-50 ps energy-transfer component ensured by spectral overlap of 685 nm fluorescence of Lhca1

Melkozernov et al. TABLE 1: Lifetimes and Relative Quantum Yields of Exponential Components Obtained from the Fluorescence Global Analysis of r-Lhca1, r-Lhca4 Monomers and r-LHC I-730 and n-LHC I-730 Heterodimers. Chl a/b ratio

τi, ns

rel quantum yield (au)a

1. r-Lhca1 monomer

3.06

2. r-Lhca4 monomer

2.48

3. r-LHC I-730 heterodimer

2.50

4. n-LHC I-730 heterodimer

2.76

0.35 1.70 3.60 0.225 0.80 1.80 0.028 0.288 0.849 2.0 0.045 0.28 0.81 2.5

0.04 0.48 0.48 0.1 0.6 0.3 0.02 0.18 0.50 0.30 0.07 0.43 0.37 0.13

protein

a Relative quantum yield calculated as A (λ)τ /∑A (λ)τ (see Material i i i i and Methods). λexc ) 650 nm; τi, lifetime of a fluorescence phase.

monomer and absorption of long wavelength spectral forms of Lhca4 monomer may be observed at room temperature. Materials and Methods Isolation of Light-Harvesting Polypeptides. Light-harvesting polypeptides of photosystem I were isolated from tomato PS I holocomplex using the detailed protocol given by Schmid et al.7 Briefly, LHC I-730 complexes were obtained by incubation of PS I holocomplexes in detergents, 1% n-dodecylβ-maltoside and 1% n-octyl-β-glucopyranoside, followed by sucrose gradient centrifugation of solution for 25 h at 270 000g. Reconstitution. Reconstituted monomeric polypeptides Lhca1 and Lhca4 and reconstituted heterodimeric complexes of LHC I-730 were obtained using the protocol of Schmid et al.7 Apoproteins of Lhca1 and Lhca4 were overexpressed in E. coli and reconstituted with HPLC purified Chl a, Chl b, and carotenoids. Chl a/b ratios in light-harvesting polypeptides were measured according to Porra et al.22 (see Table 1). Single-Photon Counting. Time-resolved fluorescence of reconstituted LHC I polypeptides were measured using the timecorrelated single-photon counting technique (SPC).23 The concentrated samples were diluted in 20 mM Tris-HCl, pH 8.0, to a final chlorophyll concentration of about 2 µg/mL. The samples were excited into the Chl b absorption band at 650 nm with 5 ps laser pulses and a repetition rate of 7.6 MHz. To avoid singlet-singlet annihilation low intensities of laser excitation of less than 1 nJ/pulse (∼1013 photons/(cm2 pulse)) were used in all experiments. The fluorescence was selected using a monochromator with slit width of 4 nm (fwhm) and detected by a Hamamatsu R2809U microchannel plate photomultiplier. The channel resolution of the time-amplitude converter was 5.13 ps. The observed kinetics measured with a 10 nm step in a 670-760 nm region were deconvoluted with an instrument response function (fwhm ≈ 30 ps) and fitted to a sum of exponentials (∑Ai exp(-t/τi). The results of 3-, 4-, or 5-component fit were judged by a χ2 parameter and residuals. The fluorescence kinetics were analyzed globally, and decay associated spectra (DAS) were constructed as a preexponential component plotted against emission wavelength with positive and negative amplitudes in DAS reflecting a fluorescence decay and a fluorescence rise, respectively. Relative quantum yields of each fluorescence phase were calculated as a ratio of integrated area under the curve φi(λ) ) Ai(λ)τi and the integrated area under the steady-state spectrum φ ) ∑Ai(λ)τi.

Energy Redistribution in LHC I-730

J. Phys. Chem. B, Vol. 102, No. 42, 1998 8185

Figure 1. Absorption spectra of r-Lhca1 (1), r-Lhca4 (2) monomers, and r-LHC I-730 complexes (3) at 295 K in 20 mM Tris HCl buffer, pH 7.8. The absorption spectrum of native LHC I-730 is similar to the spectrum of r-LHC I-730 (not shown). Spectra are normalized to maximum peak height.

Steady-State Fluorescence. Steady-state fluorescence emission spectra were recorded using a SPEX Fluorolog 2 instrument. The samples were diluted to a concentration of 2 µg of Chl/mL in 20 mM Tris HCl, pH 7.8. The excitation and emission bandwidths were 6 and 2.4 nm, respectively. Samples were excited at 440 nm. Steady-state emission spectra were corrected for the sensitivity of the photodetector. Results Absorption and Steady-State Emission Spectroscopy of Reconstituted LHC Polypeptides at Room Temperature. Absorption spectra of r-Lhca1, r-Lhca4, and r-LHC I-730 complexes recorded at room temperature in the 550-750 nm spectral region are illustrated in Figure 1. All three spectra exhibit similar asymmetric spectral profiles with maxima of the major Qy transition of Chl a around 675-677 nm. However, the bands have different fwhm of 712, 1015, and 811 cm-1 for r-Lhca1, r-Lhca4, and r-LHC I-730, respectively. Below 675 nm the differences in spectral bandwidths are due to specific contributions of different spectral forms of Chl b and Chl a in the 630-670 nm region in agreement with different Chl a/b ratios of samples listed in Table 1. In the red region, above 700 nm, the absorption spectra of r-Lhca4 (Figure 1, curve 2) and r-LHC I-730 (curve 3) show more intensity than the absorption spectrum of r-Lhca1 (curve 1). Quantitatively, the area under the absorption spectra of r-Lhca4 and r-LHC I-730 in the 700-730 nm region is respectively 7 and 4 times larger than the area under the spectrum of r-Lhca1. This indicates an increased oscillator strength of Chl a species absorbing above 700 nm in both r-Lhca4 and r-LHC I-730 complexes. The absorption spectrum of native LHC I-730 is indistinguishable within experimental error from the absorption spectrum of r-LHC I-730 and is not shown in Figure 1. Steady-state fluorescence spectra of reconstituted and native LHC I polypeptides used in this study and measured at 295 K with excitation at 440 nm are shown in Figure 2a. The steadystate emission of an Lhca1 monomer (curve 1) is characterized by a spectral band with maximum at 686 nm and fwhm of about 450 cm-1 and a broad shoulder at 740 nm with significantly reduced emission compared to a 686 nm peak. The emission maximum is 10 nm (216 cm-1) shifted as compared to the maximum of absorption spectrum (Figure 1, curve 1). In a striking contrast to Lhca1 monomer, an Lhca4 subunit exhibits a significant increase in steady-state emission in the red 700750 nm spectral region with a peak at 725 nm, a shoulder at

Figure 2. (a) Steady-state fluorescence spectra at 295 K of r-Lhca1 (1), r-Lhca4 (2), r-LHC I-730 (3), n-LHC I-730 (4), and a mixture of Lhca1 and Lhca4 (5); 2 µg of Chl per milliliter of sample in 20 mM Tris HCl buffer, pH 7.8, and excitation at 440 nm. (b) Overlap of emission spectrum of r-Lhca1 and absorption spectrum of r-Lhca4 at 295 K. Spectra are normalized to maximum peak height.

687 nm (Figure 2a, curve 2), and an unusual Stokes shift of about 50 nm (∼1000 cm-1) compared to the absorption peak at 676 nm (Figure 1, spectrum 2). If Chl a species of r-Lhca4 protein absorbing in the red region approximately around 715 nm (Figure 1) would account for a 725 nm emission (spectrum 2 in Figure 2a), then the Stokes shift would decrease to 10 nm (173 cm-1). The spectral overlap of r-Lhca1 emission and absorption of its counterpart, r-Lhca4 monomer, that could enable the donor-acceptor resonance interactions in LHC I-730 complexes is shown in Figure 2b. The emission band of r-Lhca4 polypeptide has a fwhm of about 1600 cm-1, indicating a significant inhomogeneous broadening due to the presence of a series of spectral forms in the 680-750 nm region. The issue of different spectral forms of Chl a contributing to both absorption and emission of LHC I-730 heterodimers will be addressed in a separate publication.24 Emission spectra of r-LHC I-730 (curve 3) and n-LHC I-730 (curve 4) complexes have similar shapes with two distinct spectral bands at 686 and 728 nm, indicating contributions from emission of both Lhca1 and Lhca4 monomers. However, the increased emission intensity of heterodimers at 728 nm makes the spectrum significantly different from the simple sum of emission spectra of monomers Lhca1 and Lhca4 comprising a heterodimer (curve 5 in Figure 2a). Time-Resolved Fluorescence of Reconstituted Monomers at 295 K. SPC measurements of reconstituted Lhca1 and Lhca4 monomers were performed to compare the fluorescence phases of the monomers with those of LHC I-730 heterodimers. The fluorescence decay kinetics of r-Lhca1 and r-Lhca4 were measured in the 650-750 nm spectral region with laser excitation of Chl b molecules at 650 nm. The fluorescence kinetics were analyzed globally using 3-, 4-, or 5-exponential component fits. Fluorescence DAS of r-Lhca1 polypeptide

8186 J. Phys. Chem. B, Vol. 102, No. 42, 1998

Melkozernov et al.

Figure 3. Fluorescence decay associated spectra of monomeric components of r-LHC I-730 obtained after global analysis of fluorescence kinetics at 295 K: (a) reconstituted monomeric Lhca1 protein, χ2 ) 1.16; (b) reconstituted monomeric Lhca4 protein, χ2 ) 1.10. Samples contained about 2 µg/mL Chl in 20 mM Tris HCl buffer, pH 7.8, and were excited at 650 nm.

Figure 4. Fluorescence decay associated spectra of LHC I-730 complexes from tomato obtained after global analysis of fluorescence kinetics at 295 K: (a) reconstituted LHC I-730 heterodimers, χ2 ) 1.10; (b) native LHC I-730 heterodimers, χ2 ) 1.08. Samples contained about 2 µg/mL Chl in 20 mM Tris HCl buffer, pH 7.8, and were excited at 650 nm.

measured at 295 K and low-intensity laser excitation at 650 nm are depicted in Figure 3a. Three decay processes with lifetimes of 350 ps, 1.7 ns, and 3.6 ns were found to best describe the experimental results. All three decay components have similar spectral profiles with emission maxima around 680-690 nm and significantly decreased relative amplitudes around 740 nm. A 1.7 ns DAS with peak at 685 nm dominates the fluorescence. A 350 ps DAS is the second major decaying component with emission maximum at 680 nm. A third 3.6 ns DAS has a maximum at 685 nm and a broad shoulder around 700 nm. The broader spectral shape may indicate the presence of other spectral forms; however, a 10 nm step in the DAS in this study does not allow a fine spectral resolution. The relative quantum yields of the fluorescence phases of r-Lhca1 are presented in Table 1. The fluorescence relaxation processes with lifetimes of 1.7 and 3.6 ns have similar quantum yields, while the 350 ps process has a quantum yield 10 times lower. The time-resolved fluorescence of r-Lhca4 monomer at 295 K is also characterized by three decaying components with lifetimes of 225 ps, 800 ps, and 1.8 ns (Figure 3b). The shapes of the DAS of these components are significantly different from the DAS of r-Lhca1 (Figure 3a), indicating the presence of spectral forms in the 700-750 nm region that are absent in fluorescence spectra of r-Lhca1 monomers. Although all three DAS broadly resemble the steady-state spectrum of the r-Lhca4 at 295 K (Figure 2a, curve 2), the ratios of relative amplitudes at 730 and 690 nm in these DAS are different. The dominating decay process in r-Lhca4 at 295 K is presented by a broad 800 ps DAS with a maximum at 725 nm and a shoulder at 690 nm. The relative quantum yield of this fluorescence phase is 0.6 (Table 1). In contrast to the 800 ps DAS, a 225 ps DAS has an increased relative amplitude at 690 nm. A third component with a lifetime of 1.8 ns is characterized by a very broad spectral

profile with decreased relative amplitudes as compared to other DAS. The 225 ps and 1.8 ns processes have relative quantum yields of 0.1 and 0.3, respectively (Table 1). Time-Resolved Fluorescence of the Reconstituted and Native LHC I-730 Complexes. DAS of fluorescence kinetics of reconstituted LHC I-730 and native LHC I-730 at 295 K in the 670-750 nm spectral region are plotted in Figure 4. Four exponential components with lifetimes of 28 ps, 288 ps, 849 ps, and 2 ns were found to describe the relaxation of fluorescence in r-LHC I-730 heterodimers under laser excitation at 650 nm (Figure 4a). The 288 ps, 849 ps, and 2 ns DAS have spectral profiles similar to DAS of r-Lhca4 (Figure 3b). A slight increase in relative amplitudes around 686 nm in these DAS indicates a contribution from the 686 nm emission of r-Lhca1 monomer (Figure 3a). The additional fast 28 ps decaying component was necessary to best fit the data. The shape of the 28 ps DAS with positive amplitudes around 686 nm and negative amplitudes around 735 nm clearly indicates energy transfer from a Chl a species emitting at 686 nm (fluorescence decay) to a Chl a species emitting at 735 nm (fluorescence rise). An increase in the broad 730 nm emission in the steady-state spectrum of r-LHC I-730 (curve 3 in Figure 2a) is the result of an increase of the relative amplitudes of 849 ps and 2 ns fluorescence decaying components (Figure 4a). These two components account for 80% of the integrated fluorescence (Table 1). DAS of native LHC I-730 heterodimers obtained under similar conditions are shown in Figure 4b. Again, four exponential components were necessary to fit the data. A 45 ps DAS has a positive band with maximum at 685 nm and amplitudes close to zero around 735-740 nm. Although the global analysis did not reveal negative amplitudes in the 720-

Energy Redistribution in LHC I-730 750 nm region, they were found in a single decay analysis of fluorescence kinetics measured at 740 nm (data not shown). Therefore, we attribute this component similarly to the 28 ps DAS in r-LHC I-730 (Figure 4a) to energy transfer from Chl a species emitting at 686 nm to Chl a species emitting at 735 nm. The other three decaying components have lifetimes similar to decay processes in r-LHC I-730. The relative amplitudes of long-lived components with lifetimes of 810 ps and 2.5 ns are decreased compared to that in r-LHC I-730 (Figure 4a), while the amplitude of an intermediate 280 ps component remained unchanged. This results in a decrease of the relative quantum yield of the 810 ps and 2.5 ns fluorescence and relative increase of quantum yield of the 280 ps fluorescence (Table 1). Although the spectral profiles of the 280 ps DAS in r-LHC I-730 and n-LHC I-730 are largely similar, the 280 ps DAS in native samples is characterized by a broader spectrum in the 700750 nm region. A lack of spectral resolution as well as aggregation might account for these differences. The differences in lifetime and the shape of the energy-transfer component as well as differences in relative amplitudes and relative quantum yields of decaying components in n-LHC I-730 and r-LHC I-730 heterodimers may reflect the real biochemical differences between reconstituted and native samples. Discussion Relaxation of Fluorescence in Lhca1 and Lhca4 Monomers. In this paper the excitation dynamics of LHC I-730 heterodimers obtained by picosecond fluorescence are compared with excitation dynamics of its constituent monomers, Lhca1 and Lhca4 Chl a/b-binding polypeptides. Data presented in Figures 1 and 2 clearly indicate that at room temperature the r-Lhca1 and r-Lhca4 monomers possess different spectral forms. In contrast to the r-Lhca1 protein, the r-Lhca4 protein exhibits the larger inhomogeneous broadening with fwhm of 1015 cm-1 (Figure 1, curve 2). The presence of spectrally unresolved forms that clearly can be seen in the long wavelength tail of absorption spectrum of r-Lhca4 indicates Chl a species specific for this subunit. Figure 2a demonstrates that at 295 K the steady-state fluorescence of Lhca1 and Lhca4 monomers are dominated by emission peaking at distinctly different wavelength, 686 nm in r-Lhca1 (Figure 2a, curve 1) and 725 nm in r-Lhca4 (curve 2). This is in agreement with a conclusion of Schmid et al.,7 who showed that at 77 K the shape of emission spectrum of r-Lhca1 does not change while the spectral width of r-Lhca4 emission decreases and the maximum of emission shifts to 735 nm. This low-temperature red shift of the r-Lhca4 emission is possibly due to a significant increase of a 740 nm component of the inhomogeneously broadened band.24 The spectral profiles of fluorescence DAS of intermediate and long-lived decaying components of monomers (Figure 3) are consistent with the shapes of the steady-state emission spectra. Upon laser excitation at 650 nm the following processes appear to take place in both monomers: (i) substantial excitation of Chl b, (ii) fast energy transfer to Chl a resulting in population of Chl a species, and (iii) overall relaxation of the excitation from terminal emitting states of Chl a. There is growing evidence that all light-harvesting Chl a/b-binding proteins show structural similarity due to protein sequence homology in two of the most conserved transmembrane helices.2 These transmembrane helices in Lhcb1 monomers of LHC II were identified as a structural core binding molecules of Chl a+b and carotenoids.9 It is likely that in LHC I monomers the similar structure would bind Chl a and b molecules and carotenoids. The energy transfer from Chl b to Chl a is beyond time

J. Phys. Chem. B, Vol. 102, No. 42, 1998 8187 resolution in this study. It is thought to be in the range 300600 fs for LHC II polypeptides.10,11 The only femtosecond transient anisotropy study of excitation relaxation in LHC I-730 complexes from spinach8 did not reveal, however, the rate of Chl b to Chl a energy transfer. In our study the absence of the fast energy-transfer components in time-resolved fluorescence of r-Lhca1 and r-Lhca4 (Figure 3) suggests that the unresolved fast equilibration processes could populate terminal emitting states of Chl a in both subunits. The observed difference in lifetimes of fluorescence decays in both monomers may reflect the different pigment-protein interactions, which is consistent with their different Chl a/b ratios (Table 1) and significantly different CD spectra at room temperature.7 Several decay phases in the fluorescence of monomers probably reflect the kinetic heterogeneity of excitation relaxation in these Chl a/b polypeptides. Lifetimes for overall relaxation of excitation of Chl a in bulk LHC I complexes and native LHC I-730 complexes were reported to be in the range 1-3.5 ns.8,19 The observed kinetic heterogeneity of excitation relaxation in reconstituted monomers (Figure 3) may be due to different pigment-protein interactions. However, the aggregation of Chl a/b polypeptides may contribute to this heterogeneity as it was reported for LHC II polypeptides.25,26 We have recently obtained evidence that supports the second interpretation.27 Intersubunit Energy Transfer in LHC I Heterodimer. Successful reconstitution of r-LHC I-730 complexes from r-Lhca1 and r-Lhca4 polypeptides proved the heterodimeric nature of LHC I-730 complexes and showed that formation of a heterodimer resulted from protein interactions between the two subunits.7 The authors found that at 77 K steady-state emission spectra of both r-LHC I-730 and n-LHC I-730 have similar spectral profiles dominated by emission at 732-734 nm that is different from spectra of a simple mixture of r-Lhca1 and r-Lhca4 proteins. At room temperature, steady-state emission spectra (Figure 2a, curves 3 and 4) and DAS of intermediate and long-lived fluorescence decaying components (Figure 4a,b) of heterodimeric complexes exhibit increased relative amplitudes at 686 nm. Basically, the 686 nm band in DAS of r-LHC I-730 originates from fluorescence of Chl a species of the Lhca1 half of a heterodimer. However, at 295 K there is a possibility of thermal back reaction which may populate the Chl a species fluorescing at 686 nm. Last, the slight contamination of the samples with detergent-solubilized uncoupled Chl-protein complexes cannot be ruled out. The measurements of time-resolved fluorescence of reconstituted and native LHC I-730 heterodimers revealed an additional picosecond component of fluorescence relaxation (Figure 4) which is absent in fluorescence relaxation of both Lhca1 and r-Lhca4 monomers (Figure 3). The shape of the 28 ps DAS of r-LHC I-730 (positive amplitudes at 686 nm and negative amplitudes at 720-750 nm) (Figure 4A) clearly indicates energy transfer from 686 nm emitters to red pigments emitting around 730-740 nm. We conclude from our data that the 45 ps component in DAS of native LHC I-730 is likely to represent a kinetic phase similar to the 28 ps decay phase in reconstituted LHC I-730. This energy-transfer component is most likely an intersubunit energy transfer as it appears only upon formation of a heterodimer from two monomers. Alternatively, heterodimerization might induce organizational changes of Chl(s) within the Lhca4 subunit. At room temperature, the amplitude of the fluorescence rise at the long wavelength is much less than the amplitudes of the decay at 686 nm, indicating that energy transfer to long wavelength Chl a species in vitro is not a major sink for energy from these species at room

8188 J. Phys. Chem. B, Vol. 102, No. 42, 1998 temperature. There are several reports indicating the energytransfer component from Chl a species emitting at 680 nm to Chl a species emitting at 735 nm. Mukerji and Sauer19 found a 40 ps energy-transfer component at 77 K in detergent-isolated bulk LHC I complexes. Pålsson et al.8 were unable to resolve any fast energy-transfer component in time-resolved fluorescence spectra of LHC I-730 complexes at room temperature. However, the authors resolved a 15 ps component by transient absorption spectroscopy and assigned this phase to energy transfer from pigment clusters emitting at 680 to pigments emitting at 735 nm, which is generally consistent with our measurements of the lifetime of this phase obtained by SPC. It is likely that the fast intersubunit energy-transfer component follows the Fo¨rster inductive resonance mechanism with probability of the transfer depending on spectral overlap between donor and acceptor molecules and their distance and relative orientation.28 The increased intensity in the long-wavelength tail (above 700 nm) of the major absorption band of r-Lhca4 and r-LHC I-730 (Figure 1) indicates the presence of specific spectral forms of Chl a in this region. These spectral forms are largely absent in this region in the absorption spectrum of r-Lhca1 subunit (Figure 1). The energy transfer is possibly due to spectral overlap of 686 nm emitters in r-Lhca1 subunit with Chl a species absorbing around 715 nm and residing on r-Lhca4 subunit (Figure 2b). LHC I-730 heterodimers were reported to have increased pigment/protein ratio and Chl b content compared to monomers, which implies that heterodimerization promotes some changes in pigment-protein interactions.7 These changes were shown to result in appearance of CD signals different from the CD signals of both Lhca1 and Lhca4 monomers. It is possible that the intersubunit energy transfer occurs between Chl a pigment molecules located within the interaction area of monomers comprising the heterodimer. Our data show that reconstituted and native LHC I-730 heterodimers exhibit kinetic heterogeneity of the decay processes with lifetimes falling into three groups of 150-350 ps, 0.81.8 ns, and 2-3.5 ns (Figure 4). However, the lifetimes of fluorescence components in reconstituted and native samples are similar, which indicate similar decay processes in these two samples. The relaxation times in the range 1-3.5 ns were reported earlier as lifetimes of terminal relaxation of excitation for bulk LHC I19 and the LHC I-730 subpopulation.8 This is also close to lifetimes of similar decaying states in LHC II.12,13 The kinetic heterogeneity of excitation relaxation processes in LHC I-730 probably reflects the pigment-protein interactions although the possibility that different decaying processes represent the quenching states of overall excitation relaxation due to aggregation in LHC I-730 may not be ruled out. It should be noted that both steady-state and time-resolved fluorescence measurements were performed under similar conditions with LHC I polypeptides dissolved in 20 mM Tris HCl, pH 7.8. For LHC II polypeptides there have been a series of reports25,26 that aggregation may change the lifetimes of the overall excitation. The observed increase in the relative quantum yield of the 849 ps and 2 ns decay component in r-LHC I-730 (Figure 4a and Table 1) results from increased relative amplitudes of these components which, in turn, indicates increased amount of decaying species. Unfortunately, it is difficult to determine to which extent pigment-protein complexes uncoupled due to detergent modification contribute to fluorescence decay with lifetime of 1-3 ns since fluorescence of pigment-protein complexes energetically uncoupled in the absence of RC decays with similar lifetimes.

Melkozernov et al.

Figure 5. Scheme of energy-transfer pathways in LHC I-730 heterodimers. C650, Chl b absorbing at 650 nm (solid vertical arrows); F686 and F735, Chl a emitting at 686 and 735 nm, respectively. Dashed vertical arrows show heterogeneous fluorescence relaxation. The fast energy transfer from Chl b to Chl a is not resolved in this study. See explanations in the text.

The assignment of the 150-350 ps component is of special interest. We found this component in assays of fluorescence decays kinetics in PS II-deleted thylakoids of Chlamydomonas reinhardtii.29 In LHC-deleted thylakoids the relative amplitude of this component was significantly decreased, indicating that this relaxation process is associated with the peripheral antenna. Mukerji and Sauer19 ascribed a 200 ps component of fluorescence relaxation at 77 K in bulk spinach LHC I complexes to equilibration process between Chl clusters in the complex. A similar conclusion was obtained for LHC II complexes.13 However, the aggregation study of LHC II polypeptides26 reported the 250 ps decay as a quenching state. No similar reports on LHC I polypeptides have been made. The shape of the 250 ps DAS in both monomers and heterodimers at room temperature (Figures 3 and 4) does not allow us to ascribe this component to an energy-transfer process. Whether this component represents energy equilibration step or a phase of aggregation quenching as well as Chl-protein spectral forms of LHC I complexes at low temperature needs to be investigated. In summary, our data led us to the scheme of energy redistribution in LHC I-730 complexes of LHC I (Figure 5). Excitation at 650 nm (C650 absorption) results in a fast unresolved energy transfer from Chl b to Chl a molecules with lifetime of less than 1 ps in both Lhca1 and Lhca4 halves of the complex. The energy remains in the LHC I-730 complex for 1-3 ns. This is the lifetime of terminal emitting Chl a species. Kinetic heterogeneity of excitation relaxation processes possibly reflects different pigment-protein interactions. Heterodimerization results in an assembly of more Chl b and a change in pigment-protein interactions. The 30-50 ps energytransfer component in a heterodimer which is absent in both Lhca1 and Lhca4 monomers is probably due to an intersubunit redistribution of energy from Chl a clusters residing in a contact area of interacting subunits. This energy transfer is possible because Lhca1 and Lhca4 monomers in the heterodimer possess principally different spectral forms fluorescing around 685 nm in Lhca1 and 735 nm in Lhca4. The spectral overlap of the fluorescence of Lhca1 and the absorption of long wavelength spectral forms of Chl a in Lhca4 suggests that the energy transfer is via the Fo¨rster inductive resonance mechanism. However, the quantitative estimation of the distance between the donor and acceptor molecules requires knowledge of the extent of spectral overlap and structural information about the orientation of emitting and absorbing pigments in the complex. Pålsson

Energy Redistribution in LHC I-730 et al.8 suggest that the spectral overlap factor may be small. This may be a partial explanation for this relatively slow (≈30 ps) energy transfer. In addition, either the distance between the pigments is large (>20 Å) or the transition dipole orientation must be nearly perpendicular to explain the observed slow rate of energy transfer. The former is supported by our suggestion that the 30 ps process reflects an intersubunit energy transfer in the LHC I-730 heterodimer. Acknowledgment. This work was supported by NSF Grant MCB-9727607 to R.E.B. and DOE Grant DE-FG02-96ER20213 to G.W.S. The authors thank Dr. Christian Poweleit for his assistance with the time-resolved fluorescence measurements. This is publication 354 of the Center for the Study of Early Events in Photosynthesis at Arizona State University. References and Notes (1) Jansson, S. Biochim. Biophys. Acta 1994, 1184, 1. (2) Pichersky, I.; Jansson, S. Oxygenic Photosynthesis: The Light Reactions; Ort, D. R., Yocum, C. F., Eds.; Kluwer Academic Publishers: Dordrecht, 1996; pp 507-521. (3) Haworth, P.; Watson, J. L.; Arntzen, C. J. Biochim. Biophys. Acta 1983, 724, 151. (4) Lam, E.; Ortiz, W.; Mayfield, S.; Malkin, R. Plant. Physiol. 1984, 74, 650. (5) Knoetzel, J.; Svendsen, I.; Simpson, D. J. Eur. J. Biochem. 1992, 206, 209. (6) Jansson, S.; Andersen, B.; Scheller, H. V. Plant Physiol. 1996, 112, 409. (7) Schmid, V. H. R.; Cammarata, K. V.; Bruns, B. U.; Schmidt, G. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 7667. (8) Pålsson, L.-O.; Tjus, S. E.; Andersson, B.; Gillbro, T. Biochim. Biophys. Acta 1995, 1230, 291. (9) Ku¨hlbrandt, W.; Wang, D. N.; Fujiyoshi, Y. Nature 1994, 367, 614.

J. Phys. Chem. B, Vol. 102, No. 42, 1998 8189 (10) Eads, D. D.; Castner, E. W.; Alberte, R. S.; Mets, L.; Fleming, G. R. J. Phys. Chem. 1989, 93, 8271. (11) Connelly, J. P.; Mu¨ller, M. G.; Hucke, M.; Gatzen, G.; Mullineaux, C. W.; Ruban, A. V.; Horton, P.; Holzwarth, A. R. J. Phys. Chem. B 1997, 101, 1902. (12) Ide, J. P.; Klug, D. R.; Ku¨hlbrandt, W.; Giorgi, L. B.; Porter, G. Biochim. Biophys. Acta 1987, 893, 349. (13) Mullineaux, C. W.; Pascal, A. A.; Horton, P.; Holzwarth, A. R. Biochim. Biophys. Acta 1993, 1141, 23. (14) Hemelrijk, P. W.; Kwa, S. L. S.; van Grondelle, R.; Dekker: J. P. Biochim. Biophys. Acta 1992, 1098, 159. (15) Nussberger, S.; Dekker, J. P.; Ku¨hlbrandt, W.; van Bolhuis, B. M.; van Grondelle, R.; van Amerongen, H. Biochemistry 1994, 33, 14775. (16) Zucchelli, G.; Dainese, P.; Jennings, R. C.; Breton, J.; Garlaschi, F. M.; Bassi, R. Biochemistry 1994, 33, 8982. (17) Satoh, K.; Butler, W. L. Biochim. Biophys. Acta 1978, 502, 103. (18) Mukerji, I.; Sauer, K. Current Research in Photosynthesis; Baltcheffsky, M., Ed.; Kluwer Academic Publishers: Dordrecht, 1990; Vol. II, p 321. (19) Mukerji, I.; Sauer, K. Biochim. Biophys. Acta 1993, 1142, 311. (20) Tjus, S. E.; Roobol-Boza, M.; Pålsson, L. O.; Andersson, B. Photosynth. Res. 1995, 45, 41. (21) Van Grondelle, R.; Dekker, J. P.; Gillbro, T.; Sundstrom, V. Biochim. Biophys. Acta 1994, 1187, 1. (22) Porra, R. G.; Thompson, W. A.; Kriedemann, P. E. Biochim. Biophys. Acta 1989, 975, 384. (23) Causgrove, T. P.; Brune, D. C.; Blankenship, R. E.; Olson, J. M. Photosynth. Res. 1990, 25, 1. (24) Manuscript in preparation. (25) Ruban, A. V.; Horton, P. Photosynth. Res. 1994, 40, 181. (26) Vasil′ev, S.; Irrgang, K.-D.; Schro¨tter, T.; Bergmann, A.; Eichler, H.-J.; Renger, G. Biochemistry 1997, 36, 7503. (27) Melkozernov, A. N.; Schmid, V. H. R.; Schmidt, G. W.; Blankenship, R. E. Unpublished results. (28) Fo¨rster, T. ComprehensiVe Biochemistry; Florkin, M., Stotz, A., Eds.; Elsevier: Amsterdam, 1967; Vol. 22, pp 61-80. (29) Melkozernov, A. N.; Su, Hui; Webber, A. N.; Blankenship, R. E. Photosynth. Res., in press.