Photosynthesis Research 48: 99-106, 1996. (~) 1996 KluwerAcademic Publishers. Printedin the Netherlands. Minireview

Reaction center and antenna processes in photosynthesis at low temperature T h i j s J. Aartsma & J a n A m e s z Department of Biophysics, Huygens Laboratory, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands Received 6 December 1995; accepted in revised form 29 January 1996

Key words: electron transfer, energy transfer, low temperature, photosynthesis, spectroscopy

Abstract Around 1960 experiments of Arnold and Clayton, Chance and Nishimura and Calvin and coworkers demonstrated that the primary photosynthetic electron transfer processes are not abolished by cooling to cryogenic temperatures. After a brief historical introduction, this review discusses some aspects of electron transfer in bacterial reaction centers and of optical spectroscopy of photosynthetic systems with emphasis on low-temperature experiments.

Abbreviations: (B)Chl - (bacterio)chlorophyll; (B)Phe - (bacterio)pheophytin; FMO - Fenna-Matthews-Olson; LH1, LH2 - light harvesting complexes of purple bacteria; LHC II, CP47 - light harvesting complexes of Photosystem II; P, P870 - primary electron donor; RC - reaction center

Historical aspects A landmark in the study of photosynthesis was the discovery that the primary processes are not abolished by cooling, as are 'normal' biological reactions, but can also be observed at cryogenic temperatures. The first demonstration of this kind was by Chance and Nishimura (1960), who observed that cytochrome c oxidation in the purple bacterium Chromatium vinosum occurred at 77 K with about the same efficiency as at room temperature. Inspired by these results, Arnold and Clayton (1960) applied even lower temperatures and observed in chromatophores of Rhodobacter sphaeroides a reversible absorption increase at 420 nm at 1 K upon illumination. At about the same time Calvin (1959; Sogo et al. 1959) showed that the light-induced EPR signal centered at 9 = 2.0025 (Calvin and Sogo 1957) was also present at 77 K in chromatophores of Rhodospirillum rubrum. This signal is now known to be due to the oxidized primary electron donor, P870 +. Taken together, these experiments clearly established the 'electronic nature' (Arnold and Clayton 1960) of the primary step or steps in photosynthesis, and disproved earlier theories according to which the transfer

of hydrogen or other atoms would constitute the primary photosynthetic reaction (see, e.g., Rabinowitch 1945, 1951, 1956 and Duysens 1956 for a review of the older theories). Nevertheless, the name 'hydrogen donor' has been retained for some time in the literature (e.g., Clayton 1962) to designate what are now called secondary electron donors. The above-mentioned authors recognized that the occurence of photosynthetic processes at cryogenic temperatures implied an ordered structure of the photosynthetic apparatus with well defined distances between the photosynthetic components, and properties comparable to those of solid state systems. Arnold and Clayton, as well as Calvin, explained their results in terms of a semiconductor model (Arnold and Sherwood 1957; Calvin 1959; Sogo et al. 1959; Clayton 1962; Arnold 1965), whereas Chance and Nishimura (1960) proposed that the primary reaction in purple bacteria would be the transfer of an electron from cytochrome c to bacteriochlorophyll. Unequivocal evidence for the role of P870 as primary electron donor, as proposed as early as 1956 by Duysens (1956; Duysens et al. 1956), came only with the development of

100 time-resolved laser spectroscopy (Parson 1967; Parson and Cogdell 1975). The low-temperature experiments have provided a strong impetus for theoretical studies aimed at obtaining insight in the characteristics of photosynthetic electron transfer. The first was that of DeVault and Chance (1966), who performed a detailed study of the rate of cytochrome c oxidation as a function of temperature. Below 100 K, the rate of the reaction was found to be essentially independent of the temperature (Figure 1), which was explained by electron tunneling. Recent studies of electron transfer at low temperatures will be discussed below; at this point we should mention the pioneering studies on the primary electron donors of plant photosynthesis by Witt and coworkers (1961) and Chance and coworkers (Chance and Bonner 1963; Floyd et al. 1971) and on iron-sulfur centers of Photosystem I by Malkin and Bearden (1971). During the last decades, low-temperature research of photosynthesis has developed to such an extent as to include a considerable proportion of all research of the primary processes of photosynthesis and of the optical properties of the antenna and reaction center pigments. For this reason, a comprehensive treatment within the limited space of this review is impossible. In the following sections we shall confine ourselves to a few areas that may serve to illustrate the importance of low temperature studies. The vast amount of lowtemperature EPR work will not be covered at all.

Electron transfer In the years that followed the discoveries of Arnold, Chance, Calvin and coworkers an increasing body of evidence has accumulated showing that the primary charge separation and some of the secondary electron transfer reactions in the reaction center can be observed at cryogenic temperatures. Some of the early work has been mentioned already in the previous section. Here we shall confine ourselves to more recent studies on photosynthetic bacteria. Martin and coworkers were the first to accurately measure the rate of primary electron transfer to bacteriopheophytin in reaction centers of purple bacteria, which was found to proceed with a time constant of 2.8 ps in isolated reaction centers of Rb. sphaeroides and Rhodopseudomonas viridis (Martin et al. 1986). In contrast to cytochrome c oxidation, the rate of electron transfer was found to increase upon cooling, and reached a time constant of 1.2 ps at 10 K for Rb.

sphaeroides, while the effect was even more dramatic for Rps. viridis, with a time constant of 0.7 ps at temperatures below 25 K (Breton et al. 1988; Fleming et al. 1988). A similar increase in the rate of electron transfer has been observed for secondary electron transfer from reduced BPhe to the first acceptor quinone (QA) in Rb. sphaeroides (Kirmaier et al. 1985). The same phenomenon was observed in heliobacteria, where the rate of electron transfer from the primary electron acceptor, hydroxy-Chl a (van de Meent et al. 1991) to the secondary acceptor increases three-fold upon cooling to 15 K (van Kan et al. 1989; van Kan 1991). It thus appears that an acceleration of electron transfer upon cooling is a more or less 'normal' phenomenon in bacterial reaction centers, and it may in principle be explained by the theories for electron transfer proposed by Marcus (1964) and by Jortner (1976, 1980) and Bixon and Jortner (1986). However, Kirmaier and coworkers (1985), as well as van Kan (1991), from a detailed study of the temperature dependence, concluded that these theories could not describe the rate of secondary electron transfer in Rb. sphaeroides and Heliobacterium chlorum in a quantitative way. The results could be fitted with the theory of Kakitani and Kakitani (1981) for non-adiabatic electron transfer in terms of a temperature dependent Franck-Condon factor, with reasonable fits of the free energy difference of the reaction and other parameters, while the approach of Bixon and Jortner (1986) did not yield a satisfactory fit of the data. A quantitative explanation of the temperature dependence of the primary charge separation is complicated by two more recent developments; (i) evidence indicating that the reaction is a two-step mechanism, involving the accessory BChl as an intermediate (Marcus 1987; Holzapfel et al. 1989, 1990; Arlt et al. 1993; Schmidt et al. 1994) and (ii) the recent observation that the primary charge separation is at least biphasic (Mialler et al. 1992; Duet al. 1992; Hamm et al. 1993). The first point has been invoked to explain the temperature dependence of charge separation (Lauterwasser et al. 1991). Nevertheless, the two-step mechanism is still a matter of contention (Breton et al. 1986; Martin et al. 1986; Chan et al. 1991; Kirmaier and Holten 1991; Bixon et al. 1991; Woodbury et al. 1994, 1995). The biphasicity of the charge separation manifests itself in the decay of excited P870 as well as in the formation of BPhe-. The clearest example was observed in the (M)Y210W mutant of Rb. sphaeroides where the tyrosine M210 had been replaced by tryptophan (Shochat et al. 1994; van Noort 1994) with a major time constant

101 of 33 and a minor one of 5 ps, but biphasic kinetics were also observed in other tyrosine mutants (Finkele et al. 1990; Nagarajan et al. 1990; Chan et al. 1991; Hamm et al. 1993; Jia et al. 1993), in wild type Rb. sphaeroides (Mi]ller et al. 1992; Duet al. 1992; Hamm et al. 1993) and in reaction centers of the green filamentous bacterium Chloroflexus aurantiacus (Becker et al. 1991). The biphasic kinetics of charge separation persist at low temperature (Vos et al. 1992). The biphasicity has been variously explained by a so-called 'parking state' model of electron transfer in the 'inactive' (M) chain (MOiler et al. 1992; Hamm et al. 1993), or by heterogeneity of site energies and intermolecular interactions (Nagarajan et al. 1990; DiMagno et al. 1992; Jia et al. 1993; Ogrodnik et al. 1994). As will be discussed below, accumulated photon echo measurements support the latter explanation. The implications of a static heterogeneity for the primary electron transfer in relation to the free energy differences of the states involved have recently been discussed by Bixon et al. (1995). Interestingly, the (M)Y210W mutant showed a temperature dependence that was significantly different from the wild strain: both time constants increased upon cooling (Nagarajan et al. 1993; van Noort 1994). The effect was largest for the 33 ps component, which increased to 300 ps at 10 K (Figure 2), by far the longest lifetime reported for this type of mutant so far. This temperature dependence has been explained by a change of the free energy difference between the excited primary donor and the charge separated state relative to the wild type (Nagarajan et al. 1993) together with a change in reorganization energy (van Noort 1994) and possibly involvement of the two-step electron transfer mechanism mentioned above (Nagarajan et al. 1993).

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Low temperature spectroscopy One of the most conspicuous effects of lowering the temperature on the optical spectra of photosynthetic systems is a significant decrease of the optical linewidth (Pullerits et al. 1995). The effect is illustrated in Figure 3 which shows the absorption spectrum of the purple bacterium Rps. cryptolactis, measured at 298 K and at 6 K (Kramer et al. 1995). Considerably more detail is seen in the latter spectrum, where the bands of the peripheral antenna complexes, B800-820 and B800-850, at 800, 829 and 872 nm, and of the core complex, at 893 nm, can be clearly distinguished.

Figure 2. Absorption kinetics at 545 nm in isolated quinone-depleted reaction centers of a mutant of Rb. sphaeroides, in which tyrosine at the (M)210 position, located near the primary electron donor, P870, has been replaced by tryptophan. The experiment was done at 10 K with a time resolution of 30 ps. The positive initial change is due to excited state absorption of P870, the slow downward change, fitted with a time constant of 300 ps, is due to BPhe reduction (van Noort 1994). A smaller component of 12 ps observed by Nagarajan et al. (1993) at 80 K is not resolved in this experiment.

The enhanced resolution thus can be very useful for the analysis of spectroscopic details in the optical spectra in relation to the primary processes in photosynthesis. A particularly interesting example is the isolated reaction center of purple bacteria. At low temperature,

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the Qx absorption bands of the two pheophytins in this reaction center are well resolved, with maxima at 530 nm and 545 nm. Time-resolved measurements showed that only the latter pheophytin is reduced (Kirmaler et al. 1985), and thus is located in the active (L) branch of electron transfer. This conclusion was reinforced by the effect of replacement of the active BPhe with BChl in the (M)L214H RC mutant of Rb. sphaeroides (Kirmaier et al. 1991). Together, these observations provided convincing evidence that electron transfer in the reaction center is strongly directional. The intrinsic, o r homogeneous, linewidth of an optical transition is in principle determined by the coherent lifetime of the excited state. Measurement of the homogeneous linewidth, therefore, provides information about the excited state dynamics of pigments in photosynthetic systems. At sufficiently low temperature, the homogeneous line width in such systems is determined by population relaxation, i.e. by energy transfer and charge separation (Reddy et al. 1992a). The parameters which determine the homogeneous line width at low temperature can be measured in the frequency domain by hole burning spectroscopy (Jankowiak et al. 1993), or in the time domain by accumulated photon echo measurements (Hesselink and Wiersma 1981; Aartsma et al. 1996). Both methods eliminate contributions from the site inhomogeneous broadening which dominates the line shape in conventional forms of spectroscopy. Photon echo and hole burning experiments provide information about the relaxation of the initially excited state irrespective of the relaxation path which may involve other components of the system, this in contrast to absorbance and fluorescence measurements. Thus, photon echo and hole burning measurements provide

information that is complementary, but comparable, to that obtained by the latter methods. Time-resolved spectroscopy is the method of choice to study, e.g., the rate of energy transfer between groups of pigments or antenna complexes, for example that from LH2 to LH 1 in purple bacteria (Freiberg et al. 1989; Zhang et al. 1992; Kennis et al. 1995), or between BChl a 800 and BChl a 850 in LH2 (Shreve et al. 1991; Hess et al. 1993; Kennis et al. 1996). For a recent review, see van Grondelle et al. (1994). Hole burning spectroscopy has been applied to a variety of isolated antenna complexes, such as the antenna complexes CP47 (Chang et al. 1994) and LHC II (Reddy et al. 1994) from plants, the FMO complex from green sulfur bacteria (Johnson and Small 1991), and the LH1 (Reddy et al. 1992b) and LH2 (van der Laan et al. 1990, 1993; Reddy et al. 1991, 1993; Decaro et al. 1994) antenna complexes of purple bacteria. In general, a narrow hole is only observed upon excitation in the long-wavelength edge of the Qy absorption band, whereas excitation in the middle and short-wavelength regions results in broad, featureless hole spectra. This can be explained in terms of the exciton level structure of the excited states in such systems (Johnson and Small 1991). Rapid downward exciton scattering within the manifold of exciton states leads to relatively broad homogeneous line widths, in contrast to direct excitation of the lowest exciton state. The widths of the narrow holes are determined by the (coherent) lifetime of the lowest exciton state which is typically of the order of 10 ps or longer. Of course, the energy levels and dipolar interactions are still affected by inhomogeneous broadening of the site energies of the individual molecules. According to the exciton model, the fluorescence of an isolated antenna complex at low temperature originates predominantly from the lowest exciton state. In systems with a low symmetry the various exciton transitions have widely varying orientations of the optical transition moment, and consequently a high degree of fluorescence polarization is only to be expected upon excitation of the lowest exciton state within the complex. This may even be the case in LH1 and LH2, assuming that the symmetry is - most likely - reduced by spatial and energetic disorder. Therefore, the general features of the exciton model seem to be consistent with the observation that, for example, in LH1 and LH2 at low temperature the fluorescence polarization increases significantly upon long-wavelength excitation (Kramer et al. 1984), and with the relationship between the wavelength of the fluorescence maximum

103 and that of excitation (van Mourik et al. 1992). An alternative explanation for these observations is based on the assumption that the excitations are localized on individual pigment molecules within the antenna complex characterized by site heterogeneity and inhomogeneous broadening. Energy transfer will lead to a rapid thermalization of the excited state, and polarized fluorescence is predominantly observed upon excitation of the energetically lowest pigment molecule. In view of the structure of LH2 (McDermott et al. 1995), and the conjecture that the structures of LH1 and LH2 are very similar, it seems that the exciton model should be favored: the strong dipolar interactions between the BChls (Novoderezhkin and Razjivin 1993; Kennis et al. 1996) are difficult to reconcile with localized excitations. This indicates that the concept of 'major' and 'minor' forms of BChl in LH1 and LH2 (Reddy et al. 1992b, and references therein) should not necessarily be associated with specific pigments or pigment pools within these complexes. Additional support for the exciton model has been obtained from accumulated photon echo measurements. The accumulated photon echo is essentially the time domain equivalent of spectral hole burning, and directly measures the optical dephasing time T2 (Hesselink and Wiersma 1981). This technique has been applied to investigate the excited state dynamics of isolated photosynthetic pigment-protein complexes at low temperature (Louwe and Aartsma 1994; Aartsma et al. 1996). The primary objective of these experiments was to determine the coherent lifetime of the initially excited state and the implications for energy transfer. The antenna systems studied so far comprise the FMO complex of green sulfur bacteria and the LHC II complex of green plants (Louwe and Aartsma 1995). Measurements performed on the FMO-complex at low temperatures showed decay times ranging from hundreds of picoseconds to well in the sub-picosecond domain, depending on temperature and on wavelength of excitation (Louwe and Aartsma 1994, 1995). The wavelength dependence of the amplitudes of the various time constants suggests that they are associated with discrete exciton states. The variation in lifetime is explained in terms of phonon assisted relaxation to lower lying energy levels within the exciton manifold. The temperature dependence of the different time constants in the range of 1.4-30 K can be fitted with a combination of a power law and an exponential activation of higher energy levels (Louwe and Aartsma 1995). The power law dependence arises from the dynamics of the protein matrix which behaves as a

so-called two-level system, similar to a glass (Jackson and Silbey 1983; V~51ker 1989). Experiments performed on the LHC II complex at 1.4 K showed similar decay patterns as the FMO complex with respect to the range of time constants and the wavelength dependence. At least three different time constants can be discerned in the spectral region around 680 nm, each with a specific wavelength distribution (Louwe and Aartsma 1995). These observations strongly suggest that the excited state dynamics in these antennas at low temperature should be described in terms of coherent exciton states, as opposed to the Ftirster model for energy transfer. The observed photon echo life times appear to be much longer than what would be expected on the basis of the F/Srster model taking into account the strength of the dipolar interactions. It should be realized that exciton states are the true eigenstates of the system obtained by diagonalizing the Hamiltonian including intermolecular interactions. Note that at 1.4 K vibrational motion is completely damped. It may be argued that at sufficiently high temperature the exciton states become strongly mixed by vibronic coupling, leading to a rapid localization of the excitation. Subsequent energy transfer occurs by a hopping mechanism which may be viewed as arising from phonon-induced modulation of the mixing coefficients of the exciton states, either directly or by modulation of the transition frequencies of the individual molecules. In this limit, energy transfer is determined by bath-fluctuations, rather than dipolar coupling. Hence, the mechanisms of energy dissipation at low and at room temperature appear to be fundamentally different. Accumulated photon echo experiments were also performed to probe early steps in the charge separation process in reaction center of purple bacteria (Schellenberg et al. 1995). The systems investigated included the reaction centers ofRb. sphaeroides R26 and that of the M(Y)210W mutant mentioned in the previous section. At sufficiently low temperature, pure dephasing processes are eliminated and the accumulated photon echo kinetics should reflect the decay of P870" by electron transfer. At 1.5 K, the accumulated photon echo decay of R26 showed a dominant very fast decay in addition to two slower components of about 1 and 8 ps, in reasonable agreement with those observed by time-resolved absorbance measurements under similar conditions (see previous section) and attributed to primary electron transfer (Schellenberg et al. 1995). In the (M)Y210W mutant the corresponding time constants of the photon echo decay were 18 and 120 ps. The

104 difference between the latter number and that obtained from pump-probe measurements (see above) remains to be explained; it should be noted, however, that the experimental conditions, and in particular the redox state of the first acceptor quinone (QA), were different in the two sets of experiments. The slower components in the photon echo decay are only observed in the red wing of the absorption spectrum, upon excitation of the zero-phonon transition. On the basis of these experiments one can rule out the so-called 'parking state' model as an explanation for the biexponential kinetics of charge separation. The biphasic decay of P870" is indicative of dispersive kinetics of electron transfer, most likely due to site heterogeneity. The very fast decay is attributed to rapid dephasing through vibronic relaxation upon excitation of the phonon sideband. The fact that it dominates the photon echo decay is explained by the strong electron-phonon coupling associated with excitation of P870. Therefore, it is not necessary to invoke rapid relaxation on the 100 fs time scale to some intermediate state to explain the photon echo kinetics (Meech et al. 1986). In conclusion, the various forms of lowtemperature spectroscopy have greatly contributed to our understanding of the processes and interactions in photosynthetic systems. For this reason it may be expected that such investigations will continue, and maintain a promine m place in photosynthesis research.

Acknowledgements Research in this laboratory was supported by the Foundations for Life Sciences (SLW) and for Chemical Research (SON), financed by the Netherlands Organization for Scientific Research (NWO), and by the European Community (Contract Nos. ERBCHBGCT930361 and SCI*-CT92-0796).

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