Selective quenching of the fluorescence of core chlorophyll–protein complexes by photochemistry indicates that Photosystem II is partly diffusion limited

The spectral characteristics of fluorescence quenching by open reaction centres in isolated Photosystem II membranes were determined with very high resolution and analysed. Quenching due to photochemistry is maximal near 687 nm, minimal in the chlorophyll b emission interval and displays a distinctive structure around 670 nm. The amplitude of this `quenching hole' is about 0.03 for normalised spectra. On the basis of the absorption spectra of isolated chlorophyll–protein complexes, it is shown that these quenching structures can be exactly described by assuming that photochemistry lowers the fluorescence yield of the reaction centre complex (D1/D2/cytb ₅₅₉) plus CP47, with quenching of the former complex being approximately double that of the latter complex. These data, which qualitatively indicate that there are kinetically limiting processes for primary photochemistry in the antenna, have been analysed by means of several different kinetic models. These models are derived from present structural knowledge of the arrangement of the chlorophyll–protein complexes in Photosystem II and incorporate the reversible charge separation characteristic of the exciton/radical pair equilibration model. In this way it is shown that Photosystem II cannot be considered to be purely trap limited and that exciton migration in the antenna imposes a diffusion limitation of about 30%, irrespective of the structural model assumed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: the phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster F(X)

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A(1), the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F(X) and the phylloquinone bound to either the PsaA (A(1A)) or the PsaB (A(1B)) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F(A) and F(B). The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A(1)) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F(X). A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A(1B) quinone and slightly endergonic, in the case of the A(1A) quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A(0) on both electron transfer branches and the reduction of F(A) by F(X).     

The Q(y) absorption spectrum of the light-harvesting complex II as determined by structure-based analysis of chlorophyll macrocycle deformations

The absorption spectrum of the main antenna complex of photosystem II, LHCII, has been modeled using, as starting points, the chlorophyll (chl) atomic coordinates as obtained by the LHCII crystal analysis [Liu, Z., Yan, H., Wang, K., Kuang, T., Zhang, J., Gui, L., An, X., and Chang, W. (2004) Nature 428, 287-292] of three different trimers. The chl site Q(y) transition energies have been obtained in terms of the chl macrocycle deformations influencing the energy level of the chl frontier orbitals. Using these chl site transition energy values and the entire set of interaction energies, calculated in the ideal dipole approximation, the complete Hamiltonians for the three LHCII trimers have been written and the full set of 42 eigenstates of each LHCII trimer have been calculated. With the 42 transition energies and transition dipole strengths, either unperturbed or associated to the eigenstates, the LHCII Q(y) absorption spectrum has been calculated using a chl absorption band shape. These calculations have been performed both in vacuo and in the presence of a medium. Despite the number of approximations, a good correlation with the measured absorption spectrum of a LHCII preparation is obtained. This analysis shows that, although a substantial C3 symmetry of the LHCII trimer in terms of both chl-chl distances and interaction energies is present, a marked variation among monomer subsets of site transition energies is estimated. This leads to a C3 symmetry breaking in the unperturbed chl site transition energies set and, consequently, in the trimer eigenstates. It is also concluded that interactions among chlorophylls do not significantly modify the light absorption role of LHCII in plant leaves.     

Functional Analyses of the Plant Photosystem I-Light-Harvesting Complex II Supercomplex Reveal That Light-Harvesting Complex II Loosely Bound to Photosystem II Is a Very Efficient Antenna for Photosystem I in State II

State transitions are an important photosynthetic short-term response that allows energy distribution balancing between photosystems I (PSI) and II (PSII). In plants when PSII is preferentially excited compared with PSI (State II), part of the major light-harvesting complex LHCII migrates to PSI to form a PSI-LHCII supercomplex. So far, little is known about this complex, mainly due to purification problems. Here, a stable PSI-LHCII supercomplex is purified from Arabidopsis thaliana and maize (Zea mays) plants. It is demonstrated that LHCIIs loosely bound to PSII in State I are the trimers mainly involved in state transitions and become strongly bound to PSI in State II. Specific Lhcb1-3 isoforms are differently represented in the mobile LHCII compared with S and M trimers. Fluorescence analyses indicate that excitation energy migration from mobile LHCII to PSI is rapid and efficient, and the quantum yield of photochemical conversion of PSI-LHCII is substantially unaffected with respect to PSI, despite a sizable increase of the antenna size. An updated PSI-LHCII structural model suggests that the low-energy chlorophylls 611 and 612 in LHCII interact with the chlorophyll 11145 at the interface of PSI. In contrast with the common opinion, we suggest that the mobile pool of LHCII may be considered an intimate part of the PSI antenna system that is displaced to PSII in State I.     

The Carnot efficiency and plant photosystems

The concept that the Carnot efficiency places an upper limit of 0.60–0.75 on the thermodynamic efficiency of photosynthetic primary photochemistry is examined using a PSI-LHCI preparation. The maximal quantum efficiency was determined ≈0.99 which yielded a thermodynamic efficiency of at least 0.96, a value far above that predicted on the basis of the Carnot efficiency. The commonly presented reasoning leading to the Carnot efficiency idea was therefore critically examined. It is concluded that the conventional assumption that the excited/ground state pigments are ergodic is incorrect, as is the assumption that the pigment system, under illumination, is in equilibrium with the incident light field, at a black body temperature of T _r. It is concluded that the classical reasoning used to describe the thermodynamics of heat systems is not applicable to “photonic” systems such as plant photosystems.     

Light-adapted charge-separated state of photosystem II: structural and functional dynamics of the closed reaction center

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSII_O), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSII_C). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSII_C into a light-adapted charge-separated state (PSII_L). The PSII_C-to-PSII_L transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (F_v) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSII_L requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events—pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, F_m, is associated with PSII_L rather than with PSII_C, and thus the F_v/F_m parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

The closed-state of photosystem II possesses a hitherto unrecognized structural and functional plasticity and upon illumination assumes a light-adapted charge-separated state.     

Analysis of the spin-polarized electron spin echo of the [P700+ A1-] radical pair of photosystem I indicates that both reaction center subunits are competent in electron transfer in cyanobacteria, green algae, and higher plants

The decay of the light-induced spin-correlated radical pair [P700+ A1-] and the associated electron spin echo envelope modulation (ESEEM) have been studied in either thylakoid membranes, cellular membranes, or purified photosystem I prepared from the wild-type strains of Synechocystis sp. PCC 6803, Chlamydomonas reinhardtii, and Spinaceae oleracea. The decay of the spin-correlated radical pair is described in the wild-type membrane by two exponential components with lifetimes of 2-4 and 16-25 micros. The proportions of the two components can be altered by preillumination of the membranes in the presence of reductant at temperatures lower than 220 K, which leads to the complete reduction of the iron-sulfur electron acceptors F(A), F(B), and F(X) and partial photoaccumulation of the reduced quinone electron acceptor A1A-. The "out-of-phase" (OOP) ESEEM attributed to the [P700+ A1-] radical pair has been investigated in the three species as a function of the preillumination treatment. Values of the dipolar (D) and the exchange (J) interactions were extracted by time-domain fitting of the OOP-ESEEM. The results obtained in the wild-type systems are compared with two site-directed mutants of C. reinhardtii [Santabarbara et al. (2005) Biochemistry 44, 2119-2128], in which the spin-polarized signal on either the PsaA- or PsaB-bound electron transfer pathway is suppressed so that the radical pair formed on each electron transfer branch could be monitored selectively. This comparison indicates that when all of the iron-sulfur centers are oxidized, only the echo modulation associated with the A branch [P700+ A1A-] radical pair is observed. The reduction of the iron-sulfur clusters and the quinone A1 by preillumination treatment induces a shift in the ESEEM frequency. In all of the systems investigated this observation can be interpreted in terms of different proportions of the signal associated with the [P700+ A1A-] and [P700+ A1B-] radical pairs, suggesting that bidirectionality of electron transfer in photosystem I is a common feature of all species rather than being confined to green algae.     

Involvement of uncoupled antenna chlorophylls in photoinhibition in thylakoids

Evidence is presented, by means of both fluorescence and action spectroscopy, that a small, spectroscopically heterogeneous population of both Chl a and Chl b molecules is present in isolated spinach thylakoids and is active in photoinhibition. The broadness of the action spectrum suggests that degraded or incompletely assembled pigment-protein complexes may be involved.     

The Requirement for Carotenoids in the Assembly and Function of the Photosynthetic Complexes in Chlamydomonas reinhardtii

We have investigated the importance of carotenoids on the accumulation and function of the photosynthetic apparatus using a mutant of the green alga Chlamydomonas reinhardtii lacking carotenoids. The FN68 mutant is deficient in phytoene synthase, the first enzyme of the carotenoid biosynthesis pathway, and therefore is unable to synthesize any carotenes and xanthophylls. We find that FN68 is unable to accumulate the light-harvesting complexes associated with both photosystems as well as the RC subunits of photosystem II. The accumulation of the cytochrome b₆f complex is also strongly reduced to a level approximately 10% that of the wild type. However, the residual fraction of assembled cytochrome b₆f complexes exhibits single-turnover electron transfer kinetics comparable to those observed in the wild-type strain. Surprisingly, photosystem I is assembled to significant levels in the absence of carotenoids in FN68 and possesses functional properties that are very similar to those of the wild-type complex.Carotenoids (Cars) are fundamental components of the photosynthetic apparatus (Young and Britton, 1993, and refs. therein). The vast majority of Cars are noncovalently bound to either the core or the antenna subunits of PSI or PSII (Siefermann-Harms, 1985; Bassi et al., 1993). The most abundant Car bound to the core subunits of both photosystems is β-carotene, which is found in the vast majority of oxygenic organisms (Siefermann-Harms, 1985; Bassi et al., 1993). The light-harvesting complexes (LHCs) that act as the outer antenna in plants and green algae bind a wider range of oxygenated Cars, known as xanthophylls, the most abundant of which is lutein (Siefermann-Harms, 1985; Bassi et al., 1993; Jennings et al., 1996). The stoichiometry of xanthophylls binding to LHC complexes depends on the particular complexes and often on the illumination conditions during the organism’s growth (Siefermann-Harms, 1985; Demmig-Adams, 1990; Horton et al., 1996). Intriguingly, a molecule of β-carotene (as well as a molecule of chlorophyll [Chl] a) is found also in the cytochrome (Cyt) b₆f complex (Kurisu et al., 2003; Stroebel et al., 2003).Cars have multiple functions in the photosynthetic process; they act as light-harvesting pigments (Frank and Cogdell, 1993), enlarging the optical cross section to radiation that is poorly absorbed by Chl. Moreover, Cars play a crucial role in processes such as nonphotochemical quenching that control the efficiency of light harvesting in response to the intensity of the incident radiation (for review, see Demmig-Adams, 1990; Horton et al., 1996; Niyogi, 1999). Probably the most important role of Cars in photosynthesis is the quenching of the excited triplet state of Chl (for review, see Frank and Cogdell, 1993; Giacometti et al., 2007), preventing the formation of highly reactive singlet oxygen, which represents the principal species active under high light stress (Hideg et al., 1994; Krieger-Liszkay, 2005). The importance of Cars is demonstrated by the observation that disruption of their biosynthesis through mutation, or by inhibition of a key enzyme in the pathway, leads to either lethal phenotypes or to rapid photobleaching of the photosynthetic tissue (Claes, 1957; Faludi-Dániel et al., 1968, 1970; Bolychevtseva et al., 1995; Trebst and Depka, 1997).Moreover, it has been shown that the presence of xanthophylls is absolutely necessary for refolding in vitro of LHC I and LHC II antenna complexes (Plumley and Schmidt, 1987; Paulsen et al., 1993; Sandonà et al., 1998). Such Cars, therefore, have a structural role, as well as their involvement in light harvesting, nonphotochemical quenching regulation, and the quenching of the Chl triplet state. Whether Cars also play a key structural role in the formation and stability of the core complexes of both PSI and PSII has not been systematically explored, since assembly of these complexes in vitro is not feasible. Studies in vivo using higher plants are complicated by the fact that Car deficiency is lethal and can be studied only during the early stages of greening and leaf development (Faludi-Dániel et al., 1968, 1970; Inwood et al., 2008). In these studies, it was shown that the accumulation of PSII complexes was greatly impaired in mutants of maize (Zea mays; Faludi-Dániel et al., 1968, 1970; Inwood et al., 2008), while the assembly of PSI appeared to be less sensitive to Car availability. In mutants of the cyanobacterium Synechocystis sp. PCC 6803 lacking the genes for phytoene desaturase or ζ-carotene desaturase, there was a complete loss of PSII assembly, while functional PSI complexes were assembled, albeit with slightly altered electron transfer kinetics with respect to the wild-type complex (Bautista et al., 2005). In agreement with the higher sensitivity of PSII assembly to Car availability, Trebst and Depka (1997) reported a specific effect on the synthesis of the D1 subunit of PSII RC upon treatment with phytoene desaturase inhibitors. On the other hand, it has recently been reported that in lycopene-β-cyclase mutants of Arabidopsis (Arabidopsis thaliana) that have a decreased amount of β-carotene (bound to the RC) with respect to most of the xanthophyll pool pigments (bound to the LHCs), the level of accumulation of PSI complexes, particularly that of the LHC I complement, was more affected that that of PSII, probably also because of an increased sensitivity to photodamage of mutated PSI RC (Cazzaniga et al., 2012; Fiore et al., 2012).In this investigation, we have studied the accumulation and functionality of the major chromophore-binding complexes of the photosynthetic apparatus, PSI, PSII, and Cyt b₆f, in a Car-less mutant of the green alga Chlamydomonas reinhardtii (FN68) that is blocked at the first committed step of Car biosynthesis, namely, phytoene synthesis (McCarthy et al., 2004). Although the mutant is incapable of growing under phototrophic or photomixotrophic conditions, it can grow in complete darkness on a medium supplemented with a carbon source. Here, we show that the PSII core and antenna complexes fail to accumulate in the mutant and that the Cyt b₆f complex accumulates to approximately one-tenth of the wild-type level. On the other hand, the PSI reaction center accumulates in FN68 and possesses electron transfer properties that are remarkably similar to those of wild-type PSI. Interestingly, we find that the level of PSI accumulation differs in other phytoene synthase null mutants, suggesting that additional mutations in one or other of these strains affect PSI stability. Nevertheless, our findings demonstrate that Cars are not required for either the assembly or the functionality of PSI in vivo.