Chlorophyll triplet states in thylakoid membranes of Acaryochloris marina. Evidence for a triplet state sitting on the photosystem I primary donor populated by intersystem crossing.

Photo-induced triplet states in the thylakoid membranes isolated from the cyanobacterium Acaryocholoris marina, that harbours Chlorophyll (Chl) d as its main chromophore, have been investigated by Optically Detected Magnetic Resonance (ODMR) and time-resolved Electron Paramagnetic Resonance (TR-EPR). Thylakoids were subjected to treatments aimed at poising the redox state of the terminal electron transfer acceptors and donors of Photosystem II (PSII) and Photosystem I (PSI), respectively. Under ambient redox conditions, four Chl d triplet populations were detectable, identifiable by their characteristic zero field splitting parameters, after deconvolution of the Fluorescence Detected Magnetic Resonance (FDMR) spectra. Illumination in the presence of the redox mediator N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) and sodium ascorbate at room temperature led to a redistribution of the triplet populations, with T3 (|D|= 0.0245 cm-1, |E|= 0.0042 cm-1) becoming dominant and increasing in intensity with respect to untreated samples. A second triplet population (T4, |D|= 0.0248 cm-1, |E|= 0.0040 cm-1) having an intensity ratio of about 1:4 with respect to T3 was also detectable after illumination in the presence of TMPD and ascorbate. The microwave-induced Triplet-minus-Singlet spectrum acquired at the maximum of the |D|-|E| transition (610 MHz) displays a broad minimum at 740 nm, accompanied by a set of complex spectral features that overall resemble, despite showing further fine spectral structure, the previously reported Triplet-minus-Singlet spectrum attributed to the recombination triplet of PSI reaction centre, 3 P 740 [Schenderlein M, Çetin M, Barber J, et al. Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium Acaryochloris marina. Biochim Biophys Acta 1777:1400-1408]. However, TR-EPR experiments indicate that this triplet displays an eaeaea electron spin polarisation pattern which is characteristic of triplet sublevels populated by intersystem crossing rather than recombination, for which an aeeaae polarisation pattern is expected instead. It is proposed that the observed triplet, which leads to the bleaching of the P740 singlet state, sits on the PSI reaction centre.

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 (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) 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 PSIIL 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, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm 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.

Thermodynamic Factors Controlling Electron Transfer among the Terminal Electron Acceptors of Photosystem I: Insights from Kinetic Modelling.

Photosystem I is a key component of primary energy conversion in oxygenic photosynthesis. Electron transfer reactions in Photosystem I take place across two parallel electron transfer chains that converge after a few electron transfer steps, sharing both the terminal electron acceptors, which are a series of three iron-sulphur (Fe-S) clusters known as FX, FA, and FB, and the terminal donor, P700. The two electron transfer chains show kinetic differences which are, due to their close geometrical symmetry, mainly attributable to the tuning of the physicochemical reactivity of the bound cofactors, exerted by the protein surroundings. The factors controlling the rate of electron transfer between the terminal Fe-S clusters are still not fully understood due to the difficulties of monitoring these events directly. Here we present a discussion concerning the driving forces associated with electron transfer between FX and FA as well as between FA and FB, employing a tunnelling-based description of the reaction rates coupled with the kinetic modelling of forward and recombination reactions. It is concluded that the reorganisation energy for FX- oxidation shall be lower than 1 eV. Moreover, it is suggested that the analysis of mutants with altered FA redox properties can also provide useful information concerning the upstream phylloquinone cofactor energetics.

Temperature-induced zeaxanthin overproduction in Synechococcus elongatus PCC 7942.

The exogenous crtZ gene from Brevundimonas sp. SD212, coding for a 3,3' ß-car hydroxylase, was expressed in Synechococcus elongatus PCC 7942 under the control of a temperature-inducible promoter in an attempt to engineer the carotenoid metabolic pathway, to increase the content of zeaxanthin and its further hydroxylated derivatives caloxanthin and nostoxanthin. These molecules are of particular interest due to their renowned antioxidant properties. Cultivation of the engineered strain S7942Z-Ti at 35 °C, a temperature which is well tolerated by the wild-type strain and at which the inducible expression system is activated, led to a significant redistribution of the relative carotenoid content. ß-Carotene decreased to about 10% of the pool that is an excess of a threefold decrease with respect to the control, and concomitantly, zeaxanthin became the dominant carotenoid accounting for about half of the pool. As a consequence, zeaxanthin and its derivatives caloxanthin and nostoxanthin collectively accounted for about 90% of the accumulated carotenoids. Yet, upon induction of CrtZ expression at 35 °C the S7942Z-Ti strain displayed a substantial growth impairment accompanied, initially, by a relative loss of carotenoids and successively by the appearance of chlorophyll degradation products which can be interpreted as markers of cellular stress. These observations suggest a limit to the exploitation of Synechococcus elongatus PCC 7942 for biotechnological purposes aimed at increasing the production of hydroxylated carotenoids.

Ultrafast excited state dynamics in the monomeric and trimeric photosystem I core complex of Spirulina platensis probed by two-dimensional electronic spectroscopy.

Photosystem I (PSI), a naturally occurring supercomplex composed of a core part and a light-harvesting antenna, plays an essential role in the photosynthetic electron transfer chain. Evolutionary adaptation dictates a large variability in the type, number, arrangement, and absorption of the Chlorophylls (Chls) responsible for the early steps of light-harvesting and charge separation. For example, the specific location of long-wavelength Chls (referred to as red forms) in the cyanobacterial core has been intensively investigated, but the assignment of the chromophores involved is still controversial. The most red-shifted Chl a form has been observed in the trimer of the PSI core of the cyanobacterium Spirulina platensis, with an absorption centered at ∼740 nm. Here, we apply two-dimensional electronic spectroscopy to study photoexcitation dynamics in isolated trimers and monomers of the PSI core of S. platensis. By means of global analysis, we resolve and compare direct downhill and uphill excitation energy transfer (EET) processes between the bulk Chls and the red forms, observing significant differences between the monomer (lacking the most far red Chl form at 740 nm) and the trimer, with the ultrafast EET component accelerated by five times, from 500 to 100 fs, in the latter. Our findings highlight the complexity of EET dynamics occurring over a broad range of time constants and their sensitivity to energy distribution and arrangement of the cofactors involved. The comparison of monomeric and trimeric forms, differing both in the antenna dimension and in the extent of red forms, enables us to extract significant information regarding PSI functionality.

Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms.

Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

On wavelength-dependent exciton lifetime distributions in reconstituted CP29 antenna of the photosystem II and its site-directed mutants.

To provide more insight into the excitonic structure and exciton lifetimes of the wild type (WT) CP29 complex of photosystem II, we measured high-resolution (low temperature) absorption, emission, and hole burned spectra for the A2 and B3 mutants, which lack chlorophylls a612 and b614 (Chls), respectively. Experimental and modeling results obtained for the WT CP29 and A2/B3 mutants provide new insight on the mutation-induced changes at the molecular level and shed more light on energy transfer dynamics. Simulations of the A2 and B3 optical spectra, using the second-order non-Markovian theory, and comparison with improved fits of WT CP29 optical spectra provide more insight into their excitonic structure, mutation induced changes, and frequency-dependent distributions of exciton lifetimes (T1). A new Hamiltonian obtained for WT CP29 reveals that deletion of Chls a612 or b614 induces changes in the site energies of all remaining Chls. Hamiltonians obtained for A2 and B3 mutants are discussed in the context of the energy landscape of chlorophylls, excitonic structure, and transfer kinetics. Our data suggest that the lowest exciton states in A2 and B3 mutants are contributed by a611(57%), a610(17%), a615(15%) and a615(58%), a611(20%), a612(15%) Chls, respectively, although other compositions of lowest energy states are also discussed. Finally, we argue that the calculated exciton decay times are consistent with both the hole-burning and recent transient absorption measurements. Wavelength-dependent T1 distributions offer more insight into the interpretation of kinetic traces commonly described by discrete exponentials in global analysis/global fitting of transient absorption experiments.

Preliminary Investigation on Phytoplankton Dynamics and Primary Production Models in an Oligotrophic Lake from Remote Sensing Measurements.

The aim of this study is to test a series of methods relying on hyperspectral measurements to characterize phytoplankton in clear lake waters. The phytoplankton temporal evolutions were analyzed exploiting remote sensed indices and metrics linked to the amount of light reaching the target (EPAR), the chlorophyll-a concentration ([Chl-a]OC4) and the fluorescence emission proxy. The latter one evaluated by an adapted version of the Fluorescence Line Height algorithm (FFLH). A peculiar trend was observed around the solar noon during the clear sky days. It is characterized by a drop of the FFLH metric and the [Chl-a]OC4 index. In addition to remote sensed parameters, water samples were also collected and analyzed to characterize the water body and to evaluate the in-situ fluorescence (FF) and absorbed light (FA). The relations between the remote sensed quantities and the in-situ values were employed to develop and test several phytoplankton primary production (PP) models. Promising results were achieved replacing the FA by the EPAR or FFLH in the equation evaluating a PP proxy (R2 > 0.65). This study represents a preliminary outcome supporting the PP monitoring in inland waters by means of remote sensing-based indices and fluorescence metrics.

Ultrafast excited-state dynamics in land plants Photosystem I core and whole supercomplex under oxidised electron donor conditions.

The kinetics of excited-state energy migration were investigated by femtosecond transient absorption in the isolated Photosystem I-Light-Harvesting Complex I (PSI-LHCI) supercomplex and in the isolated PSI core complex of spinach under conditions in which the terminal electron donor P700 is chemically pre-oxidised. It is shown that, under these conditions, the relaxation of the excited state is characterised by lifetimes of about 0.4 ps, 4.5 ps, 15 ps, 35 ps and 65 ps in PSI-LHCI and 0.15 ps, 0.3 ps, 6 ps and 16 ps in the PSI core complex. Compartmental spectral-kinetic modelling indicates that the most likely mechanism to explain the absence of long-lived (ns) excited states is the photochemical population of a radical pair state, which cannot be further stabilised and decays non-radiatively to the ground state with time constants in the order of 6-8 ps.

Modelling electron transfer in photosystem I: limits and perspectives.

Uncovering the parameters underlying the electron transfer (ET) in photosynthetic reaction centres is of importance for understanding the molecular mechanisms underpinning their functionality. The reductive nature of most cofactors involved in photosynthetic ET makes the direct estimation of their properties difficult. Photosystem I (PSI) operates in a highly reducing regime, making the assessment of cofactor properties even more difficult. Kinetic modelling coupled to a non-adiabatic description of ET is a useful approach in overcoming this hindrance. Here we review the theory and modelling approaches that have been used in assessing parameters associated with ET reactions in PSI, with particular attention to ET reactions involving the phylloquinones and the iron-sulphur clusters. In most modelling studies, the goal is to estimate the driving force of ET, which is usually associated with the cofactor midpoint potentials. The driving force is sensitive to many factors, which define the ET rate, i.e. the reorganisation energy, the coupling with nuclear modes and the electronic matrix elements, which are explored and discussed here. The importance of an inclusive modelling of both forward and reverse ET processes is discussed and highlighted. It is shown that although estimates are indeed sensitive to the exact parameter sets employed in the modelling, a general consensus is still attained, pointing to a scenario where Δ G A 1 A → F X 0 / Δ G A 1 B → F X 0 is weakly endergonic/exergonic, respectively. It is emphasised that to further refine those estimates, it will require a joint effort between computational modelling and more wide-ranging experimental studies.

Comparative excitation-emission dependence of the FV /FM ratio in model green algae and cyanobacterial strains.

The emission spectra collected under conditions of open (F0 ) and closed (FM ) photosystem II (PSII) reaction centres are close-to-independent from the excitation wavelength in Chlamydomonas reinhardtii and Chlorella sorokiniana, whereas a pronounced dependence is observed in Synechocystis sp. PCC6803 and Synechococcus PCC7942, instead. The differences in band-shape between the F0 and FM emission are limited in green algae, giving rise only to a minor trough in the FV /FM spectrum in the 705-720 nm range, irrespectively of the excitation. More substantial variations are observed in cyanobacteria, resulting in marked dependencies of the measured FV /FM ratios on both the excitation and the detection wavelengths. In cyanobacteria, the maximal FV /FM values (0.5-0.7), observed monitoring at approximately 684 nm and exciting Chl a preferentially, are comparable to those of green algae; however, FV /FM decreases sharply below approximately 660 nm. Furthermore, in the red emission tail, the trough in the FV /FM spectrum is more pronounced in cyanobacteria with respect to green algae, corresponding to FV /FM values of 0.25-0.4 in this spectral region. Upon direct phycobilisomes excitation (i.e. >520 nm), the FV /FM value detected at 684 nm decreases to 0.3-0.5 and is close-to-negligible (approximately 0.1) below 660 nm. At the same time, the FV spectra are, in all species investigated, almost independent on the excitation wavelength. It is concluded that the excitation/emission dependencies of the FV /FM ratio arise from overlapped contributions from the three independent emissions of PSI, PSII and a fraction of energetically uncoupled external antenna, excited in different proportions depending on the respective optical cross-section and fluorescence yield.

Isolation and characterization of CAC antenna proteins and photosystem I supercomplex from the cryptophytic alga Rhodomonas salina.

In the present paper, we report an improved method combining sucrose density gradient with ion-exchange chromatography for the isolation of pure chlorophyll a/c antenna proteins from the model cryptophytic alga Rhodomonas salina. Antennas were used for in vitro quenching experiments in the absence of xanthophylls, showing that protein aggregation is a plausible mechanism behind non-photochemical quenching in R. salina. From sucrose gradient, it was also possible to purify a functional photosystem I supercomplex, which was in turn characterized by steady-state and time-resolved fluorescence spectroscopy. R. salina photosystem I showed a remarkably fast photochemical trapping rate, similar to what recently reported for other red clade algae such as Chromera velia and Phaeodactylum tricornutum. The method reported therefore may also be suitable for other still partially unexplored algae, such as cryptophytes.

Non-endogenous ketocarotenoid accumulation in engineered Synechocystis sp. PCC 6803.

The cyanobacterium Synechocystis sp. PCC 6803 is a model species commonly employed for biotechnological applications. It is naturally able to accumulate zeaxanthin (Zea) and echinenone (Ech), but not astaxanthin (Asx), which is the highest value carotenoid produced by microalgae, with a wide range of applications in pharmaceutical, cosmetics, food and feed industries. With the aim of finding an alternative and sustainable biological source for the production of Asx and other valuable hydroxylated and ketolated intermediates, the carotenoid biosynthetic pathway of Synechocystis sp. PCC 6803 has been engineered by introducing the 4,4' ß-carotene oxygenase (CrtW) and 3,3' ß-carotene hydroxylase (CrtZ) genes from Brevundimonas sp. SD-212 under the control of a temperature-inducible promoter. The expression of exogenous CrtZ led to an increased accumulation of Zea at the expense of Ech, while the expression of exogenous CrtW promoted the production of non-endogenous canthaxanthin and an increase in the Ech content with a concomitant strong reduction of ß-carotene (ß-car). When both Brevundimonas sp. SD-212 genes were coexpressed, significant amounts of non-endogenous Asx were obtained accompanied by a strong decrease in ß-car content. Asx accumulation was higher (approximately 50% of total carotenoids) when CrtZ was cloned upstream of CrtW, but still significant (approximately 30%) when the position of genes was inverted. Therefore, the engineered strains constitute a useful tool for investigating the ketocarotenoid biosynthetic pathway in cyanobacteria and an excellent starting point for further optimisation and industrial exploitation of these organisms for the production of added-value compounds.

Iron Binding Properties of Recombinant Class A Protein Disulfide Isomerase from Arabidopsis thaliana.

The protein disulfide isomerase (PDI) family comprises a wide set of enzymes mainly involved in thiol-disulfide exchange reactions in the endoplasmic reticulum. Class A PDIs (PDI-A) constitute the smallest members of the family, consisting of a single thioredoxin (TRX) module without any additional domains. To date, their catalytic activity and cellular function are still poorly understood. To gain insight into the role of higher-plant class A PDIs, the biochemical properties of rAtPDI-A, the recombinant form of Arabidopsis thaliana PDI-A, have been investigated. As expressed, rAtPDI-A has only little oxidoreductase activity, but it appears to be capable of binding an iron-sulfur (Fe-S) cluster, most likely a [2Fe-2S] center, at the interface between two protein monomers. A mutational survey of all cysteine residues of rAtPDI-A indicates that only the second and third cysteines of the CXXXCKHC stretch, containing the putative catalytic site CKHC, are primarily involved in cluster coordination. A key role is also played by the lysine residue. Its substitution with glycine, which restores the canonical PDI active site CGHC, does not influence the oxidoreductase activity of the protein, which remains marginal, but strongly affects the binding of the cluster. It is therefore proposed that the unexpected ability of rAtPDI-A to accommodate an Fe-S cluster is due to its very unique CKHC motif, which is conserved in all higher-plant class A PDIs, differentiating them from all other members of the PDI family.

Spectral dependence of irreversible light-induced fluorescence quenching: Chlorophyll forms with maximal emission at 700-702 and 705-710nm as spectroscopic markers of conformational changes in the core complex.

The spectral dependence of the irreversible non-photochemical fluorescence quenching associated with photoinhibition in vitro has been comparatively investigated in thylakoid membranes, PSII enriched particles and PSII core complexes isolated from spinach. The analysis of the fluorescence emission spectra of dark-adapted and quenched samples as a function of the detection temperature in the 280-80K interval, indicates that Chlorophyll spectral forms having maximal emission in the 700-702nm and 705-710nm ranges gain relative intensity in concomitance with the establishment of irreversible light-induced quenching, acting thereby as spectroscopic markers. The relative enhancement of the 700-702nm and 705-710nm forms emission could be due either to an increase of their stoichiometric abundance or to their intrinsically low fluorescence quantum yields. These two factors, that can also coexist, need to be promoted by light-induced alterations in chromophore-protein as well as chromophore-chromophore interactions. The bands centred at about 701 and 706nm are also observed in the PSII core complex, suggesting their, at least partial, localisation in proximity to the reaction centre, and the occurrence of light-induced conformational changes in the core subunits.

High photochemical trapping efficiency in Photosystem I from the red clade algae Chromera velia and Phaeodactylum tricornutum.

In the present work, we report the first comparative spectroscopic investigation between Photosystem I (PSI) complexes isolated from two red clade algae. Excitation energy transfer was measured in PSI from Chromera velia, an alga possessing a split PsaA protein, and from the model diatom Phaeodactylum tricornutum. In both cases, the estimated effective photochemical trapping time was in the 15-25ps range, i.e. twice as fast as higher plants. In contrast to green phototrophs, the trapping time was rather constant across the whole emission spectrum. The weak wavelength dependence was attributed to the limited presence of long-wavelength emitting chlorophylls, as verified by low temperature spectroscopy. As the trapping kinetics of C. velia PSI were barely distinguishable from those of P. tricornutum PSI, it was concluded that the scission of PsaA protein had no significant impact on the overall PSI functionality. In conclusion, the two red clade algae analysed here, carried amongst the most efficient charge separation so far reported for isolated Photosystems.

Biochemical and Spectroscopic Characterization of Highly Stable Photosystem II Supercomplexes from Arabidopsis.

Photosystem II (PSII) is a large membrane supercomplex involved in the first step of oxygenic photosynthesis. It is organized as a dimer, with each monomer consisting of more than 20 subunits as well as several cofactors, including chlorophyll and carotenoid pigments, lipids, and ions. The isolation of stable and homogeneous PSII supercomplexes from plants has been a hindrance for their deep structural and functional characterization. In recent years, purification of complexes with different antenna sizes was achieved with mild detergent solubilization of photosynthetic membranes and fractionation on a sucrose gradient, but these preparations were only stable in the cold for a few hours. In this work, we present an improved protocol to obtain plant PSII supercomplexes that are stable for several hours/days at a wide range of temperatures and can be concentrated without degradation. Biochemical and spectroscopic properties of the purified PSII are presented, as well as a study of the complex solubility in the presence of salts. We also tested the impact of a large panel of detergents on PSII stability and found that very few are able to maintain the integrity of PSII. Such new PSII preparation opens the possibility of performing experiments that require room temperature conditions and/or high protein concentrations, and thus it will allow more detailed investigations into the structure and molecular mechanisms that underlie plant PSII function.

Kinetics and heterogeneity of energy transfer from light harvesting complex II to photosystem I in the supercomplex isolated from Arabidopsis.

State transitions are a phenomenon that maintains the excitation balance between photosystem II (PSII) and photosystem I (PSI-LHCI) by controlling their relative absorption cross-sections. Under light conditions exciting PSII preferentially, a trimeric LHCII antenna moves from PSII to PSI-LHCI to form the PSI-LHCI-LHCII supercomplex. In this work, the excited state dynamics in the PSI-LHCI and PSI-LHCI-LHCII supercomplexes isolated from Arabidopsis have been investigated by picosecond time-resolved fluorescence spectroscopy. The excited state decays were analysed using two approaches based on either (i) a sum of discrete exponentials or (ii) a continuous distribution of lifetimes. The results indicate that the energy transfer from LHCII to the bulk of the PSI antenna occurs with an average macroscopic transfer rate in the 35-65 ns-1 interval. Yet, the most satisfactory description of the data is obtained when considering a heterogeneous population containing two PSI-LHCI-LHCII supercomplexes characterised by a transfer time of ∼15 and ∼60 ns-1, likely due to the differences in the strength and orientation of LHCII harboured to PSI. Both these values are of the same order of magnitude of those estimated for the average energy transfer rates from the low energy spectral forms of LHCI to the bulk of the PSI antenna (15-40 ns-1), but they are slower than the transfer from the bulk antenna of PSI to the reaction centre (>150 ns-1), implying a relatively small kinetics bottleneck for the energy transfer from LHCII. Nevertheless, the kinetic limitation imposed by excited state diffusion has a negligible impact on the photochemical quantum efficiency of the supercomplex, which remains about 98% in the case of PSI-LHCI.

Carotenoid triplet states in photosystem II: coupling with low-energy states of the core complex.

The photo-excited triplet states of carotenoids, sensitised by triplet-triplet energy transfer from the chlorophyll triplet states, have been investigated in the isolated Photosystem II (PSII) core complex and PSII-LHCII (Light Harvesting Complex II) supercomplex by Optically Detected Magnetic Resonance techniques, using both fluorescence (FDMR) and absorption (ADMR) detection. The absence of Photosystem I allows us to reach the full assignment of the carotenoid triplet states populated in PSII under steady state illumination at low temperature. Five carotenoid triplet ((3)Car) populations were identified in PSII-LHCII, and four in the PSII core complex. Thus, four (3)Car populations are attributed to ß-carotene molecules bound to the core complex. All of them show associated fluorescence emission maxima which are relatively red-shifted with respect to the bulk emission of both the PSII-LHCII and the isolated core complexes. In particular the two populations characterised by Zero Field Splitting parameters |D|=0.0370-0.0373 cm(-1)/|E|=0.00373-0.00375 cm(-1) and |D|=0.0381-0.0385 cm(-1)/|E|=0.00393-0.00389 cm(-1), are coupled by singlet energy transfer with chlorophylls which have a red-shifted emission peaking at 705 nm. This observation supports previous suggestions that pointed towards the presence of long-wavelength chlorophyll spectral forms in the PSII core complex. The fifth (3)Car component is observed only in the PSII-LHCII supercomplex and is then assigned to the peripheral light harvesting system.

Comparative kinetic and energetic modelling of phyllosemiquinone oxidation in Photosystem I.

The oxidation kinetics of phyllo(semi)quinone (PhQ), which acts as an electron transfer (ET) intermediate in the Photosystem I reaction centre, are described by a minimum of two exponential phases, characterised by lifetimes in the 10-30 ns and 150-300 ns ranges. The fastest phase is considered to be dominated by the oxidation of the PhQ molecule coordinated by the PsaB reaction centre subunit (PhQB), and the slowest phase is dominated by the oxidation of the PsaA coordinated PhQ (PhQA). Testing different energetic schemes within a unified theory-based kinetic modelling approach provides reliable limit-values for some of the physical-chemical parameters controlling these ET reactions: (i) the value of ΔG(0) associated with PhQA oxidation is smaller than ∼+30 meV; (ii) the value of the total reorganisation energy (λt) likely exceeds 0.7 eV; (iii) different mean nuclear modes are coupled to PhQB and PhQA oxidation, the former being larger, and both being ≥100 cm(-1).