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.

Role of pheophytin a in the primary charge separation of photosystem I from Acaryochloris marina: Femtosecond optical studies of excitation energy and electron transfer reactions.

Photosystem I (PSI) of the cyanobacterium Acaryochloris marina is capable of performing an efficient photoelectrochemical conversion of far-red light due to its unique suite of cofactors. Chlorophyll d (Chl-d) has been long known as the major antenna pigment in the PSI from A. marina, while the exact cofactor composition of the reaction centre (RC) was established only recently by cryo-electron microscopy. The RC consists of four Chl-d molecules, and, surprisingly, two molecules of pheophytin a (Pheo-a), which provide a unique opportunity to resolve, spectrally and kinetically, the primary electron transfer reactions. Femtosecond transient absorption spectroscopy was here employed to observe absorption changes in the 400-860 nm spectral window occurring in the 0.1-500 ps timescale upon unselective antenna excitation and selective excitation of the Chl-d special pair P740 in the RC. A numerical decomposition of the absorption changes, including principal component analysis, allowed the identification of P740(+)Chld2(-) as the primary charge separated state and P740(+)Pheoa3(-) as the successive, secondary, radical pair. A remarkable feature of the electron transfer reaction between Chld2 and Pheoa3 is the fast, kinetically unresolved, equilibrium with an estimated ratio of 1:3. The energy level of the stabilised ion-radical state P740(+)Pheoa3(-) was determined to be ~60 meV below that of the RC excited state. In this regard, the energetics and the structural implications of the presence of Pheo-a in the electron transfer chain of PSI from A. marina are discussed, also in comparison with those of the most diffused Chl-a binding RC.

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.

Impact of energy limitations on function and resilience in long-wavelength Photosystem II.

Photosystem II (PSII) uses the energy from red light to split water and reduce quinone, an energy-demanding process based on chlorophyll a (Chl-a) photochemistry. Two types of cyanobacterial PSII can use chlorophyll d (Chl-d) and chlorophyll f (Chl-f) to perform the same reactions using lower energy, far-red light. PSII from Acaryochloris marina has Chl-d replacing all but one of its 35 Chl-a, while PSII from Chroococcidiopsis thermalis, a facultative far-red species, has just 4 Chl-f and 1 Chl-d and 30 Chl-a. From bioenergetic considerations, the far-red PSII were predicted to lose photochemical efficiency and/or resilience to photodamage. Here, we compare enzyme turnover efficiency, forward electron transfer, back-reactions and photodamage in Chl-f-PSII, Chl-d-PSII, and Chl-a-PSII. We show that: (i) all types of PSII have a comparable efficiency in enzyme turnover; (ii) the modified energy gaps on the acceptor side of Chl-d-PSII favour recombination via PD1+Phe- repopulation, leading to increased singlet oxygen production and greater sensitivity to high-light damage compared to Chl-a-PSII and Chl-f-PSII; (iii) the acceptor-side energy gaps in Chl-f-PSII are tuned to avoid harmful back reactions, favouring resilience to photodamage over efficiency of light usage. The results are explained by the differences in the redox tuning of the electron transfer cofactors Phe and QA and in the number and layout of the chlorophylls that share the excitation energy with the primary electron donor. PSII has adapted to lower energy in two distinct ways, each appropriate for its specific environment but with different functional penalties.

Algae, plants and cyanobacteria perform a process called photosynthesis, in which carbon dioxide and water are converted into oxygen and energy-rich carbon compounds. The first step of this process involves an enzyme called photosystem II, which uses light energy to extract electrons from water to help capture the carbon dioxide. If the photosystem absorbs too much light, compounds known as reactive oxygen species are produced in quantities that damage the photosystem and kill the cell. To ensure that the photosystem works efficiently and to protect it from damage, about half of the energy from the absorbed light is dissipated as heat, while the rest of the energy is stored in the products of photosynthesis. The standard form of photosystem II uses the energy of visible light, but some cyanobacteria contain different types of photosystem II, which do the same chemical reactions using lower energy far-red light. One type of far-red photosystem II is found in Acaryochloris marina, a cyanobacterium living in stable levels of far-red light, shaded from visible light. The other type is found in a cyanobacterium called Chroococcidiopsis thermalis, which can switch between using its far-red photosystem II when shaded from visible light and using its standard photosystem II when exposed to it. Being able to work with less energy, the two types of far-red photosystem II appear to be more efficient than the standard one, but it has been unclear if there were any downsides to this trait. Viola et al. compared the standard photosystem II with the far-red photosystem II types from C. thermalis and A. marina by measuring the efficiency of these enzymes, the quantity of reactive oxygen species produced, and the resulting light-induced damage. The experiments revealed that the far-red photosystem II of A. marina is highly efficient but produces elevated levels of reactive oxygen species if exposed to high light conditions. On the other hand, the far-red photosystem II of C. thermalis is less efficient in collecting and using far-red light, but is more robust, producing fewer reactive oxygen species. Despite these tradeoffs, engineering crop plants or algae that could use far-red photosynthesis may help boost food and biomass production. A better understanding of the trade-offs between efficiency and resilience in the two types of far-red photosystem II could determine which features would be beneficial, and under what conditions. This work also improves our knowledge of how the standard photosystem II balances light absorption and damage limitation to work efficiently in a variable environment.

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.

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.

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.

Direct Evidence for Excitation Energy Transfer Limitations Imposed by Low-Energy Chlorophylls in Photosystem I-Light Harvesting Complex I of Land Plants.

The overall efficiency of photosynthetic energy conversion depends both on photochemical and excitation energy transfer processes from extended light-harvesting antenna networks. Understanding the trade-offs between increase in the antenna cross section and bandwidth and photochemical conversion efficiency is of central importance both from a biological perspective and for the design of biomimetic artificial photosynthetic complexes. Here, we employ two-dimensional electronic spectroscopy to spectrally resolve the excitation energy transfer dynamics and directly correlate them with the initial site of excitation in photosystem I-light harvesting complex I (PSI-LHCI) supercomplex of land plants, which has both a large antenna dimension and a wide optical bandwidth extending to energies lower than the peak of the reaction center chlorophylls. Upon preferential excitation of the low-energy chlorophylls (red forms), the average relaxation time in the bulk supercomplex increases by a factor of 2-3 with respect to unselective excitation at higher photon energies. This slowdown is interpreted in terms of an excitation energy transfer limitation from low-energy chlorophyll forms in the PSI-LHCI. These results aid in defining the optimum balance between the extension of the antenna bandwidth to the near-infrared region, which increases light-harvesting capacity, and high photoconversion quantum efficiency.

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.

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.

A Comparison of Constitutive and Inducible Non-Endogenous Keto-Carotenoids Biosynthesis in Synechocystis sp. PCC 6803.

The model cyanobacterium Synechocystis sp. PCC 6803 has gained significant attention as an alternative and sustainable source for biomass, biofuels and added-value compounds. The latter category includes keto-carotenoids, which are molecules largely employed in a wide spectrum of industrial applications in the food, feed, nutraceutical, cosmetic and pharmaceutical sectors. Keto-carotenoids are not naturally synthesized by Synechocystis, at least in any significant amounts, but their accumulation can be induced by metabolic engineering of the endogenous carotenoid biosynthetic pathway. In this study, the accumulation of the keto-carotenoids astaxanthin and canthaxanthin, resulting from the constitutive or temperature-inducible expression of the CrtW and CrtZ genes from Brevundimonas, is compared. The benefits and drawbacks of the two engineering approaches are discussed.

Kinetics and Energetics of Phylloquinone Reduction in Photosystem I: Insight From Modeling of the Site Directed Mutants.

Two phylloquinone molecules (A 1), one being predominantly coordinated by PsaA subunit residues (A 1A) the other by those of PsaB (A 1B), act as intermediates in the two parallel electron transfer chains of Photosystem I. The oxidation kinetics of the two phyllosemiquinones by the iron-sulfur cluster FX differ by approximately one order of magnitude, with A 1 A - being oxidized in about 200 ns and A 1 B - in about 20 ns. These differences are generally explained in terms of asymmetries in the driving force for FX reduction on the two electron transfer chains. Site directed mutations of conserved amino acids composing the A 1 binding site have been engineered on both reaction center subunits, and proved to affect selectively the oxidation lifetime of either A 1 A - , for PsaA mutants, or A 1 B - , for PsaB mutants. The mutation effects are here critically reviewed, also by novel modeling simulations employing the tunneling formalism to estimate the electron transfer rates. Three main classes of mutation effects are in particular addressed: (i) those leading to an acceleration, (ii) those leading to a moderated slowing (~5-folds), and (iii) those leading to a severe slowing (>20-folds) of the kinetics. The effect of specific amino acid perturbations contributing to the poising of the phylloquinones redox potential and, in turn, to PSI functionality, is discussed.

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.

Excitation and emission wavelength dependence of fluorescence spectra in whole cells of the cyanobacterium Synechocystis sp. PPC6803: Influence on the estimation of Photosystem II maximal quantum efficiency.

The fluorescence emission spectrum of Synechocystis sp. PPC6803 cells, at room temperature, displays: i) significant bandshape variations when collected under open (F0) and closed (FM) Photosystem II reaction centre conditions; ii) a marked dependence on the excitation wavelength both under F0 and FM conditions, due to the enhancement of phycobilisomes (PBS) emission upon their direct excitation. As a consequence: iii) the ratio of the variable and maximal fluorescence (FV/FM), that is a commonly employed indicator of the maximal photochemical quantum efficiency of PSII (Φpc, PSII), displays a significant dependency on both the excitation and the emission (detection) wavelength; iv) the FV/FM excitation/emission wavelength dependency is due, primarily, to the overlap of PSII emission with that of supercomplexes showing negligible changes in quantum yield upon trap closure, i.e. PSI and a PBS fraction which is incapable to transfer the excitation energy efficiently to core complexes. v) The contribution to the cellular emission and the relative absorption-cross section of PSII, PSI and uncoupled PBS are extracted using a spectral decomposition strategy. It is concluded that vi) Φpc, PSII is generally underestimated from the FV/FM measurements in this organism and, the degree of the estimation bias, which can exceed 50%, depends on the measurement conditions. Spectral modelling based on the decomposed emission/cross-section profiles were extended to other processes typically monitored from steady-state fluorescence measurements, in the presence of an actinic illumination, in particular non-photochemical quenching. It is suggested that vii) the quenching extent is generally underestimated in analogy to FV/FM but that viii) the location of quenching sites can be discriminated based on the combined excitation/emission spectral analysis.

Photochemistry beyond the red limit in chlorophyll f-containing photosystems.

Photosystems I and II convert solar energy into the chemical energy that powers life. Chlorophyll a photochemistry, using red light (680 to 700 nm), is near universal and is considered to define the energy "red limit" of oxygenic photosynthesis. We present biophysical studies on the photosystems from a cyanobacterium grown in far-red light (750 nm). The few long-wavelength chlorophylls present are well resolved from each other and from the majority pigment, chlorophyll a. Charge separation in photosystem I and II uses chlorophyll f at 745 nm and chlorophyll f (or d) at 727 nm, respectively. Each photosystem has a few even longer-wavelength chlorophylls f that collect light and pass excitation energy uphill to the photochemically active pigments. These photosystems function beyond the red limit using far-red pigments in only a few key positions.

Structure-Based Exciton Hamiltonian and Dynamics for the Reconstituted Wild-type CP29 Protein Antenna Complex of the Photosystem II.

We provide an analysis of the pigment composition of reconstituted wild type CP29 complexes. The obtained stoichiometry of 9 ± 0.6 Chls a and 3 ± 0.6 Chls b per complex, with some possible heterogeneity in the carotenoid binding, is in agreement with 9 Chls a and 3.5 Chls b revealed by the modeling of low-temperature optical spectra. We find that ∼50% of Chl b614 is lost during the reconstitution/purification procedure, whereas Chls a are almost fully retained. The excitonic structure and the nature of the low-energy (low-E) state(s) are addressed via simulations (using Redfield theory) of 5 K absorption and fluorescence/nonresonant hole-burned (NRHB) spectra obtained at different excitation/burning conditions. We show that, depending on laser excitation frequency, reconstituted complexes display two (independent) low-E states (i.e., the A and B traps) with different NRHB and emission spectra. The red-shifted state A near 682.4 nm is assigned to a minor (∼10%) subpopulation (sub. II) that most likely originates from an imperfect local folding occurring during protein reconstitution. Its lowest energy state A (localized on Chl a604) is easily burned with λB = 488.0 nm and has a red-shifted fluorescence origin band near 683.7 nm that is not observed in native (isolated) complexes. Prolonged burning by 488.0 nm light reveals a second low-E trap at 680.2 nm (state B) with a fluorescence origin band at ∼681 nm, which is also observed when using a direct low-fluence excitation near 650 nm. The latter state is mostly delocalized over the a611, a612, a615 Chl trimer and corresponds to the lowest energy state of the major (∼90%) subpopulation (sub. I) that exhibits a lower hole-burning quantum yield. Thus, we suggest that major sub. I correspond to the native folding of CP29, whereas the red shift of the Chl a604 site energy observed in the minor sub. II occurs only in reconstituted complexes.