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.

Comparative Absorption Dynamics of the Singlet Excited States of Chlorophylls a and d.

Transient absorption dynamics of chlorophylls a and d dissolved in tetrahydrofuran was measured by the broadband femtosecond laser pump-probe spectroscopy in a spectral range from 400 to 870 nm. The absorption spectra of the excited S1 singlet states of chlorophylls a and d were recorded, and the dynamics of the of the Qy band shift of the stimulated emission (Stokes shift of fluorescence) was determined in a time range from 60 fs to 4 ps. The kinetics of the intramolecular conversion Qx→Qy (electronic transition S2→S1) was measured; the characteristic relaxation time was 54 ± 3 and 45 ± 9 fs for chlorophylls a and d, respectively.

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.

Cyclic electron transfer around Photosystem I mediated by 2,3-dichloro-1,4-naphtoquinone and ascorbate.

In this work, we investigated electron transport around the photosynthetic pigment-protein complex of Photosystem I (PS I) mediated by external high-potential electron carrier 2,3-dichloro-1,4-naphtoquinone (Cl2 NQ) and ascorbate. It has been demonstrated that the oxidized species of Cl2 NQ and ascorbate serve as intermediates capable of accepting electrons from the iron-sulfur cluster FX of PS I. Reduced species of Cl2 NQ and ascorbate are oxidized by photooxidized PS I primary donor P700+ and/or by molecular oxygen. We have found the synergistic effect of Cl2 NQ and ascorbate on the rate of P700+ reduction. Accelerated electron flow to P700+, observed in the presence of both Cl2 NQ and ascorbate, is explained by an increase in the reduced species of Cl2 NQ due to electron transfer from ascorbate.

Mechanism of adiabatic primary electron transfer in photosystem I: Femtosecond spectroscopy upon excitation of reaction center in the far-red edge of the QY band.

The ultrafast primary charge separation in Photosystem I (PS I) excited by femtosecond pulses centered at 720 and 760nm was studied by pump-to-probe laser spectroscopy. The absorbance in the red edge of PS I absorption spectrum has an unusual exponential dependence on wavelength. The cutoff of short wavelength components of 760nm pulse allows direct excitation of reaction center chlorophyll molecules without involvement of light-harvesting antenna. The transient spectrum manifests the features of the primary ion-radical pair P700+A0- at time delay <180fs, followed by formation of the secondary pair P700+A1- with a characteristic time of 26ps. The obtained data are rationalized in the framework of adiabatic three-state model that includes the chlorophyll dimer P700 and two symmetrically arranged nearest chlorophyll molecules of A0. The arrangement of chlorophylls results in strong electronic coupling between P700 and A0. Excitation in the maximum of P700 absorption generates electronic states with the highest contribution from P700*, whereas excitation in the far-red edge predominantly generates charge transfer state P700+A0- in both branches of redox-cofactors. The three-level model accounts for a flat-bottomed potential surface of the excited state and adiabatic character of electron transfer between P700 and A0, providing a microscopic explanation of the ultrafast formation of P700+A0- and exponential decline of PS I absorption.

Interaction of various types of photosystem I complexes with exogenous electron acceptors.

Interaction of photosystem I (PS I) complexes from cyanobacteria Synechocystis sp. PCC 6803 containing various quinones in the A1-site (phylloquinone PhQ in the wild-type strain (WT), and plastoquinone PQ or 2,3-dichloronaphthoquinone Cl 2 NQ in the menB deletion strain) and different numbers of Fe4S4 clusters (intact WT and FX-core complexes depleted of FA/FB centers) with external acceptors has been studied. The efficiency of interaction was estimated by measuring the light-induced absorption changes at 820 nm due to the reduction of the special pair of chlorophylls (P700+) by an external acceptor(s). It was shown that externally added Cl 2 NQ is able to effectively accept electrons from the terminal iron-sulfur clusters of PS I. Moreover, the efficiency of Cl 2 NQ as external acceptor was higher than the efficiency of the commonly used artificial electron acceptor, methylviologen (MV) for both the intact WT PS I and for the FX-core complexes. The comparison of the efficiency of MV interaction with different types of PS I complexes revealed gradual decrease in the following order: intact WT > menB > FX-core. The effect of MV on the recombination kinetics in menB complexes of PS I with Cl 2 NQ in the A1-site differed significantly from all other PS I samples. The obtained effects are considered in terms of kinetic efficiency of electron acceptors in relation to thermodynamic and structural characteristics of PS I complexes.

Kinetic modeling of electron transfer reactions in photosystem I complexes of various structures with substituted quinone acceptors.

The reduction kinetics of the photo-oxidized primary electron donor P700 in photosystem I (PS I) complexes from cyanobacteria Synechocystis sp. PCC 6803 were analyzed within the kinetic model, which considers electron transfer (ET) reactions between P700, secondary quinone acceptor A1, iron-sulfur clusters and external electron donor and acceptors - methylviologen (MV), 2,3-dichloro-naphthoquinone (Cl2NQ) and oxygen. PS I complexes containing various quinones in the A1-binding site (phylloquinone PhQ, plastoquinone-9 PQ and Cl2NQ) as well as F X-core complexes, depleted of terminal iron-sulfur F A/F B clusters, were studied. The acceleration of charge recombination in F X-core complexes by PhQ/PQ substitution indicates that backward ET from the iron-sulfur clusters involves quinone in the A1-binding site. The kinetic parameters of ET reactions were obtained by global fitting of the P700+ reduction with the kinetic model. The free energy gap ΔG 0 between F X and F A/F B clusters was estimated as -130 meV. The driving force of ET from A1 to F X was determined as -50 and -220 meV for PhQ in the A and B cofactor branches, respectively. For PQ in A1A-site, this reaction was found to be endergonic (ΔG 0 = +75 meV). The interaction of PS I with external acceptors was quantitatively described in terms of Michaelis-Menten kinetics. The second-order rate constants of ET from F A/F B, F X and Cl2NQ in the A1-site of PS I to external acceptors were estimated. The side production of superoxide radical in the A1-site by oxygen reduction via the Mehler reaction might comprise ≥0.3% of the total electron flow in PS I.

O2 reduction by photosystem I involves phylloquinone under steady-state illumination.

O2 reduction was investigated in photosystem I (PSI) complexes isolated from cyanobacteria Synechocystis sp. PCC 6803 wild type (WT) and menB mutant strain, which is unable to synthesize phylloquinone and contains plastoquinone at the quinone-binding site A1. PSI complexes from WT and menB mutant exhibited different dependencies of O2 reduction on light intensity, namely, the values of O2 reduction rate in WT did not reach saturation at high intensities, in contrast to the values in menB mutant. The obtained results suggest the immediate phylloquinone involvement in the light-induced O2 reduction by PSI.