Roles of ApcD and orange carotenoid protein in photoinduction of electron transport upon dark-light transition in the Synechocystis PCC 6803 mutant deficient in flavodiiron protein Flv1.

Flavodiiron proteins Flv1/Flv3 accept electrons from photosystem (PS) I. In this work we investigated light adaptation mechanisms of Flv1-deficient mutant of Synechocystis PCC 6803, incapable to form the Flv1/Flv3 heterodimer. First seconds of dark-light transition were studied by parallel measurements of light-induced changes in chlorophyll fluorescence, P700 redox transformations, fluorescence emission at 77 K, and OCP-dependent fluorescence quenching. During the period of Calvin cycle activation upon dark-light transition, the linear electron transport (LET) in wild type is supported by the Flv1/Flv3 heterodimer, whereas in Δflv1 mutant activation of LET upon illumination is preceded by cyclic electron flow that maintains State 2. The State 2-State 1 transition and Orange Carotenoid Protein (OCP)-dependent non-photochemical quenching occur independently of each other, begin in about 10 s after the illumination of the cells and are accompanied by a short-term re-reduction of the PSI reaction center (P700+). ApcD is important for the State 2-State 1 transition in the Δflv1 mutant, but S-M rise in chlorophyll fluorescence was not completely inhibited in Δflv1/ΔapcD mutant. LET in Δflv1 mutant starts earlier than the S-M rise in chlorophyll fluorescence, and the oxidation of plastoquinol (PQH2) pool promotes the activation of PSII, transient re-reduction of P700+ and transition to State 1. An attempt to induce state transition in the wild type under high intensity light using methyl viologen, highly oxidizing P700 and PQH2, was unsuccessful, showing that oxidation of intersystem electron-transport carriers might be insufficient for the induction of State 2-State 1 transition in wild type of Synechocystis under high light.

State of the phycobilisome determines effective absorption cross-section of Photosystem II in Synechocystis sp. PCC 6803.

Quenching of excess excitation energy is necessary for the photoprotection of light-harvesting complexes. In cyanobacteria, quenching of phycobilisome (PBS) excitation energy is induced by the Orange Carotenoid Protein (OCP), which becomes photoactivated under high light conditions. A decrease in energy transfer efficiency from the PBSs to the reaction centers decreases photosystem II (PS II) activity. However, quantitative analysis of OCP-induced photoprotection in vivo is complicated by similar effects of both photochemical and non-photochemical quenching on the quantum yield of the PBS fluorescence overlapping with the emission of chlorophyll. In the present study, we have analyzed chlorophyll a fluorescence induction to estimate the effective cross-section of PS II and compared the effects of reversible OCP-dependent quenching of PBS fluorescence with reduction of PBS content upon nitrogen starvation or mutations of key PBS components. This approach allowed us to estimate the dependency of the rate constant of PS II primary electron acceptor reduction on the amount of PBSs in the cell. We found that OCP-dependent quenching triggered by blue light affects approximately half of PBSs coupled to PS II, indicating that under normal conditions, the concentration of OCP is not sufficient for quenching of all PBSs coupled to PS II.

Lack of Excitation Energy Transfer from the Bacteriochlorophyll Soret Band to Carotenoids in Photosynthetic Complexes of Purple Bacteria.

The excitation energy transfer (EET) from the bacteriochlorophyll (BChl) Soret band to the second excited state(s) (S2) of carotenoids in pigment-protein complexes of purple bacteria was investigated. The efficiency of EET was determined, based on fluorescence excitation and absorption spectra of chromatophores, peripheral light-harvesting complexes (LH2), core complexes (LH1-RC), and pigments in solution. Carotenoid-containing and carotenoid-less samples were compared: LH1-RC and LH2 from Allochromatium minutissimum, Ectothiorhodospira haloalkaliphila, and chromatophores from Rhodobacter sphaeroides and Rhodospirillum rubrum wild type and carotenoid-free strains R-26 and G9. BChl-to-carotenoid EET was absent, or its efficiency was less than the accuracy of the measurements of ∼5%. Quantum chemical calculations support the experimental results: The transition dipole moments of spatially close carotenoid/BChl pairs were found to be nearly orthogonal. The structural arrangements suggest that Soret EET may be lacking for the studied systems, however, EET from carotenoids to Qx appears to be possible.

Deficiency in flavodiiron protein Flv3 promotes cyclic electron flow and state transition under high light in the cyanobacterium Synechocystis sp. PCC 6803.

Photosynthetic organisms adjust their activity to changes in irradiance by different ways, including the operation of cyclic electron flow around photosystem I (PSI) and state transitions that redistribute amounts of light energy absorbed by PSI and PSII. In dark-acclimated wild type cells of Synechocystis PCC 6803, linear electron transport was activated after the first 500 ms of illumination, while cyclic electron flow around PSI was long predominant in the mutant deficient in flavodiiron protein Flv3. Chlorophyll P700 oxidation associated with activation of linear electron flow extended in the Flv3- mutant to several tens of seconds and included a P700+ re-reduction phase. Parallel monitoring of chlorophyll fluorescence and the redox state of P700 indicated that, at low light intensity both in wild type and in the Flv3- mutant, the transient re-reduction step coincided in time with S-M fluorescence rise, which reflected state 2-state 1 transition (Kana et al., 2012). Despite variations in the initial redox state of plastoquinone pool, the oxidases-deficient mutant, succinate dehydrogenase-deficient mutant, and wild type cells did not show the S-M rise under high-intensity light until additional Flv3- mutation was introduced in these strains. Thus, the lack of available electron acceptor for PSI was the main cause for the appearance of S-M fluorescence rise under high light. It is concluded that the lack of Flv3 protein promotes cyclic electron flow around PSI and facilitates the subsequent state 2-state 1 transition in the absence of strict relation to the dark-operated pathways of plastoquinone reduction or oxidation.

Engineering the photoactive orange carotenoid protein with redox-controllable structural dynamics and photoprotective function.

Photosynthesis requires various photoprotective mechanisms for survival of organisms in high light. In cyanobacteria exposed to high light, the Orange Carotenoid Protein (OCP) is reversibly photoswitched from the orange (OCPO) to the red (OCPR) form, the latter binds to the antenna (phycobilisomes, PBs) and quenches its overexcitation. OCPR accumulation implicates restructuring of a compact dark-adapted OCPO state including detachment of the N-terminal extension (NTE) and separation of protein domains, which is reversed by interaction with the Fluorescence Recovery Protein (FRP). OCP phototransformation supposedly occurs via an intermediate characterized by an OCPR-like absorption spectrum and an OCPO-like protein structure, but the hierarchy of steps remains debatable. Here, we devise and analyze an OCP variant with the NTE trapped on the C-terminal domain (CTD) via an engineered disulfide bridge (OCPCC). NTE trapping preserves OCP photocycling within the compact protein structure but precludes functional interaction with PBs and especially FRP, which is completely restored upon reduction of the disulfide bridge. Non-interacting with the dark-adapted oxidized OCPCC, FRP binds reduced OCPCC nearly as efficiently as OCPO devoid of the NTE, suggesting that the low-affinity FRP binding to OCPO is realized via NTE displacement. The low efficiency of excitation energy transfer in complexes between PBs and oxidized OCPCC indicates that OCPCC binds to PBs in an orientation suboptimal for quenching PBs fluorescence. Our approach supports the presence of the OCPR-like intermediate in the OCP photocycle and shows effective uncoupling of spectral changes from functional OCP photoactivation, enabling redox control of its structural dynamics and function.

Hybrid complexes of photosynthetic reaction centers and quantum dots in various matrices: resistance to UV irradiation and heating.

The effects of ultraviolet (UV) irradiation (up to 0.6 J/cm2) and heating (65 °C, 20 min) on the absorption spectra and electron transfer in dehydrated film samples of photosynthetic reaction centers (RCs) from purple bacterium Rhodobacter (Rb.) sphaeroides, as well as in hybrid structures consisting of RCs and quantum dots (QDs), have been studied. The samples were placed in organic matrices containing the stabilizers of protein structure-polyvinyl alcohol (PVA) and trehalose. UV irradiation led to partially irreversible oxidation of some RCs, as well as to transformation of some fraction of the bacteriochlorophyll (BChl) molecules into bacteriopheophytin (BPheo) molecules. In addition, UV irradiation causes degradation of some BChl molecules that is accompanied by formation of 3-acetyl-chlorophyll a molecules. Finally, UV irradiation destroys the RCs carotenoid molecules. The incorporation of RCs into organic matrices reduced pheophytinization. Trehalose was especially efficient in reducing the damage to the carotenoid and BChl molecules caused by UV irradiation. Hybrid films containing RC + QD were more stable to pheophytinization upon UV irradiation. However, the presence of QDs in films did not affect the processes of carotenoid destruction. The efficiency of the electronic excitation energy transfer from QD to P865 also did not change under UV irradiation. Heating led to dramatic destruction of the RCs structure and bacteriochlorins acquired the properties of unbound molecules. Trehalose provided strong protection against destruction of the RCs and hybrid (RC + QD) complexes.

Role of the PB-loop in ApcE and phycobilisome core function in cyanobacterium Synechocystis sp. PCC 6803.

The phycobilisome (PBS) is a giant highly-structured pigment-protein antenna of cyanobacteria and red algae. PBS is composed of the phycobiliproteins and several linker polypeptides. The large core-membrane linker protein (LCM or ApcE) influences many features and functions of PBS and consists of several domains including the chromophorylated PB-domain. Being homologous to the phycobiliprotein α-subunits this domain includes a so-called PB-loop insertion whose functions are still unknown. We have created the photoautotrophic mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with lacking PB-loop. Using various spectral techniques we have demonstrated that this mutation does not destroy the PBS integrity and the internal PBS excitation energy transfer pathways. At the same time, the deletion of the PB-loop leads to the decrease of connectivity between the PBS and thylakoid membrane and to the compensatory increase of the relative photosystem II content. Mutation provokes the violation of the thylakoid membranes arrangement, the inability to perform state transitions, and diminishing of the OCP-dependent non-photochemical PBS quenching. In essence, even such a minute mutation of the PBS polypeptide component, like the PB-loop deletion, becomes important for the concerted function of the photosynthetic apparatus.

Functional interaction of low-homology FRPs from different cyanobacteria with Synechocystis OCP.

Photosynthesis requires a balance between efficient light harvesting and protection against photodamage. The cyanobacterial photoprotection system uniquely relies on the functioning of the photoactive orange carotenoid protein (OCP) that under intense illumination provides fluorescence quenching of the light-harvesting antenna complexes, phycobilisomes. The recently identified fluorescence recovery protein (FRP) binds to the photoactivated OCP and accelerates its relaxation into the basal form, completing the regulatory circle. The molecular mechanism of FRP functioning is largely controversial. Moreover, since the available knowledge has mainly been gained from studying Synechocystis proteins, the cross-species conservation of the FRP mechanism remains unexplored. Besides phylogenetic analysis, we performed a detailed structural-functional analysis of two selected low-homology FRPs by comparing them with Synechocystis FRP (SynFRP). While adopting similar dimeric conformations in solution and preserving binding preferences of SynFRP towards various OCP variants, the low-homology FRPs demonstrated distinct binding stoichiometries and differentially accentuated features of this functional interaction. By providing clues to understand the FRP mechanism universally, our results also establish foundations for upcoming structural investigations necessary to elucidate the FRP-dependent regulatory mechanism.

Phycobilisomes from the mutant cyanobacterium Synechocystis sp. PCC 6803 missing chromophore domain of ApcE.

Phycobilisome (PBS) is a giant photosynthetic antenna associated with the thylakoid membranes of cyanobacteria and red algae. PBS consists of two domains: central core and peripheral rods assembled of disc-shaped phycobiliprotein aggregates and linker polypeptides. The study of the PBS architecture is hindered due to the lack of the data on the structure of the large ApcE-linker also called LCM. ApcE participates in the PBS core stabilization, PBS anchoring to the photosynthetic membrane, transfer of the light energy to chlorophyll, and, very probably, the interaction with the orange carotenoid protein (OCP) during the non-photochemical PBS quenching. We have constructed the cyanobacterium Synechocystis sp. PCC 6803 mutant lacking 235 N-terminal amino acids of the chromophorylated PBLCM domain of ApcE. The altered fluorescence characteristics of the mutant PBSs indicate that the energy transfer to the terminal emitters within the mutant PBS is largely disturbed. The PBSs of the mutant become unable to attach to the thylakoid membrane, which correlates with the identified absence of the energy transfer from the PBSs to the photosystem II. At the same time, the energy transfer from the PBS to the photosystem I was registered in the mutant cells and seems to occur due to the small cylindrical CpcG2-PBSs formation in addition to the conventional PBSs. In contrast to the wild type Synechocystis, the OCP-mediated non-photochemical PBS quenching was not registered in the mutant cells. Thus, the PBLCM domain takes part in formation of the OCP binding site in the PBS.

Fluorescence quenching of the phycobilisome terminal emitter LCM from the cyanobacterium Synechocystis sp. PCC 6803 detected in vivo and in vitro.

The fluorescence emission of the phycobilisome (PBS) core-membrane linker protein (L(CM)) can be directly quenched by photoactivated orange carotenoid protein (OCP) at room temperature both in vitro and in vivo, which suggests the crucial role of the OCP-L(CM) interaction in non-photochemical quenching (NPQ) of cyanobacteria. This implication was further supported (i) by low-temperature (77K) fluorescence emission and excitation measurements which showed a specific quenching of the corresponding long-wavelength fluorescence bands which belong to the PBS terminal emitters in the presence of photoactivated OCP, (ii) by systematic investigation of the fluorescence quenching and recovery in wild type and L(CM)-less cells of the model cyanobacterium Synechocystis sp. PCC 6803, and (iii) by the impact of dephosphorylation of isolated PBS on the quenching. The OCP binding site within the PBS and the most probable geometrical arrangement of the OCP-allophycocyanin (APC) complex was determined in silico using the crystal structures of OCP and APC. Geometrically modeled attachment of OCP to the PBS core is not at variance with the OCP-L(CM) interaction. It was concluded that besides being a very central element in the PBS to reaction center excitation energy transfer and PBS assembly, L(CM) also has an essential role in the photoprotective light adaptation processes of cyanobacteria.

Fluorescence changes accompanying short-term light adaptations in photosystem I and photosystem II of the cyanobacterium Synechocystis sp. PCC 6803 and phycobiliprotein-impaired mutants: State 1/State 2 transitions and carotenoid-induced quenching of phycobilisomes.

The features of the two types of short-term light-adaptations of photosynthetic apparatus, State 1/State 2 transitions, and non-photochemical fluorescence quenching of phycobilisomes (PBS) by orange carotene-protein (OCP) were compared in the cyanobacterium Synechocystis sp. PCC 6803 wild type, CK pigment mutant lacking phycocyanin, and PAL mutant totally devoid of phycobiliproteins. The permanent presence of PBS-specific peaks in the in situ action spectra of photosystem I (PSI) and photosystem II (PSII), as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 690 nm (PSII) and 725 nm (PSI) showed that PBS are constitutive antenna complexes of both photosystems. The mutant strains compensated the lack of phycobiliproteins by higher PSII content and by intensification of photosynthetic linear electron transfer. The detectable changes of energy migration from PBS to the PSI and PSII in the Synechocystis wild type and the CK mutant in State 1 and State 2 according to the fluorescence excitation spectra measurements were not registered. The constant level of fluorescence emission of PSI during State 1/State 2 transitions and simultaneous increase of chlorophyll fluorescence emission of PSII in State 1 in Synechocystis PAL mutant allowed to propose that spillover is an unlikely mechanism of state transitions. Blue-green light absorbed by OCP diminished the rout of energy from PBS to PSI while energy migration from PBS to PSII was less influenced. Therefore, the main role of OCP-induced quenching of PBS is the limitation of PSI activity and cyclic electron transport under relatively high light conditions.

Conductive wiring of immobilized photosynthetic reaction center to electrode by cytochrome C.

The photosynthetic reaction center (RC) found in photosynthetic bacteria is one of the most advanced photoelectronic devices developed by nature. However, after immobilization on the electrode surface, the efficiency of electron transfer (ET) between the RC and the electrode is relatively low. This inefficiency has limited the possibility of using the RC for technological applications. Here we show that photoinduced electron transfer between the immobilized RC and a gold electrode can be increased by several tens-fold by incorporation of cytochrome c into the RC-self-assembled monolayer (SAM)-electrode complex. The effect does not depend on the initial redox state of the cytochrome and seems to be the result of the formation of a complex between the RC and the cytochrome c serving as an ET wire. This observation opens the possibility for electrochemical analysis of the special pair in the RC protein that is deeply buried inside the protein globe and is barely electrically addressable from the electrode surface.