Cyanobacterial flv4-2 operon-encoded proteins optimize light harvesting and charge separation in photosystem II

Photosystem II (PSII) complexes drive the water splitting reaction necessary to transform sunlight into chemical energy. However, too much light can damage and disrupt PSII. In cyanobacteria, the flv4-2 operon encodes three proteins (Flv2, Flv4 and Sll0218), which safeguard PSII activity under air-level CO2 and in high-light conditions. However, the exact mechanism of action of these proteins has not been clarified yet. We demonstrate that the PSII electron transfer properties are influenced by the flv4-2 operon-encoded proteins. Accelerated secondary charge separation kinetics was observed upon expression/overexpression of the flv4-2 operon. This is likely induced by docking of the Flv2/Flv4 heterodimer in the vicinity of the QB pocket of PSII which, in turn, increases the QB redox potential and consequently stabilizes forward electron transfer. The alternative electron transfer route constituted by Flv2/Flv4 sequesters electrons from QB - guaranteeing the dissipation of excess excitation energy in PSII under stressful conditions. In addition, we demonstrate that in the absence of the flv4-2 operonencoded proteins about 20% of the phycobilisome antenna becomes detached from the reaction centers, thus decreasing light harvesting. Phycobilisome detachment is a consequence of a decreased relative content of PSII dimers, a feature observed in the absence of the Sll0218 protein

Dissecting the Photoprotective Mechanism Encoded by the flv4-2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization

In Synechocystis sp. PCC 6803, the flv4-2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4-2 operon mutants. In the sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with sll0218-flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the sll0218 and sll0218-flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218-lacking mutants. The YFP-tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA-defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the flv4-2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting. This work clarifies and dissects the roles of the flv4-2 operon-encoded proteins, Flv2/Flv4 heterodimer and the elusive Sll0218, in photoprotection of the photosynthetic apparatus in Synechosystis. While Flv2/Flv4 heterodimer is involved in an alternative electron transfer route, the Sll0218 protein is localized to specific cell compartments where photosynthetic complexes are assembled, and it is involved in the stabilization of Photosystem II complexes

Cyanobacterial Light-Harvesting Phycobilisomes Uncouple From Photosystem I During Dark-To-Light Transitions

Photosynthetic organisms cope with changes in light quality by balancing the excitation energy flow between photosystems I (PSI) and II (PSII) through a process called state transitions. Energy redistribution has been suggested to be achieved by movement of the light-harvesting phycobilisome between PSI and PSII, or by nanometre scale rearrangements of the recently discovered PBSPSII- PSI megacomplexes. The alternative ‘spillover’ model, on the other hand, states that energy redistribution is achieved by mutual association/dissociation of PSI and PSII. State transitions have always been studied by changing the redox state of the electron carriers using electron transfer inhibitors, or by applying illumination conditions with different colours. However, the molecular events during natural dark-to-light transitions in cyanobacteria have largely been overlooked and still remain elusive. Here we investigated changes in excitation energy transfer from phycobilisomes to the photosystems upon dark-light transitions, using picosecond fluorescence spectroscopy. It appears that megacomplexes are not involved in these changes, and neither does spillover play a role. Instead, the phycobilisomes partly energetically uncouple from PSI in the light but hardly couple to PSII

Flavodiiron proteins Flv1 and Flv3 enable cyanobacterial growth and photosynthesis under fluctuating light

Cyanobacterial flavodiiron proteins (FDPs; A-type flavoprotein, Flv) comprise, besides the beta-lactamase-like and flavodoxin domains typical for all FDPs, an extra NAD(P)H:flavin oxidoreductase module and thus differ from FDPs in other Bacteria and Archaea. Synechocystis sp. PCC 6803 has four genes encoding the FDPs. Flv1 and Flv3 function as an NAD(P) H: oxygen oxidoreductase, donating electrons directly to O-2 without production of reactive oxygen species. Here we show that the Flv1 and Flv3 proteins are crucial for cyanobacteria under fluctuating light, a typical light condition in aquatic environments. Under constant-light conditions, regardless of light intensity, the Flv1 and Flv3 proteins are dispensable. In contrast, under fluctuating light conditions, the growth and photosynthesis of the Delta flv1(A) and/or Delta flv3(A) mutants of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 become arrested, resulting in cell death in the most severe cases. This reaction is mainly caused by malfunction of photosystem I and oxidative damage induced by reactive oxygen species generated during abrupt short-term increases in light intensity. Unlike higher plants that lack the FDPs and use the Proton Gradient Regulation 5 to safeguard photosystem I, the cyanobacterial homolog of Proton Gradient Regulation 5 is shown not to be crucial for growth under fluctuating light. Instead, the unique Flv1/Flv3 heterodimer maintains the redox balance of the electron transfer chain in cyanobacteria and provides protection for photosystem I under fluctuating growth light. Evolution of unique cyanobacterial FDPs is discussed as a prerequisite for the development of oxygenic photosynthesis

Spectrally decomposed dark-to-light transitions in Synechocystis sp. PCC 6803

Photosynthetic activity and respiration share the thylakoid membrane in cyanobacteria. We present a series of spectrally resolved fluorescence experiments where whole cells of the cyanobacterium Synechocystis sp. PCC 6803 and mutants thereof underwent a dark-to-light transition after different dark-adaptation (DA) periods. Two mutants were used: (i) a PSI-lacking mutant (ΔPSI) and (ii) M55, a mutant without NAD(P)H dehydrogenase type-1 (NDH-1). For comparison, measurements of the wild-type were also carried out. We recorded spectrally resolved fluorescence traces over several minutes with 100 ms time resolution. The excitation light was at 590 nm so as to specifically excite the phycobilisomes. In ΔPSI, DA time has no influence, and in dichlorophenyl-dimethylurea (DCMU)-treated samples we identify three main fluorescent components: PB–PSII complexes with closed (saturated) RCs, a quenched or open PB–PSII complex, and a PB–PSII ‘not fully closed.’ For the PSI-containing organisms without DCMU, we conclude that mainly three species contribute to the signal: a PB–PSII–PSI megacomplex with closed PSII RCs and (i) slow PB → PSI energy transfer, or (ii) fast PB → PSI energy transfer and (iii) complexes with open (photochemically quenched) PSII RCs. Furthermore, their time profiles reveal an adaptive response that we identify as a state transition. Our results suggest that deceleration of the PB → PSI energy transfer rate is the molecular mechanism underlying a state 2 to state 1 transition