FLAVODIIRON2 and FLAVODIIRON4 Proteins Mediate an Oxygen-Dependent Alternative Electron Flow in Synechocystis sp. PCC 6803 under CO2-Limited Conditions

This study aims to elucidate the molecular mechanism of an alternative electron flow (AEF) functioning under suppressed (CO₂-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II Y(II)], reaches a maximum shortly after the onset of actinic illumination. Thereafter, Y(II) transiently decreases concomitantly with a decrease in the photosynthetic oxygen evolution rate and then recovers to a rate that is close to the initial maximum. These results show that CO₂ limitation suppresses photosynthesis and induces AEF. In contrast to the wild type, Synechocystis sp. PCC 6803 mutants deficient in the genes encoding FLAVODIIRON2 (FLV2) and FLV4 proteins show no recovery of Y(II) after prolonged illumination. However, Synechocystis sp. PCC 6803 mutants deficient in genes encoding proteins functioning in photorespiration show AEF activity similar to the wild type. In contrast to Synechocystis sp. PCC 6803, the cyanobacterium Synechococcus elongatus PCC 7942 has no FLV proteins with high homology to FLV2 and FLV4 in Synechocystis sp. PCC 6803. This lack of FLV2/4 may explain why AEF is not induced under CO₂-limited photosynthesis in S. elongatus PCC 7942. As the glutathione S-transferase fusion protein overexpressed in Escherichia coli exhibits NADH-dependent oxygen reduction to water, we suggest that FLV2 and FLV4 mediate oxygen-dependent AEF in Synechocystis sp. PCC 6803 when electron acceptors such as CO₂ are not available.In photosynthesis, photon energy absorbed by PSI and PSII in thylakoid membranes oxidizes the reaction center chlorophylls (Chls), P700 in PSI and P680 in PSII, and drives the photosynthetic electron transport (PET) system. In PSII, water is oxidized to oxygen as the oxidized P680 accepts electrons from water. These electrons then reduce the cytochrome b₆/f complex through plastoquinone (PQ) in the thylakoid membranes. Photooxidized P700 in PSI accepts electrons from the reduced cytochrome b₆/f complex through plastocyanin or cytochrome c₆. Electrons released in the photooxidation of P700 are used to produce NADPH through ferredoxin and ferredoxin NADP⁺ reductase. Thus, electrons flow from water to NADPH in the so-called photosynthetic linear electron flow (LEF). Importantly, LEF induces a proton gradient across the thylakoid membranes, which provides the driving force for ATP production by ATP synthases in the thylakoid membranes. NADPH and ATP serve as chemical energy donors in the photosynthetic carbon reduction cycle (Calvin cycle).It recently has been proposed that, in cyanobacteria, the photorespiratory carbon oxidation cycle (photorespiration) functions simultaneously with the Calvin cycle to recover carbon for the regeneration of ribulose-1,5-bisphosphate, one of the substrates of Rubisco (Hagemann et al., 2013). Rubisco catalyzes the primary reactions of carbon reduction as well as oxidation cycles. However, the presence of a specific carbon concentration mechanism (CCM) in cyanobacteria had been thought to prevent the operation of photorespiration. CCM maintains a high concentration of CO₂ around Rubisco so that the oxygenase activity of Rubisco is suppressed (Badger and Price, 1992). However, recent studies on mutants deficient in photorespiration enzymes have shown that photorespiration functions, particularly under CO₂-limited conditions, in cyanobacteria as it does in higher plants (Eisenhut et al., 2006, 2008).Decreased consumption of NADPH under CO₂-limited or high-light conditions causes electrons to accumulate in the PET system. As a result, the photooxidation and photoreduction cycles of the reaction center Chls in PSI and PSII become uncoupled from the production of NADPH, inducing alternative electron flow (AEF) pathways (Mullineaux, 2014). In cyanobacteria, several AEFs that differ from those in higher plants are proposed to function as electron sinks (Mullineaux, 2014). Electrons accumulated in the PET system flow to oxygen through FLAVODIIRON1 (FLV1) and FLV3 proteins in PSI and the terminal oxidase, cytochrome c oxidase complex, and cytochrome bd-quinol oxidase (Pils and Schmetterer, 2001; Berry et al., 2002; Helman et al., 2003; Nomura et al., 2006; Lea-Smith et al., 2013). Cyanobacterial FLV comprises a diiron center, a flavodoxin domain with an FMN-binding site, and a flavin reductase domain (Vicente et al., 2002). In Synechocystis sp. PCC 6803, Helman et al. (2003) identified four genes encoding FLV1 to FLV4 and showed that FLV1 and FLV3 were essential for the photoreduction of oxygen by PSI. FLV1 and FLV3 were proposed to function as a heterodimer (Allahverdiyeva et al., 2013). FLV2/4 have been proposed to function in energy dissipation associated with PSII (Zhang et al., 2012). In addition, hydrogenases convert H⁺ to H₂ with NADPH as an electron donor (Appel et al., 2000). Furthermore, Flores et al. (2005) suggested that the nitrate assimilation pathway functions in AEF when the cells live in medium containing nitrate.To elucidate the physiological functions of these AEFs, evaluation of the presence and capacity of each AEF pathway is required. Therefore, in vivo analyses of electron fluxes are essential. We had found that an electron flow uncoupled from photosynthetic oxygen evolution functioned under suppressed (CO₂-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803 but not in Synechococcus elongatus PCC 7942 (Hayashi et al., 2014), indicating that an AEF operated in Synechocystis sp. PCC 6803. This AEF was induced in high-CO₂]-grown Synechocystis sp. PCC 6803 during the transition from CO₂-saturated photosynthesis to CO₂-limited photosynthesis (Hayashi et al., 2014). In contrast, in Synechocystis sp. PCC 6803 grown at ambient CO₂ concentration, AEF was detected immediately following the transition to CO₂-limited photosynthesis (Hayashi et al., 2014), suggesting that AEF was already induced under ambient atmospheric conditions.The expression of the AEF activity observed under CO₂-limited photosynthesis required the presence of oxygen in Synechocystis sp. PCC 6803 (Hayashi et al., 2014). In Synechocystis sp. PCC 6803, FLV1/3 were proposed to catalyze the photoreduction of oxygen (Helman et al., 2003). However, Hayashi et al. (2014) found no evidence that FLV1/3 operated under CO₂-limited photosynthesis: a mutant Synechocystis sp. PCC 6803 deficient in FLV1/3 maintained almost constant electron flux under CO₂-limited photosynthesis after the transition from CO₂-saturated conditions. Thus, the postulated photoreduction of oxygen by FLV1/3 was not responsible for the electron flux observed under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803.In this study, we aimed to elucidate the molecular mechanism of the oxygen-dependent AEF functioning under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. The possibility that FLV2 and FLV4 catalyze the photoreduction of oxygen under CO₂-limited photosynthesis could not be excluded, given that AEF in high-CO₂]-grown Synechocystis sp. PCC 6803 was induced following the transition to CO₂-limited photosynthesis (Hayashi et al., 2014). Both FLV2 and FLV4 are predicted to possess oxidoreductase motifs, similar to FLV1 and FLV3 (Helman et al., 2003; Zhang et al., 2012). Furthermore, the expression of two FLV genes (flv2 and flv4) was enhanced under low-CO₂] conditions (Zhang et al., 2009). Zhang et al. (2012) proposed that FLV2 and FLV4 did not donate electrons to oxygen on the basis of the finding that the Synechocystis sp. PCC 6803 mutants deficient in FLV1/3 showed no light-dependent oxygen uptake (Helman et al., 2003). However, Helman et al. (2003) cultivated Synechocystis sp. PCC 6803 strains deficient in FLV1 and FLV3 proteins under high-CO₂] conditions, and we cannot exclude the possibility that the FLV2 and FLV4 proteins were not produced in the studied cells. Taken together, it seems plausible that FLV2 and FLV4 mediate oxygen-dependent AEF following the transition to CO₂-limited photosynthesis. To evaluate this possibility, we constructed Synechocystis sp. PCC 6803 mutants deficient in flv2 and flv4 and measured their oxygen evolution and Chl fluorescence simultaneously. The mutants showed suppressed LEF after transition to CO₂-limited photosynthesis, similar to S. elongatus PCC 7942. We also tested the possibility that photorespiration functions as an electron sink under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. A recent study revealed photorespiratory oxygen uptake in a flv1/3 mutant under CO₂-depleted conditions (Allahverdiyeva et al., 2011). In this study, we found that the quantum yield of photosystem II Y(II)] of mutants deficient in genes encoding proteins that function in photorespiration was similar to that of wild-type Synechocystis sp. PCC 6803. Thus, FLV2 and FLV4 appear to function in the oxygen-dependent AEF under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. This inference is further supported by the lack of FLV2 and FLV4 homologs in the genome of S. elongatus PCC 7942 (Bersanini et al., 2014). In addition, we found oxygen-reducing activities of recombinant glutathione S-transferase (GST)-FLV4 fusion protein, similar to those of recombinant FLV3 protein (Vicente et al., 2002). In light of these results, we discuss the molecular mechanism of the oxygen-dependent AEF under CO₂-limited photosynthesis and the physiological function of FLV proteins in Synechocystis sp. PCC 6803.