The repair of photosystem II (PSII) is particularly sensitive to oxidative stress and the inhibition of repair is associated with oxidative damage to the translational elongation system in the cyanobacterium sp. PCC 6803. However, the molecular mechanisms underlying this inhibition are unknown. We previously demonstrated in vitro that EF-Tu, a translation factor that delivers aminoacyl-tRNA to the ribosome, is inactivated by reactive oxygen species via oxidation of the Cys residue Cys-82. In this study, we examined the physiological role of the oxidation of EF-Tu in Under strong light, EF-Tu was rapidly oxidized to yield oxidized monomers in vivo. We generated a transformant that expressed mutated EF-Tu in which Cys-82 had been replaced with a Ser residue. Under strong light, the de novo synthesis of proteins that are required for PSII repair, such as D1, was enhanced in the transformant and photoinhibition of PSII was alleviated. However, photodamage to PSII, measured in the presence of lincomycin, was similar between the transformant and wild-type cells, suggesting that expression of mutated EF-Tu might enhance the repair of PSII. Alleviating photoinhibition through mutation of EF-Tu did not alter cell growth under strong light, perhaps due to the enhanced production of reactive oxygen species. These observations suggest that the oxidation of EF-Tu under strong light inhibits PSII repair, resulting in the stimulation of photoinhibition.

Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP-and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H 2 O 2 , whereas GDPbound EF-Tu was resistant to H 2 O 2 . The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H 2 O 2 , confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.

Carotenoids are important components of antioxidative systems in photosynthetic organisms. We investigated the roles of zeaxanthin and echinenone in the protection of PSII from photoinhibition in Synechocystis sp. PCC 6803, using mutants of the cyanobacterium that lack these carotenoids. The activity of PSII in mutant cells deficient in either zeaxanthin or echinenone was more sensitive to strong light than the activity in wild-type cells, and the activity in mutant cells deficient in both carotenoids was hypersensitive to strong light, indicating that the absence of these carotenoids increased the extent of photoinhibition. Nonetheless, the rate of photodamage to PSII, as measured in the presence of chloramphenicol, which blocks the repair of PSII, was unaffected by the absence of either carotenoid, suggesting that these carotenoids might act by protecting the repair of PSII. Knockout of the gene for the so-called orange carotenoid protein (OCP), in which the 3'-hydroxyechinenone cofactor, a derivative of echinenone, is responsible for the thermal dissipation of excitation energy, increased the extent of photoinhibition but did not affect photodamage, suggesting that thermal dissipation also protects the repair of PSII. In mutant cells lacking OCP, as well as those lacking zeaxanthin and echinenone, the production of singlet oxygen was stimulated and the synthesis de novo of various proteins, including the D1 protein, was markedly suppressed under strong light. These observations suggest that the carotenoids and thermal dissipation might protect the repair of photodamaged PSII by depressing the levels of singlet oxygen that inhibits protein synthesis.

In photosynthetic organisms, the repair of photosystem II (PSII) is enhanced after acclimation to strong light, with the resultant mitigation of photoinhibition of PSII. We previously reported that oxidation of translation elongation factor EF-Tu, which delivers aminoacyl-tRNA to the ribosome, depresses the repair of PSII in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we investigated the role of EF-Tu in the repair of PSII after acclimation of Synechocystis to strong light. In cells that had been grown under strong light, both the repair of PSII and the synthesis of proteins de novo were enhanced under strong light, with the resultant mitigation of photoinhibition of PSII. Moreover, levels of EF-Tu were elevated, whereas levels of other components of the translation machinery, such as translation factor EF-G and ribosomal proteins L2 and S12, did not change significantly. The expression of the gene for EF-Tu was induced by light, as monitored at the transcriptional level. Elevation of the level of EF-Tu was strongly correlated with the subsequent enhancement of PSII repair in cells that had been grown under light at various intensities. Furthermore, overexpression of EF-Tu in Synechocystis enhanced protein synthesis and PSII repair under strong light, even after cell culture under nonacclimating conditions. These observations suggest that elevation of the level of EF-Tu might be a critical factor in enhancing the capacity for repair of PSII that develops during acclimation to strong light.

Free fatty acids (FFA) generated in cyanobacterial cells can be utilized for the biodiesel that is required for our sustainable future. The combination of FFA and strong light induces severe photoinhibition of photosystem II (PSII), which suppresses the production of FFA in cyanobacterial cells. In the present study, we examined the effects of exogenously added FFA on the photoinhibition of PSII in Synechocystis sp. PCC 6803. The addition of lauric acid (12:0) to cells accelerated the photoinhibition of PSII by inhibiting the repair of PSII and the de novo synthesis of D1. α-Linolenic acid (18:3) affected both the repair of and photodamage to PSII. Surprisingly, palmitic (16:0) and stearic acids (18:0) enhanced the repair of PSII by accelerating the de novo synthesis of D1 with the mitigation of the photoinhibition of PSII. Our results show chemical potential of FFA in the regulation of PSII without genetic manipulation.