Regulation of Translation by the Redox State of Elongation Factor G in the Cyanobacterium Synechocystis sp. PCC 6803

Elongation factor G (EF-G), a key protein in translational elongation, was identified as a primary target of inactivation by reactive oxygen species within the translational machinery of the cyanobacterium Synechocystis sp. PCC 6803 (Kojima, K., Oshita, M., Nanjo, Y., Kasai, K., Tozawa, Y., Hayashi, H., and Nishiyama, Y. (2007) Mol. Microbiol. 65, 936–947). In the present study, we found that inactivation of EF-G (Slr1463) by H₂O₂ was attributable to the oxidation of two specific cysteine residues and formation of a disulfide bond. Substitution of these cysteine residues by serine residues protected EF-G from inactivation by H₂O₂ and allowed the EF-G to mediate translation in a translation system in vitro that had been prepared from Synechocystis. The disulfide bond in oxidized EF-G was reduced by thioredoxin, and the resultant reduced form of EF-G regained the activity to mediate translation in vitro. Western blotting analysis showed that levels of the oxidized form of EF-G increased under strong light in a mutant that lacked NADPH-thioredoxin reductase, indicating that EF-G is reduced by thioredoxin in vivo. These observations suggest that the translational machinery is regulated by the redox state of EF-G, which is oxidized by reactive oxygen species and reduced by thioredoxin, a transmitter of reducing signals generated by the photosynthetic transport of electrons.Reactive oxygen species (ROS)² are produced as inevitable by-products of the light-driven reactions of photosynthesis. The superoxide radical, hydrogen peroxide (H₂O₂), and the hydroxyl radical are produced as a result of the photosynthetic transport of electrons, whereas singlet state oxygen (singlet oxygen) is produced by the transfer of excitation energy (1). Exposure of the photosynthetic machinery to strong light promotes the production of ROS and gives rise to oxidative stress (1).Strong light rapidly inactivates photosystem II (PSII). This phenomenon is referred to as photoinhibition (2–4), and it occurs when the rate of photodamage to PSII exceeds the rate of the repair of photodamaged PSII (5). The actions of ROS in the photoinhibition of PSII have been studied extensively, and several mechanisms for photoinhibition have been proposed (5). Recent studies of the effects of ROS on photodamage and repair have revealed that ROS act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly (5–9). Such studies have also shown that photodamage to PSII is an exclusively light-dependent event; photodamage is initiated by disruption of the manganese cluster of the oxygen-evolving complex upon absorption of light, in particular UV and blue light, with subsequent damage to the reaction center upon absorption of visible light by chlorophylls (10–12).Inhibition of the repair of PSII has been attributed to the suppression, by ROS, of the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein, which forms a heterodimer with the D2 protein in the reaction center, in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) (6, 7), in Chlamydomonas (13), and in plants (14, 15). Analysis of polysomes in Synechocystis has demonstrated that ROS inhibit the synthesis de novo of proteins primarily at the elongation step of translation, suggesting that some proteins involved in translational elongation might be the targets of inactivation by ROS (6, 7).A translation system in vitro was successfully prepared from Synechocystis, and biochemical investigations using this translation system have revealed that elongation factor G (EF-G), a GTP-binding protein that catalyzes the translocation of peptidyl-tRNA (16), is a primary target of inactivation by ROS (17). EF-G is reversibly inactivated by ROS in a redox-dependent manner; it is inactive in the oxidized form and active in the reduced form (17). Moreover, it has been proposed that changes in the activity of EF-G might depend on and be regulated by the redox states of cysteine residues within EF-G (17). However, the specific cysteine residues within EF-G that might be the targets of ROS and might be responsible for redox regulation remain to be determined.In the present study, we investigated the redox state of Slr1463, the EF-G that is phylogenetically closest to chloroplast EF-G among three homologs of EF-G in Synechocystis (17). We determined that two specific cysteine residues in the EF-G of Synechocystis were targets of oxidation by ROS. The resultant disulfide bond between the two cysteine residues was efficiently reduced by thioredoxin. In addition, we observed that EF-G was reduced by thioredoxin in vivo. Our findings revealed the mechanism of the ROS-induced inactivation of EF-G and suggested a mechanism for the redox regulation of translation by electrons generated during photosynthesis.