We examined the translational activity of EF-Tu using the PURE system, an E.coli-derived in vitro translation system that contains all components essential for translation (17) . Purified wild-type EF-Tu from Synechocystis that had been treated with various concentrations of H2O2 was added to the translation system, which was constructed without EF-Tu, and observed the synthesis of DHFR. The results revealed that DHFR synthesis was significantly reduced as the H2O2 concentration increased (Fig. 1A). Only EF-Tu treated with 0.1 mM H2O2 can inactivate translational activity by approximately 80% compared to reduced EF-Tu (Fig. 1B). The results indicated that the translational activity of EF-Tu is hypersensitive to H2O2. On the contrary, when we monitored the in vitro translation of the mutated EF-Tu, which has the substitution of Cys82 with serine (cysteine ​​isosteric), on the sensitivity to oxidation by H2O2 we showed that the synthesis of DHFR was not affected in the presence of H2O2 (Figure 1C). Even though we added the concentration of H2O2 to 2 mM in the Cys82 mutant protein, the translational activity was quite constant, whereas the translational activity of wild-type EF-Tu was inactivation (Fig. 1B and 1D). These results suggested that replacement of Cys82 with serine rendered EF-Tu insensitive to inactivation by H2O2 in the translational machinery. We monitored the redox state of Cys82 of EF-Tu by thiol modification assay by modifying the free thiol group with maleimidyl reagent (molecular mass medium, 5 kDa). Changing the thiol group of reduced EF-Tu with this reagent resulted in a change in the electrophoretic mobility of EF-Tu on a non-reducing SDS-PAGE by an increase in molecular mass when co... in the center of the paper. ....wn as the G domain. It is involved in the formation of numerous hydrogen bonds with water molecules which coordinates the Mg2+ ion in the nucleotide binding pocket (22). Furthermore, Cys81 is present near the EF-Tu interaction site with the 3' end and 5' end of the aminoacyl-tRNA molecule (23,24). Supporting data previously revealed that chemical modification of Cys82 by N-tosyl-L-phenylalanylchloromethane (TosPheCH2Cl) in Thermus thermophilus EF-Tu (25,26), equivalent to Cys 81 of EF-Tu from E.coli (27,28) and Bacillus stearothermophilus (29), interfere with the formation of an appropriate pocket on EF-Tu to accommodate the aa-tRNA for the ternary complex. Replacement of Cys82 with alanine prevents alkylation of EF-Tu by TosPheCH2Cl and allows interaction with aa-tRNA, suggesting that Cys82 plays a more specific role in the former conformation.