Regeneration of ergothioneine after reaction with singlet oxygen

Ergothioneine (ET), an imidazole-2-thione derivative of histidine betaine, is generally considered an antioxidant. Important antioxidants are typically regenerated from their oxidized products, to prevent the interceptors from being lost after a single chemical reaction with a reactive oxygen species. However, no mechanism for the complete regeneration of ET has yet been uncovered. Here we define a non-enzymatic multi-step cycle for the regeneration of ET after reaction with singlet oxygen (O-1(2)). All reaction steps were verified by density functional theory computations. Four molecules of GSH are used per turn to detoxify O-1(2) to water. Pure O-1(2) was generated by thermolysis at 37 degrees C of the endoperoxide DHPNO2. Addition of 1 mM ET to 10 mM DHPNO2 and 10 mM GSH increased the production of oxidized GSH (GSSG), measured by LC-MS/MS, by a factor of 26 (water) and 28 (D2O), respectively. In the same assay, the ring of ET alone was able to drive the cycle at equal speed; thus, the zwitterionic amino acid backbone was not involved. Our data suggest that ET reacts at least 4-fold faster with O-1(2) than ascorbic acid. ET must now be viewed as tightly linked with the GSH/GSSG redox couple. The necessary thiol foundation is present in all mammalian and vertebrate cells, and also in all species that generate ET, such as cyanobacteria, mycobacteria, and fungi. Regeneration provides a decisive advantage for ET over other reactive, but non-recoverable, compounds. Our findings substantiate the importance of ET for the eradication of noxious O-1(2).