Tetrathionate hydrolase (TTH) is a key enzyme for oxidation of reduced inorganic sulfur compounds (RISCs) with S4I pathway, which is distributed in autotrophic or facultative auto-trophic sulfur-oxidizing bacteria and archaea. In this study, the enzyme TTHMc from the aci-dothermophilic archaeon Metallosphaera cuprina Ar-4T, encoded by mcup_1281 and belonging to the pyrroloquinoline quinone (PQQ) family, has been shown to possess tetrathionate hydrolysis ac-tivity. The molecular mass of the single subunit of TTHMc was determined to be 57 kDa. TTHMc is proved to locate in the cytoplasm, periplasmic space, and membrane, the activity of them ac-counted for 72.3%, 24.0%, and 3.7% of the total activity. Optimal activity was observed at tem-peratures above 95 °C and pH 6.0, the kinetic constants Km and Vmax were 0.35 mmol/L and 8.63 μmol/L, respectively. The presence of 0.01 mol/L Mg2+ enhances the activity of TTHMc, while 0.01 mol/L Ca2+ inhibits its activity. The hydrolysis of tetrathionate (TT) by TTHMc results in the pro-duction of thiosulfate, pentathionate, and hexathionate. This study represents the first description of TTH in the genus Metallosphaera, providing new theoretical insights into the study of sul-fur-oxidizing proteins in acidothermophilic archaea.

Tetrathionate hydrolase (TTH) is a key enzyme for the oxidation of reduced inorganic sulfur compounds (RISCs) with the S₄I pathway, which is distributed in autotrophic or facultative autotrophic sulfur-oxidizing bacteria and archaea. In this study, the enzyme TTH_Mc from the acidothermophilic archaeon Metallosphaera cuprina Ar-4^T, encoded by mcup_1281 and belonging to the pyrroloquinoline quinone (PQQ) family, has been shown to possess tetrathionate hydrolysis activity. The molecular mass of the single subunit of TTH_Mc was determined to be 57 kDa. TTH_Mc is proved to be located in the cytoplasm, periplasmic space, and membrane, and the activity of them accounted for 72.3%, 24.0%, and 3.7% of the total activity. Optimal activity was observed at temperatures above 95 °C and pH 6.0, and the kinetic constants K_m and V_max were 0.35 mmol/L and 86.3 μmol/L, respectively. The presence of 0.01 mol/L Mg²⁺ enhances the activity of TTH_Mc, while 0.01 mol/L Ca²⁺ inhibits its activity. The hydrolysis of tetrathionate (TT) by TTH_Mc results in the production of thiosulfate, pentathionate, and hexathionate. This study represents the first description of TTH in the genus Metallosphaera, providing new theoretical insights into the study of sulfur-oxidizing proteins in acidothermophilic archaea.