H₂-dependent reduction of rubredoxin by FrhAGB hydrogenase in Thermococcus onnurineus NA1

The oxidative stress defense mechanism in the order Thermococcales functions through an electron-transfer cascade that transfers electrons from NAD(P)H to various antioxidant enzymes, utilizing rubredoxin (Rd) as the electron carrier. Thermococcus onnurineus NA1 possesses a unique soluble hydrogenase encoded by the frhAGB genes. This hydrogenase has been reported to mediate direct electron transfer from H₂ oxidation through protein-protein interactions. In this study, it was found that the FrhAGB hydrogenase can directly reduce Rd using H₂ as an electron source. Additionally, its subcomplex, FrhAG, also reduced Rd. Binding affinity, as measured by surface plasmon resonance, showed strong interactions between both FrhAGB and FrhAG with Rd, with similar binding constants. In the structural model predicted by AlphaFold 3, the [4Fe-4S] clusters in FrhG and the mononuclear Fe center in Rd are arranged at distances allowing for sequential electron transfer. This research identifies a novel pathway for supplying electrons to Rd and elucidates the role of FrhAGB hydrogenase in the oxidative stress defense system. KEY POINTS: • Identification of direct electron transfer between FrhAGB hydrogenase and rubredoxin. • Quantitative analysis of protein-protein interactions between FrhAGB and rubredoxin. • The role of hydrogenase in a H₂-driven oxidative stress defense mechanism.