Reactivation and long-term stabilization of the [NiFe] Hox hydrogenase of Synechocystis sp. PCC6803 by glutathione after oxygen exposure.

Hydrogenases are key enzymes forming or consuming hydrogen. The inactivation of these transition metal biocatalysts with oxygen limits their biotechnological applications. Oxygen-sensitive hydrogenases are distinguished from oxygen-insensitive (tolerant) ones by their initial hydrogen turnover rates influenced by oxygen. Several hydrogenases, such as the oxygen-sensitive bidirectional [NiFe] Hox hydrogenase (Hox) of the unicellular cyanobacterium Synechocystis sp. PCC6803, are reactivated after oxygen-induced deactivation by redox mechanisms. In cyanobacteria, the glutathione (GSH) redox buffer majorly controls intracellular redox potentials. The relationship between Hox turnover rates and the redox potential in its natural reaction environment is not fully understood. We thus determined hydrogen oxidation rates as activities of Hox in cell-free extracts of Synechocystis using benzyl viologen as artificial electron acceptor. We found that GSH modulates Hox hydrogen oxidation rates under oxygen-free conditions. After oxygen exposure, it influences the maximal turnover rate and aids in the reactivation of Hox. Moreover, GSH stabilizes the long-term Hox activity under anoxic conditions and attenuates oxygen-induced deactivation of Hox in a concentration dependent manner, probably by fostering reactivation. Conversely, oxidized GSH (GSSG) negatively affects Hox activity and oxygen insensitivity. Using Blue Native PAGE followed by mass spectrometry, we showed that oxygen affects Hox complex integrity. The in-silico predicted structure of the Hox complex and complexome analyses reveal the formation of various Hox subcomplexes under different conditions. Our findings refine our current classification of oxygen-hydrogenase interactions beyond sensitive and insensitive, which is particularly important for understanding hydrogenase function under physiological conditions in future.