Hydrogenases are ubiquitous metalloenzymes facilitating both hydrogen (H2) oxidation and H2 formation. However, their biotechnological applicability is hampered by oxygen sensitivity, particularly in aerobic or oxygen-evolving organisms, e.g., the unicellular cyanobacterium Synechocystis sp. PCC6803 standing out as a promising candidate for photosynthetic H2 production. It harbors Hox hydrogenase, a heteromultimeric oxygen-sensitive [NiFe] hydrogenase encoded by the hox operon. Hox is gradually inactivated by increasing oxygen concentrations and reactivated under a specific redox potential threshold or by electron mediators. Intracellularly, glutathione (GSH) and its oxidized form (GSSG) play a significant role in shaping the cellular redox potential.
Given that, we hypothesize an interplay between GSH and GSSG in the regulation of Hox activity, both in the presence and absence of oxygen. This was investigated through analyzing the impact of oxygen on the Hox complex via blue native PAGE by activity staining and complexome analysis. Additionally, H2-driven benzyl viologen reduction activity in cell-free extracts of Synechocystis was analyzed in vitro. Oxygen exposure and different additives were applied to assess their influence on activity.
Activity staining in native gels revealed that oxygen exposure led to the formation of lower molecular weight Hox subcomplexes compared to anoxic samples. GSH addition had no impact on the staining pattern. However, at the activity level, GSH and GSSG modulated the H2 oxidation activity of Hox in a reverse manner. While GSSG addition reduced maximal activity, GSH displayed a reactivating effect on Hox after oxygen exposure in a concentration dependent manner and increased the maximal activity. Alternative thiol agents dithiothreitol and cysteine exhibited a comparable effect to GSH on Hox activity in both anoxic and oxygen-exposed samples.
In summary, our results show that GSH and GSSG play a significant role in modulating the maximal activity and oxygen tolerance of the oxygen-sensitive Hox hydrogenase of Synechocystis in vitro, which enhances our understanding of the redox buffering function of GSH in vivo.