The in-vitro enhancement of FeFe hydrogenase activity by superoxide dismutase

H₂ producing micro-algae such as Chlamydomonas reinhardtii (C. reinhardtii), express the strictly anaerobic enzyme FeFe-hydrogenase (HydA). In this study, we examined whether superoxide dismutase (SOD), an antioxidant, can protect HydA under aerobic conditions. We conducted in-vitro assays using purified enzymes, to analyze the activity of HydA in the presence or absence of SOD. We observed that SOD enhances HydA activity both in the presence and absence of oxygen, showing that the SOD effect on HydA activity is not strictly oxygen dependent. Furthermore, we found that SOD boosts Ferredoxin-NADP⁺-oxidoreductase (FNR) NDAP⁺ reduction but not NADPH oxidation i.e. diaphorase activity. Thus, suggesting a mechanism involving proton transfer rather than electron transfer. Based on these findings, we constructed a HydA-SOD fusion protein that further boosted hydrogen production by HydA. The HydA-SOD photosynthetic activity was enhanced by 300%, reaching 700 μmol H₂ (mg [chl] hr)⁻¹, which is the fastest photosynthetic rate ever reported for an algal HydA.