Biochemical and Mechanistic Insights into O2 Scavenging and Stability of the Soluble Hydrogenase from Hydrogenophaga pseudoflava.

Bidirectional hydrogenases are unique enzymes capable of regenerating costly cofactors such as NAD-(P)H using H2, making them highly attractive for biotechnological applications. The most studied example, the soluble hydrogenase fromCupriavidus necator-(CnSH), is limited by low stability, and poor activity for cofactors beyond NAD+. In this study, we produced, purified, and characterized the soluble hydrogenase from the aerobic carboxydotrophic Knallgas bacteriumHydrogenophaga pseudoflava-(HpSH) and benchmarked it against CnSH. HpSH exhibits a broader cofactor spectrum than CnSH, including H2-driven reduction of NADP+, FMN, and FAD. It also demonstrates superior stability, retaining its activity for over 72 h at 30 °C. We investigated the effect of O2 on HpSH activity and found that it is an O2-tolerant enzyme, as it catalytically scavenges O2 before initiating the reduction of the cofactor. This reactivation mechanism is distinct, requiring both H2 and NADH for effective O2 detoxification. We validated the utility of HpSH in cofactor regeneration, achieving a total turnover number (TTN) of 2.23 × 105, underscoring its promise for biotechnological applications, particularly in microaerobic and anaerobic settings.