Biochemical and Mechanistic Insights into O ₂ Scavenging and Stability of the Soluble Hydrogenase from <i>Hydrogenophaga pseudoflava</i>

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 from<i>Cupriavidus necator</i>-(<i>Cn</i>SH), 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 bacterium<i>Hydrogenophaga pseudoflava</i>-(<i>Hp</i>SH) and benchmarked it against <i>Cn</i>SH. <i>Hp</i>SH exhibits a broader cofactor spectrum than <i>Cn</i>SH, 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 <i>Hp</i>SH 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 <i>Hp</i>SH 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.