Abstract
Bidirectional hydrogenases are unique enzymes capable of regenerating costly cofactors such as NAD-(P)H using H(2), 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 H(2)-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 O(2) on HpSH activity and found that it is an O(2)-tolerant enzyme, as it catalytically scavenges O(2) before initiating the reduction of the cofactor. This reactivation mechanism is distinct, requiring both H(2) and NADH for effective O(2) detoxification. We validated the utility of HpSH in cofactor regeneration, achieving a total turnover number (TTN) of 2.23 × 10(5), underscoring its promise for biotechnological applications, particularly in microaerobic and anaerobic settings.