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

Year: 2025

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide 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 from Cupriavidus necator ( Cn 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 Hydrogenophaga pseudoflava ( Hp SH) and benchmarked it against Cn SH. Hp SH exhibits a broader cofactor spectrum than Cn SH, 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 Hp SH 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 Hp SH 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.

Source: ACS Omega

DOI: 10.1021/acsomega.5c09877

Authors: Dominik L. Siebert, Simon Grieshaber, Bastian Blombach, Volker Sieber, Ammar Al-Shameri

Institution: Technical University of Munich

Language: English

Topic: Metalloenzymes and iron-sulfur proteins

Subfield: Renewable Energy, Sustainability and the Environment

Field: Energy

Domain: Physical Sciences

SDG: Affordable and clean energy

Open Access status: gold

Estimated APC paid: $1,935