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

## TL;DR

Researchers studied a hydrogenase enzyme from Hydrogenophaga pseudoflava and found it to be more stable and versatile than a similar enzyme from Cupriavidus necator.

## Contribution

The study introduces a more stable and O2-tolerant hydrogenase with broader cofactor activity for biotech applications.

## Key findings

- HpSH shows broader cofactor activity than CnSH, including NADP+, FMN, and FAD.
- HpSH retains activity for over 72 hours at 30°C, indicating superior stability.
- HpSH catalytically scavenges O2 using H2 and NADH, enabling O2 tolerance and cofactor regeneration.

## Abstract

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.