From oxygen detoxification to peroxide supply: Microbial NADH oxidases in redox physiology and H₂O₂-coupled biocatalysis.

NADH oxidases (NOXs) are flavin adenine dinucleotide (FAD)-dependent flavoproteins widely distributed in bacteria, archaea, and other microorganisms, where they couple NADH oxidation to oxygen reduction and contribute to redox homeostasis, oxygen adaptation, and peroxide metabolism. H₂O-forming NOX has traditionally been preferred for cofactor regeneration, whereas H₂O₂-forming NOX is increasingly recognized as a dual-function catalyst for NAD⁺ regeneration and in situ H₂O₂ production. This review summarizes the catalytic mechanisms, structural basis, and physiological functions of NOX, focusing on the biological and engineering potential of H₂O₂-forming NOX for in situ H₂O₂ generation and controlled utilization. It also discusses strategies for enhancing stability through protein engineering and process integration, particularly in peroxide-dependent cascade systems, using the H₂O₂-forming NOX/unspecific peroxygenase (UPO) cascade as a representative model. Finally, it highlights how native microbial redox functions can inspire NOX-centered H₂O₂-coupled biocatalytic systems that integrate cofactor regeneration, oxidant supply, and peroxide-flux control.