Mechanistic Basis and Multiscale Engineering of [FeFe]-Hydrogenases for Aerobic Biocatalysis

[FeFe]-hydrogenases are among nature’s most efficient catalysts for hydrogen production, operating at high turnover frequencies under mild conditions. However, their extreme sensitivity to oxygen has limited their practical application. In this review, we comprehensively examine the molecular basis of oxygen sensitivity in [FeFe]-hydrogenases, highlighting recent advances in understanding the innate tolerance mechanisms discovered in rare homologs, such as those from Clostridium beijerinckii ( Cb A5H) and Thermosediminibacter oceani ( To HydA). These enzymes employ dynamic structural adaptations including flexible peptide loops (e.g., TSC-loop), hydrophobic barriers, and sacrificial residues to reversibly transition into oxygen-protected states (H inact ). We also evaluate innovative engineering strategies aimed at enhancing oxygen stability, such as rational design of gas-diffusion channels, domain shuffling, and encapsulation within protective matrices. Finally, we discuss the potential of emerging computational design, de novo design, and ancestral sequence reconstruction techniques on constructing O 2 -tolerant [FeFe]- hydrogenases, along with emerging applications in biocatalysis and bioenergy. By integrating mechanistic insights with protein engineering and materials science, this review outlines a roadmap for developing robust [FeFe]-hydrogenases for real-world applications. • [FeFe]-hydrogenases show ultra-high H₂ catalysis but are severely limited by O₂ sensitivity. • Decoding O₂ inactivation reveals the fundamental limits of [FeFe]-hydrogenases and guides engineering and biomaterial protection strategies. • Combined approaches enable stable aerobic hydrogenase operation, unlocking synthesis and biomedicine applications.