LAUSR

[FeFe]-hydrogenases are complex metalloenzymes, key to microbial energy metabolism in numerous organisms. During anaerobic metabolism, they dissipate excess reducing equivalents by using protons from water as terminal electron acceptors, leading to hydrogen production. This reaction is coupled to reoxidation of specific redox partners [ferredoxins, NAD(P)H or cytochrome c3], that can be used either individually or simultaneously (via flavin-based electron bifurcation). [FeFe]-hydrogenases also serve additional physiological functions such as H2 uptake (oxidation), H2 sensing, and CO2 fixation. This broad functional spectrum is enabled by a modular architecture and vast genetic diversity, which is not fully explored and understood. This Mini Review summarises recent advancements in identifying and characterising novel [FeFe]-hydrogenases, which has led to expanding our understanding of their multiple roles in metabolism and functional mechanisms. For example, while numerous well-known [FeFe]-hydrogenases are irreversibly damaged by oxygen, some newly discovered enzymes display intrinsic tolerance. These findings demonstrate that oxygen sensitivity varies between different [FeFe]-hydrogenases: in some cases, protection requires the presence of exogenous compounds such as carbon monoxide or sulphide, while in other cases it is a spontaneous built-in mechanism that relies on a reversible conformational change. Overall, it emerges that additional research is needed to characterise new [FeFe]-hydrogenases as this will reveal further details on the physiology and mechanisms of these enzymes that will enable potential impactful applications.