Structure, function and biosynthesis of O2-tolerant hydrogenases

Key Points Molecular hydrogen (H 2 ) is used as a valuable energy source or a way to deposit excess reducing power by a wide range of microorganisms. The reversible oxidation of H 2 into protons and electrons is mediated by ancient metalloenzymes denoted hydrogenases. Most hydrogenases are highly sensitive to molecular oxygen (O 2 ) and rapidly inactivated on exposure to O 2 . However, a subgroup of [NiFe]-hydrogenases that are mainly found in aerobic H 2 -oxidizing bacteria has developed the ability to carry out H 2 cycling at ambient O 2 levels, thereby driving the chemolithoautotrophic lifestyle of their hosts. The remarkable O 2 tolerance of membrane-bound hydrogenase (MBH) proteins largely depends on a unique [4Fe–3S] centre that is coordinated by six Cys residues. The unusual redox property of this cofactor involves the storage of two electrons, thereby providing an electron-rich environment at the active site; this environment is crucial for the enzyme to recover rapidly from O 2 attack. Biochemical, spectroscopic and electrochemical analyses, as well as comparative genome screening, have revealed that some organisms encoding putatively O 2 -tolerant MBH proteins are either strict or facultative anaerobes, including Escherichia coli . The role of these proteins under anoxic conditions remains to be elucidated. Apart from dedicated electron transfer relays supporting the detoxification of O 2 , further modifications enable certain hydrogenases to function under aerobic conditions. These include customized gas diffusion, water movement and proton transfer pathways. O 2 is also a challenge for hydrogenase biosynthesis, including metal centre assembly, cofactor incorporation and membrane translocation. Therefore, MBH proteins undergo a particularly complex maturation process involving specific chaperones that protect the metal cofactors against the detrimental effects of O 2 . Robust O 2 -tolerant hydrogenases are promising biotechnological tools for H 2 -based applications such as H 2 fuel cells, sunlight-driven H 2 production, cofactor regeneration in NADH-dependent enzymatic redox reactions, and isotopic labelling of proteins and fine chemicals. The reversible oxidation of H 2 into protons and electrons is mediated by metalloenzymes known as hydrogenases. Here, Fritsch, Lenz and Friedrich discuss recent progress in our understanding of the structure, function and biosynthesis of a subtype of [NiFe]-hydrogenases mainly found in H 2 -oxidizing bacteria