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Engineering an [FeFe]-hydrogenase: do accessory clusters influence O$_2$ resistance and catalytic bias ?

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Caserta, Giorgio | Papini, Cécilia | Adamska-Venkatesh, Agnieszka | Pecqueur, Ludovic | Sommer, Constanze | Reijerse, Edward | Lubitz, Wolfgang | Gauquelin, Charles | Meynial Salles, Isabelle | Pramanik, Debajyoti | Artero, Vincent | Atta, Mohamed | del Barrio, Melisa | Faivre, Bruno | Fourmond, Vincent | Léger, Christophe | Fontecave, Marc

Edité par CCSD ; American Chemical Society -

International audience. [FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H$_2$. However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from $Megasphaera\ elsdenii$ (MeHydA) displays significant resistance to O$_2$. Further-more, we produced a shorter version of this enzyme (MeH-HydA), lacking the N-terminal domain harbouring the accessory FeS clusters. As shown by detailed spectroscopic and biochemical characterization, MeH-HydA displays the following interesting properties. First, a functional active site can be assembled in MeH-HydA $in\ vitro$, providing the enzyme with excellent hydrogenase activity. Second, the resistance of MeHydA to O$_2$ is conserved in MeH-HydA. Third, MeH-HydA is more biased towards proton reduction than MeHydA, as the result of the truncation changing the rate limiting steps in catalysis. This work shows that it is possible to engineer HydA to generate an active hydrogenase that combines the resistance of the most resistant HydAs and the simplicity of algal HydAs, containing only the H-cluster.

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