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Engineering an [FeFe]-Hydrogenase: Do Accessory Clusters Influence O 2 Resistance and Catalytic Bias?

2018; American Chemical Society; Volume: 140; Issue: 16 Linguagem: Inglês

10.1021/jacs.8b01689

1943-2984

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

Metal-Catalyzed Oxygenation Mechanisms

[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H2. 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 O2. Furthermore, we produced a shorter version of this enzyme (MeH-HydA), lacking the N-terminal domain harboring 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 O2 is conserved in MeH-HydA. Third, MeH-HydA is more biased toward 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.