Atypical effect of temperature tuning on the insertion of the catalytic iron−sulfur center in a recombinant [FeFe]-hydrogenase

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The expression of recombinant [FeFe]-hydrogenases is an important step for the production of large amount of these enzymes for their exploitation in biotechnology and for the characterization of the protein-metal cofactor interactions. The correct assembly of the organometallic catalytic site, named H-cluster, requires a dedicated set of maturases that must be coexpressed in the microbial hosts or used forin vitroassembly of the active enzymes. In this work, the effect of the post-induction temperature on the recombinant expression of CaHydA [FeFe]-hydrogenase inE. coliis investigated. The results show a peculiar behavior: the enzyme expression is maximum at lower temperatures (20°C), while the specific activity of the purified CaHydA is higher at higher temperature (30°C), as a consequence of improved protein folding and active site incorporation.

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