Evidence for an oxygen-sensitive iron–sulfur cluster in an immature large subunit species of Escherichia coli [NiFe]-hydrogenase 2

Endoprotease-specific C-terminal processing is required to complete the maturation of the large subunit of [NiFe]-hydrogenases. This happens only after synthesis and insertion of the NiFe(CN)2CO cofactor by the Hyp maturases has occurred. It is assumed that in the absence of maturation the unprocessed species of the large subunit lacks cofactors. In this study we isolated a variant of the hydrogenase 2 large subunit, HybC, containing a fused C-terminal pentapeptide. The polypeptide could not be processed and was unable to associate with the small subunit to deliver an active enzyme. The His6-HybC variant protein isolated was brown and had sub-stoichiometric amounts of an oxygen-sensitive Iron–sulfur cluster, which could be chemically reconstituted to a [4Fe–4S] cluster. This cluster was coordinated by the conserved cysteinyl residues that normally ligate the NiFe(CN)2CO cofactor. Our findings provide evidence for temporary promiscuity of cofactor-binding sites.

► A processed [NiFe]-hydrogenase large subunit normally has only a NiFe-cofactor.
► A five amino acid C-terminal extension prevents processing of the large subunit.
► Evidence for an FeS cluster in the unprocessed large subunit species is presented.
► The FeS cluster is oxygen-sensitive but can be fully reconstituted.
► The same cysteinyl residues ligate the NiFe-cofactor and the FeS cluster.