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) 2 CO 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 His 6 -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) 2 CO cofactor. Our findings provide evidence for temporary promiscuity of cofactor-binding sites.