Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation

The interaction of the hydrogenase maturation endopeptidase HycI with its s ubstrate, the precursor of the large subunit, was studied. Replacement of c onserved amino-acid residues in HycI, which have been shown to bind a cadmi um ion from the crystallization buffer in crystals of HybD (endopeptidase f or hydrogenase 2), abolished or strongly reduced processing activity. Atomi c absorption spectroscopy of purified HycI and HybD proteins showed the abs ence of nickel. In vitro processing assays showed that the reaction require s nickel to be bound to the precursor and the protease does not have a func tion in nickel delivery to the substrate. Radioactive labelling of cells wi th Ni-63, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydro genases as a recognition motif.