Nickel incorporation into hydrogenase 3 from Escherichia coli requires the precursor form of the large subunit

Abstract

A mutant derivative of hycE, the gene for the large subunit of hydrogenase 3 from Escherichia coli, was constructed that lacks the 3′-terminal part encoding the C-terminal portion of the HycE polypeptide, which is proteolytically removed during maturation of the hydrogenase. The truncated gene was transferred to the in situ position on the chromosome. Although the mutant possessed HycE in its "mature" form, it was devoid of hydrogenase 3 activity. The activity was not restored by high nickel concentrations in the medium. The mutated HycE was not associated with detectable radioactivity when the strain was grown in the presence of ⁶³Ni²⁺. These results indicate that the C-terminal extension in the precursor form of the large subunit keeps the protein in a conformation required for the coordination of the metal.