Fidelity of metal insertion into hydrogenases

The fidelity of metal incorporation into the active center of hydrogenase 3 from Escherichia coli was studied by analyzing the inhibition of the matur ation pathway by zinc and other transition metals. Hydrogenase maturation o f wild-type cells was significantly affected only by concentrations of zinc or cadmium higher than 200 muM, whereas a mutant,vith a lesion in the nick el uptake system displayed a total blockade of the proteolytic processing o f the precursor form into the mature form of the large subunit after growth in the presence of 10 muM Zn2+. The precursor could not be processed in vi tro by the maturation endopeptidase even in the presence of an excess of ni ckel ions. Evidence is presented that zinc does not interfere with the inco rporation of iron into the metal center. Precursor of the large subunit acc umulated in nickel proficient cells formed a transient substrate complex wi th the cognate endoprotease HycI whereas that of zinc-supplemented cells di d not. The results show that zinc can intrude the nickel-dependent maturati on pathway only when nickel uptake is blocked, Under this condition zinc ap pears to be incorporated at the nickel site of the large subunit and delive rs a precursor not amenable to proteolytic processing since the interaction with the endoprotease is blocked. (C) 2001 Federation of European Biochemi cal Societies, Published by Elsevier Science B.V. All rights reserved.