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 maturation pathway by zinc and other transition metals. Hydrogenase maturation of wild-type cells was significantly affected only by concentrations of zinc or cadmium higher than 200 microM, whereas a mutant with a lesion in the nickel uptake system displayed a total blockade of the proteolytic processing of the precursor form into the mature form of the large subunit after growth in the presence of 10 microM Zn(2+). The precursor could not be processed in vitro by the maturation endopeptidase even in the presence of an excess of nickel ions. Evidence is presented that zinc does not interfere with the incorporation of iron into the metal center. Precursor of the large subunit accumulated in nickel proficient cells formed a transient substrate complex with the cognate endoprotease HycI whereas that of zinc-supplemented cells did not. The results show that zinc can intrude the nickel-dependent maturation pathway only when nickel uptake is blocked. Under this condition zinc appears to be incorporated at the nickel site of the large subunit and delivers a precursor not amenable to proteolytic processing since the interaction with the endoprotease is blocked.
Magalon
A
A
Lehrstuhl für Mikrobiologie der Ludwig-Maximilians-Universität München, Maria-Ward-Strasse 1a, D-80638, Munich, Germany.
Blokesch
M
M
Zehelein
E
E
Böck
A
A
D001667:Binding, Competitive; D015153:Blotting, Western; D002104:Cadmium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D004792:Enzyme Precursors; D008670:Metals; D009097:Multienzyme Complexes; D002104:Cadmium; D009532:Nickel; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase; D015032:Zinc