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J. Biol. Chem., Vol. 275, Issue 28, 21114-21120, July 14, 2000
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From the Lehrstuhl für Mikrobiologie der Universität
München, Maria-Ward-Strasse 1a,
80638 München, Germany
The formation of a complex between the specific
chaperone-type protein HypC and the precursor form of the large subunit
HycE in the maturation pathway of hydrogenase 3 from Escherichia
coli has been studied by targeted replacement of amino acids in
both proteins. HypC and its homologs contain the motif
MC(L/I/V)(G/A)(L/I/V)P at the amino terminus, from which the methionine
residue is post-translationally removed. The exchange of the cysteine
residue led to complete loss of the ability to interact with the
precursor form of HycE, but replacement of the proline residue had no
effect. Site-directed replacement of the conserved cysteine residues in
HycE involved in nickel binding was also performed. Exchange of
Cys241 resulted in the inability of the HycE variant to
interact with HypC and to incorporate nickel. The variants of HycE in
which Cys244 and Cys531 were replaced by
alanine residues were unable to incorporate nickel, although the
mutated proteins could interact with HypC. Intriguingly, the precursor
of HycE in which the Cys534 residue was exchanged could
form the complex with HypC, could incorporate nickel, and was
C-terminally processed, but it delivered an inactive enzyme. Our
findings are in favor of a model in which binding of HypC masks
Cys241; Cys244 and Cys531 bind the
iron and nickel moieties, respectively; and C534 closes the bridge
between the two metals after C-terminal processing has taken place.
Analysis of the HypC-HycE Complex, a Key Intermediate in the
Assembly of the Metal Center of the Escherichia coli
Hydrogenase 3*
and
*
This work was funded by grants from the Alexander von
Humboldt Foundation (to A. M.) and from the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to
A. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 89-2180-6134;
Fax: 89-2180-6122; E-mail: Axel.Magalon@lrz.uni-muenchen.de.
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