Analysis of the HypC-hycE complex, a key intermediate in the assembly of the metal center of the Escherichia coli hydrogenase 3.
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 Cys(241) resulted in the inability of the HycE variant to interact with HypC and to incorporate nickel. The variants of HycE in which Cys(244) and Cys(531) 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 Cys(534) 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 Cys(241); Cys(244) and Cys(531) bind the iron and nickel moieties, respectively; and C534 closes the bridge between the two metals after C-terminal processing has taken place.
Magalon
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Lehrstuhl für Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, 80638 München, Germany. Axel.Magalon@lrz.uni-muenchen.de
Bock
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C108874:HypC protein, Bacteria; C491587:HypC protein, E coli; D011994:Recombinant Proteins; C086230:hycE protein, E coli; D006864:Hydrogenase; D003545:Cysteine