Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel.
The hyp operon encodes accessory proteins that are required for the maturation of the [NiFe] hydrogenase enzymes and, in some organisms, for the production of urease enzymes as well. HypA or a homologous protein is required for nickel insertion into the hydrogenase precursor proteins. In this study, recombinant HypA from Escherichia coli was purified and characterized in vitro. Metal analysis was used to demonstrate that HypA simultaneously binds stoichiometric Zn(2+) and stoichiometric Ni(2+). Competition experiments with a metallochromic indicator reveal that HypA binds zinc with nanomolar affinity. Spectroscopic analysis of cobalt-containing HypA provides evidence for a tetrathiolate coordination sphere, suggesting that the zinc site has a structural role. In addition, HypA can exist as several oligomeric complexes and the zinc content modulates the quaternary structure of the protein. Fluorescence titration experiments demonstrate that HypA binds nickel with micromolar affinity and that the presence of zinc does not dramatically affect the nickel-binding activity. Finally, complex formation between HypA and HypB, another accessory protein required for nickel insertion, was observed. These experiments suggest that HypA is an architectural component of the hydrogenase metallocenter assembly pathway and that it may also have a direct role in the delivery of nickel to the hydrogenase large subunit.
Department of Chemistry, University of Toronto, Lash Miller Chemical Laboratories, Ontario, Canada.
Anelia
A
Atanassova
Deborah B
DB
Zamble
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D017124:Conserved Sequence; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014510:Urease / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D001059:Apoproteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C499625:HypA protein, E coli; D047908:Intracellular Signaling Peptides and Proteins; D003035:Cobalt; D014510:Urease; D015032:Zinc