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Journal of Bacteriology, July 2005, p. 4689-4697, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.4689-4697.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Escherichia coli HypA Is a Zinc Metalloprotein with a Weak Affinity for Nickel

Anelia Atanassova and Deborah B. Zamble*

Department of Chemistry, University of Toronto, Lash Miller Chemical Laboratories, 80 St. George St., Toronto, Ontario M5S 3H6, Canada

Received 17 February 2005/ Accepted 5 April 2005

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 Zn2+ and stoichiometric Ni2+. 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.

* Corresponding author. Mailing address: Department of Chemistry, University of Toronto, Lash Miller Chemical Laboratories, 80 St. George St., Toronto, Ontario M5S 3H6, Canada. Phone and Fax: (416) 978-3568. E-mail: dzamble{at}chem.utoronto.ca.

Journal of Bacteriology, July 2005, p. 4689-4697, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.4689-4697.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Leach, M. R., Zhang, J. W., Zamble, D. B. (2007). The Role of Complex Formation between the Escherichia coli Hydrogenase Accessory Factors HypB and SlyD. J. Biol. Chem. 282: 16177-16186 [Abstract] [Full Text]  
  • Gasper, R., Scrima, A., Wittinghofer, A. (2006). Structural Insights into HypB, a GTP-binding Protein That Regulates Metal Binding. J. Biol. Chem. 281: 27492-27502 [Abstract] [Full Text]  


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