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Journal of Bacteriology, May 2004, p. 2603-2611, Vol. 186, No. 9
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.9.2603-2611.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

HybF, a Zinc-Containing Protein Involved in NiFe Hydrogenase Maturation

Melanie Blokesch, Michaela Rohrmoser, Sabine Rode, and August Böck*

Department Biology I, Microbiology, University of Munich, Munich, Germany

Received 13 November 2003/ Accepted 28 January 2004

HypA and HypB are maturation proteins required for incorporation of nickel into the hydrogenase large subunit. To examine the functions of these proteins in nickel insertion, the hybF gene, which is a homolog of hypA essential for maturation of hydrogenases 1 and 2 from Escherichia coli, was overexpressed, and the product was purified. This protein behaves like a monomer in gel filtration and contains stoichiometric amounts of zinc but insignificant or undetectable amounts of nickel and iron. In filter binding assays radioactively labeled nickel binds to HybF with a KD of 1.87 µM and in a stoichiometric ratio. To identify amino acid residues of HybF involved in nickel and/or zinc binding, variants in which conserved residues were replaced were studied. An H2Q replacement eliminated both in vivo activity and in vitro binding of nickel. The purified protein, however, contained zinc at the level characteristic of the wild-type protein. When E3 was replaced by Q, activity was retained, but an E3L exchange was detrimental. Replacement of each of the four conserved cysteine residues of a zinc finger motif reduced the cellular amount of HybF protein without a loss of in vivo activity, indicating that these residues play a purely structural role. A triple mutant deficient in the synthesis or activity of HypA, HybF, and HypB was constructed, and it exhibited the same responsiveness for phenotypic complementation by high nickel as mutants with a single lesion in one of the genes exhibited. The results are interpreted in terms of a concerted action of HypB and HybF in nickel insertion in which HybF (as well as its homolog, HypA) functions as a metallochaperone and HypB functions as a regulator that controls the interaction of HybF with the target protein.

* Corresponding author. Mailing address: Department of Biology I, Microbiology, University of Munich, Maria Ward Strasse 1a, 80638 Munich, Germany. Phone: 49 (089) 2180 6116. Fax: 49 (089) 2180 63857. E-mail: august.boeck{at}lrz.uni-muenchen.de.

Journal of Bacteriology, May 2004, p. 2603-2611, Vol. 186, No. 9
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.9.2603-2611.2004
Copyright © 2004, 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]  
  • Atanassova, A., Zamble, D. B. (2005). Escherichia coli HypA Is a Zinc Metalloprotein with a Weak Affinity for Nickel. J. Bacteriol. 187: 4689-4697 [Abstract] [Full Text]  
  • Zhang, J. W., Butland, G., Greenblatt, J. F., Emili, A., Zamble, D. B. (2005). A Role for SlyD in the Escherichia coli Hydrogenase Biosynthetic Pathway. J. Biol. Chem. 280: 4360-4366 [Abstract] [Full Text]  
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