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Journal of Bacteriology, November 2002, p. 6280-6288, Vol. 184, No. 22
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.22.6280-6288.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

Tanja Burgdorf,1 Antonio L. De Lacey,2 and Bärbel Friedrich1*

Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany,1 Instituto de Catalisis (CSIC), Campus Universidad Autonoma de Madrid, 28049 Madrid, Spain2

Received 20 May 2002/ Accepted 16 August 2002

The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD+ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H2-oxidizing activity. In one of these isolates (HoxH I64A), H2 binding was impaired. Class II mutants revealed a high D2/H+ exchange rate relative to a low H2-oxidizing activity. A representative (HoxH H16L) displayed D2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O2-sensitive growth on hydrogen due to an O2-sensitive SH protein.

* Corresponding author. Mailing address: Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany. Phone: (49) 30 2093 8101. Fax: (49) 30 2093 8102. E-mail: baerbel.friedrich{at}rz.hu-berlin.de.

Journal of Bacteriology, November 2002, p. 6280-6288, Vol. 184, No. 22
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.22.6280-6288.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Burgdorf, T., van der Linden, E., Bernhard, M., Yin, Q. Y., Back, J. W., Hartog, A. F., Muijsers, A. O., de Koster, C. G., Albracht, S. P. J., Friedrich, B. (2005). The Soluble NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha H16 Consists of Six Subunits and Can Be Specifically Activated by NADPH. J. Bacteriol. 187: 3122-3132 [Abstract] [Full Text]  
  • Dementin, S., Burlat, B., De Lacey, A. L., Pardo, A., Adryanczyk-Perrier, G., Guigliarelli, B., Fernandez, V. M., Rousset, M. (2004). A Glutamate Is the Essential Proton Transfer Gate during the Catalytic Cycle of the [NiFe] Hydrogenase. J. Biol. Chem. 279: 10508-10513 [Abstract] [Full Text]  


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