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J Bacteriol, March 1998, p. 1023-1029, Vol. 180, No. 5
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Subforms and In Vitro Reconstitution of the NAD-Reducing Hydrogenase of Alcaligenes eutrophus

Christian Massanz, Silke Schmidt, and Bärbel Friedrich*

Institut für Biologie, Humboldt-Universität zu Berlin, D-10115 Berlin, Germany

Received 22 September 1997/Accepted 23 December 1997

The cytoplasmic, NAD-reducing hydrogenase (SH) of Alcaligenes eutrophus H16 is a heterotetrameric enzyme which contains several cofactors and undergoes a complex maturation during biogenesis. HoxH is the Ni-carrying subunit, and together with HoxY it forms the hydrogenase dimer. HoxF and HoxU represent the flavin-containing diaphorase moiety, which is closely related to NADH:ubiquinone oxidoreductase and mediates NADH oxidation. A variety of mutations were introduced into the four SH structural genes to obtain mutant enzymes composed of monomeric and dimeric forms. A deletion removing most of hoxF, hoxU, and hoxY led to the expression of a HoxH monomer derivative which was proteolytically processed at the C terminus like the wild-type polypeptide. While the hydrogenase dimer, produced by a strain deleted of hoxF and hoxU, displayed H2-dependent dye-reducing activity, the monomeric form did not mediate the activation of H2, although nickel was incorporated into HoxH. Deletion of hoxH and hoxY led to the production of HoxFU dimers which displayed NADH:oxidoreductase activity. Mixing the hydrogenase and the diaphorase moieties in vitro reconstituted the structure and catalytic function of the SH holoenzyme.

* Corresponding author. Mailing address: Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany. Phone: 49-30-20938100. Fax: 49-30-20938102. E-mail: baerbel=friedrich{at}rz.hu-berlin.de.




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