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Journal of Bacteriology, October 2001, p. 5491-5495, Vol. 183, No. 19
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.19.5491-5495.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Properties of a Thermostable Nitrate Reductase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum

Sepideh Afshar,1 Eric Johnson,1 Simon de Vries,2 and Imke Schröder1,*

Department of Microbiology and Molecular Genetics, University of California, Los Angeles, California 90095-1489,1 and Kluyver Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands2

Received 12 July 2000/Accepted 1 July 2001

The nitrate reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofactors. The P. aerophilum nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (Vmax with nitrate, 1,162 s-1 (326 U/mg); Vmax with chlorate, 1,348 s-1 (378 U/mg) [assayed at 75°C]). The Km values for nitrate and chlorate were 58 and 140 µM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was >95°C. When incubated at 100°C, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, 1602 Molecular Sciences Building, University of California, Los Angeles CA 90095-1489. Phone: (310) 825-8085. Fax: (310) 206-5231. E-mail: imkes{at}microbio.ucla.edu.

Journal of Bacteriology, October 2001, p. 5491-5495, Vol. 183, No. 19
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.19.5491-5495.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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