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Journal of Bacteriology, March 1999, p. 1831-1837, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Aspartate 205 in the Catalytic Domain of Naphthalene Dioxygenase Is Essential for Activity

Rebecca E. Parales,* Juanito V. Parales, and David T. Gibson

Department of Microbiology and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242

Received 17 September 1998/Accepted 21 December 1998

The naphthalene dioxygenase enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The crystal structure of naphthalene dioxygenase (B. Kauppi, K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund, and S. Ramaswamy, Structure 6:571-586, 1998) indicates that aspartate 205 may provide the most direct route of electron transfer between the Rieske [2Fe-2S] center of one alpha  subunit and mononuclear iron in the adjacent alpha  subunit. In this study, we constructed four site-directed mutations that changed aspartate 205 to alanine, glutamate, asparagine, or glutamine to test whether this residue is essential for naphthalene dioxygenase activity. The mutant proteins were very inefficient in oxidizing naphthalene to cis-naphthalene dihydrodiol, and oxygen uptake in the presence of naphthalene was below detectable levels. The purified mutant protein with glutamine in place of aspartate 205 had identical spectral properties to wild-type naphthalene dioxygenase and was reduced by NADH in the presence of catalytic amounts of ferredoxinNAP and reductaseNAP. Benzene, an effective uncoupler of oxygen consumption in purified naphthalene dioxygenase, did not elicit oxygen uptake by the mutant protein. These results indicate that electron transfer from NADH to the Rieske center in the mutant oxygenase is intact, a finding consistent with the proposal that aspartate 205 is a necessary residue in the major pathway of electron transfer to mononuclear iron at the active site.

* Corresponding author. Mailing address: Department of Microbiology, 3-730 BSB, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7982. Fax: (319) 335-9999. E-mail: rebecca-parales{at}uiowa.edu.

Journal of Bacteriology, March 1999, p. 1831-1837, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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