HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 38, 35861-35868, September 19, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/38/35861    most recent M300857200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by de Vries, S. Articles by Schröder, I. Search for Related Content PubMed PubMed Citation Articles by de Vries, S. Articles by Schröder, I. Social Bookmarking                      What's this?

Purification and Characterization of the MQH₂:NO Oxidoreductase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum^*

Simon de Vries  , Marc J. F. Strampraad , Shen Lu ¶, Pierre Moënne-Loccoz ¶ and Imke Schröder ||

From the Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands, the ^¶Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering at Oregon Health and Science University, Beaverton, Oregon 97006, and the ^||Department of Microbiology and Molecular Genetics, University of California, Los Angeles, California 90095-1489

The membrane-bound NO reductase from the hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum was purified to homogeneity. The enzyme displays MQH₂:NO oxidoreductase (qNOR) activity, consists of a single subunit, and contains heme and nonheme iron in a 2:1 ratio. The combined results of EPR, resonance Raman, and UV-visible spectroscopy show that one of the hemes is bis-His-coordinated low spin (g_z = 3.015; g_y = 2.226; g_x = 1.45), whereas the other heme adopts a high spin configuration. The enzyme also contains one nonheme iron center, which in the oxidized enzyme is antiferromagnetically coupled to the high spin heme. This binuclear high spin heme/nonheme iron center is EPR-silent and the site of NO reduction. The reduced high spin heme is bound to a neutral histidine and can bind CO to form of a low spin complex. The oxidized high spin heme binds NO, yielding a ferric nitrosyl complex, the intermediate causing the commonly found substrate inhibition in NO reductases (K_i(NO) = 7 µM). The qNOR as present in the membrane is, in contrast to the purified enzyme, quite thermostable, incubation at 100 °C for 86 min leading to 50% inhibition. The pure enzyme lacks heme b and instead contains stoichiometric amounts of hemes O_p1 and O_p2, ethenylgeranylgeranyl and hydroxyethylgeranylgeranyl derivatives of heme b, respectively. The archaeal qNOR is the first example of a NO reductase, which contains modified hemes reminiscent of cytochrome bo₃ and aa₃ oxidases. This report is the first describing the purification and structural and spectroscopic properties of a thermostable NO reductase.

Received for publication, January 27, 2003 , and in revised form, May 27, 2003.

^* This work was supported by Nederlandse organisatie voor energie en milieu (Novem) project number 375001/0060 (to S. d. V.), by National Science Foundation Award MCB0091351 (to I. S.), and by the National Institutes of Health Grant GM-18865 (to P. M.-L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 31-15-2785139; Fax: 31-15-2782355; E-mail: S.deVries{at}tnw.tudelft.nl.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A. E. Cozen, M. T. Weirauch, K. S. Pollard, D. L. Bernick, J. M. Stuart, and T. M. Lowe Transcriptional Map of Respiratory Versatility in the Hyperthermophilic Crenarchaeon Pyrobaculum aerophilum J. Bacteriol., February 1, 2009; 191(3): 782 - 794. [Abstract] [Full Text] [PDF]

				L. F. Feinberg and J. F. Holden Characterization of Dissimilatory Fe(III) versus NO3- Reduction in the Hyperthermophilic Archaeon Pyrobaculum aerophilum J. Bacteriol., January 15, 2006; 188(2): 525 - 531. [Abstract] [Full Text] [PDF]

				A. Busch, A. Pohlmann, B. Friedrich, and R. Cramm A DNA Region Recognized by the Nitric Oxide-Responsive Transcriptional Activator NorR Is Conserved in {beta}- and {gamma}-Proteobacteria J. Bacteriol., December 1, 2004; 186(23): 7980 - 7987. [Abstract] [Full Text] [PDF]