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J. Biol. Chem., Vol. 282, Issue 28, 20292-20300, July 13, 2007

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Formation of a Dinitrosyl Iron Complex by NorA, a Nitric Oxide-binding Di-iron Protein from Ralstonia eutropha H16^*

Katja Strube, Simon de Vries, and Rainer Cramm¹

From the Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany and the Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands

In Ralstonia eutropha H16, two genes, norA and norB, form a dicistronic operon that is controlled by the NO-responsive transcriptional regulator NorR. NorB has been identified as a membrane-bound NO reductase, but the physiological function of NorA is unknown. We found that, in a NorA deletion mutant, the promoter activity of the norAB operon was increased 3-fold, indicating that NorA attenuates activation of NorR. NorA shows limited sequence similarity to the oxygen carrier hemerythrin, which contains a di-iron center. Indeed, optical and EPR spectroscopy of purified NorA revealed the presence of a di-iron center, which binds oxygen in a similar way as hemerythrin. Diferrous NorA binds two molecules of NO maximally. Unexpectedly, binding of NO to the diferrous NorA required an external reductant. Two different NorA-NO species could be resolved. A minor species (up to 20%) showed an S = EPR signal with g = 2.041, and g_|| = 2.018, typical of a paramagnetic dinitrosyl iron complex. The major species was EPR-silent, showing characteristic signals at 420 nm and 750 nm in the optical spectrum. This species is proposed to represent a novel dinitrosyl iron complex of the form , i.e. NO is bound as NO^–. The NO binding capacity of NorA in conjunction with its high cytoplasmic concentration (20 µM) suggests that NorA regulates transcription by lowering the free cytoplasmic concentration of NO.

Received for publication, March 7, 2007 , and in revised form, April 23, 2007.

^* This work was supported by the Deutsche Forschungsgemeinschaft. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed. Tel.: 49-30-2093-8111; Fax: 49-30-2093-8102; E-mail: rainer.cramm{at}rz.hu-berlin.de.

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