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J. Biol. Chem., Vol. 281, Issue 28, 18909-18913, July 14, 2006

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Detection of Sulfide Release from the Oxygen-sensing [4Fe-4S] Cluster of FNR^*

Jason C. Crack¹, Jeffrey Green, Nick E. Le Brun, and Andrew J. Thomson²

From the Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom and the Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom

The Escherichia coli FNR protein regulates the transcription of >100 genes in response to environmental O₂, thereby coordinating the response to anoxia. Under O₂-limiting conditions, FNR binds a [4Fe-4S]²⁺ cluster through four cysteine residues (Cys²⁰, Cys²³, Cys²⁹, Cys¹²²). The acquisition of the [4Fe-4S]²⁺ cluster converts FNR into the transcriptionally active dimeric form. Upon exposure to O₂, the cluster converts to a [2Fe-2S]²⁺ form, generating FNR monomers that no longer bind DNA with high affinity. The mechanism of the cluster conversion reaction and the nature of the released iron and sulfur are of considerable current interest. Here, we report the application of a novel in vitro method, involving 5,5'-dithiobis-(2-nitrobenzoic acid), for determining the oxidation state of the sulfur atoms released during FNR cluster conversion following the addition of O₂. Conversion of [4Fe-4S]²⁺ to [2Fe-2S]²⁺ clusters by O₂ for both native and reconstituted FNR results in the release of 2 sulfide ions per [4Fe-4S]²⁺ cluster. This demonstrates that the reaction between O₂ and the [4Fe-4S]²⁺ cluster does not require sulfide oxidation and hence must entail iron oxidation.

Received for publication, February 22, 2006 , and in revised form, May 4, 2006.

^* This work was supported by Biotechnology and Biological Sciences Research Council Grant BB/C500360/1 and by a Wellcome Trust award from the Joint Infrastructure Fund for equipment. 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.

¹ To whom correspondence may be addressed. Tel.: 44-1603-593830; Fax: 44-1603-592003; E-mail: J.Crack{at}uea.ac.uk. ² To whom correspondence may be addressed. Tel.: 44-1603-593051; Fax: 44-1603-593045; E-mail: A.Thomson{at}uea.ac.uk.

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