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J Biol Chem, Vol. 275, Issue 7, 4679-4686, February 18, 2000

Regulation of Cytochrome bd Expression in the Obligate Aerobe Azotobacter vinelandii by CydR (Fnr)
SENSITIVITY TO OXYGEN, REACTIVE OXYGEN SPECIES, AND NITRIC OXIDE^*

Guanghui Wu, Hugo Cruz-Ramos, Susan Hill^§, Jeff Green, Gary Sawers^§, and Robert K. Poole^¶

From the  Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, University of Sheffield, Sheffield S10 2TN and the ^§ Nitrogen Fixation Laboratory, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant nitrogen fixation requires cytochrome bd. Regulation of cytochrome bd expression is achieved by CydR (an Fnr homologue), which represses transcription of the oxidase genes cydAB. cydAB mRNA was mapped by primer extension; the transcriptional start site was determined, and putative 10 and 35 regions were deduced. Two "CydR boxes," one at the +1 site and one upstream of the 35 region, were identified. Transcriptionally inactive, purified CydR was converted, by adding NifS, cysteine, and Fe²⁺, into an active form possessing acid-labile sulfide and spectra suggesting a [4Fe-4S]²⁺ cluster. Reconstituted CydR specifically bound both CydR boxes cooperatively, with higher affinity for the nearer consensus +1 site. Low concentrations of O₂ or NO ([O₂]/[[CydR] or [NO]/[CydR] = 0.1-0.6) elicited loss of the 420 nm absorbance attributed to the [4Fe-4S]²⁺ cluster, formation of a 315 nm species, and loss of ability to retard DNA migration. Retardation by reconstituted CydR was enhanced by superoxide dismutase and/or catalase, suggesting a role for reactive oxygen species in CydR inactivation. The role of CydR in regulating cydAB expression in the supposedly anoxic cytoplasm of A. vinelandii and similarities to cydAB regulation by Fnr in Escherichia coli are discussed.

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