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J. Bacteriol. doi:10.1128/JB.01865-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization of the signaling domain of the NO-responsive regulator NorR from Ralstonia eutropha H16 by site-directed mutagenesis

^* To whom correspondence should be addressed. Email: rainer.cramm{at}rz.hu-berlin.de.

	Abstract

In Ralstonia eutropha H16, the NO-responsive transcriptional activator NorR controls expression of a dicistronic operon that encodes a membrane-bound NO reductase NorB and a protein of unknown function, NorA. The N-terminal domain (NTD) of NorR is responsible for perception of the signal molecule nitric oxide. Thirteen out of twenty-seven conserved residues of the NTD were exchanged by site-directed mutagenesis. Replacement of R63, R72, D93, D96, C112, D130, or F137 strongly decreased NorR-dependent promoter activation, while exchange of Y95 or H110 led to an increase in promoter activity compared to the wild type. A purified truncated NorR comprising only the NTD (NorR-NTD) contained one iron atom per molecule and was able to bind NO in the as-isolated state. Based on the iron content of NorR-NTD proteins with single acid replacements, residues R72, D93, D96, C112, and D130 are likely candidates for iron ligands. Residues R63, Y95, and H110 appear not to be involved in NO binding but may take part in subsequent steps of the signal transduction mechanism of NorR.

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