Characterization of the nitric oxide-reactive transcriptional activator NorR

The prokaryotic transcriptional regulator NorR is unusual in that it utilizes a mononuclear ferrous iron center rather than a heme moiety as a means of sensing nitric oxide (NO). Binding of NO to the nonheme iron center in the amino-terminal GAF domain of NorR results in formation of a mononitrosyl iron complex and relieves intramolecular repression within NorR, allowing this regulatory protein, a member of the σ54-dependent family of enhancer-binding proteins, to activate expression of genes required for NO detoxification. This chapter describes detailed protocols for measuring transcriptional activation by Escherichia coli NorR in vivo and in vitro. It also details spectroscopic methods for analysis of the interaction of NO with the nonheme iron center and determination of the NO-binding affinity constant.