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Bacterial cells tightly regulate the intracellular concentrationsof essential transition metal ions by deploying a panel of metal-regulatedtranscriptional repressors and activators that bind to operator-promoterregions upstream of regulated genes. Like other zinc uptake regulator（Zur） proteins, Acinetobacter baumannii Zur represses transcription of its regulon when ZnII isreplete and binds more weakly to DNA when ZnII is limiting.Previous studies established that Zur proteins are homodimeric andharbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy （XAS） of the Cd2Zn2AbZur metalloderivative with CdII bound to the allosteric sites reveals a S-（N/O）3 first coordination shell. Site-directed mutagenesis suggests thatH89 and C100 from the N-terminal DNA binding domain and H107 and E122from the C-terminal dimerization domain comprise the regulatory metalsite. KZn for this allosteric site is6.0 （±2.2） × 1012 M–1 witha functional “division of labor” among the four metalligands. N-terminal domain ligands H89 and C100 contribute far moreto KZn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measuredby an in vitro transcription assay. The heterotropicallosteric coupling free energy, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailableZnII set-point of ≈6 × 10–14 M. Small-angle X-ray scattering （SAXS） experiments reveal that onlythe wild-type Zn homodimer undergoes allosteric switching, while theC100S AbZur fails to switch. These data collectivelysuggest that switching to a high affinity DNA-binding conformationinvolves a rotation/translation of one protomer relative to the otherin a way that is dependent on the integrity of C100. We place thesefindings in the context of other Zur proteins and Fur family repressorsmore broadly.
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