Characterization of the metal receptor sites in Escherichia coli Zur, an ultrasensitive zinc(II) metalloregulatory protein

The Escherichia coli Zur protein is a Fur homologue that regulates expressi on of Zn(II) uptake systems. The zinc-loaded form of Zur is proposed to bin d DNA and repress transcription of the znuABC genes. Recent in vitro data i ndicate that the transcriptional activity of Zur is half-maximal when free Zn(II) concentrations are in the sub-femtomolar range, making it the most s ensitive Zn(II) metalloregulatory protein reported to date. Previous result s indicate that Zur binds at least one zinc; however, little else is known about Zn(II) binding. We have purified E. coli Zur to homogeneity and found that it has two Zn(II) binding sites per monomer with different coordinati on environments. Using Zn(II) binding assays, ICP-AES analysis, and Zn EXAF S analysis, we show that one zinc is tightly bound in an S-3(N/O) coordinat ion environment. Both Co(II) and Zn(II) were substituted into the second me tal binding site and probed by EXAFS and UV-visible absorption spectroscopy . These studies indicate that Co(II) is bound in an S(N/O)(3) coordination environment with tetrahedral geometry. The Zn(II) EXAFS of Zn(2)Zur, which is consistent with the results for both sites, indicates an average coordin ation environment of S-2(N/O)(2), presumably due to one S(N/O)(3) site and one S-3(N/O) site. These studies reveal the coordination environments that confer such exceptional zinc sensitivity and may provide the foundation for understanding the molecular basis of metal ion selectivity. A comparison o f the metal binding sites in Zur with its Fe(II)-sensing homologue Fur prov ides clues as to why these two proteins with similar structures respond to two very different metal ions.