Format

Send to

Choose Destination
Biochemistry. 2001 Sep 4;40(35):10417-23.

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

Author information

1
Department of Chemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.

Abstract

The Escherichia coli Zur protein is a Fur homologue that regulates expression of Zn(II) uptake systems. The zinc-loaded form of Zur is proposed to bind DNA and repress transcription of the znuABC genes. Recent in vitro data indicate that the transcriptional activity of Zur is half-maximal when free Zn(II) concentrations are in the sub-femtomolar range, making it the most sensitive Zn(II) metalloregulatory protein reported to date. Previous results 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 coordination environments. Using Zn(II) binding assays, ICP-AES analysis, and Zn EXAFS analysis, we show that one zinc is tightly bound in an S(3)(N/O) coordination environment. Both Co(II) and Zn(II) were substituted into the second metal 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 coordination 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 of the metal binding sites in Zur with its Fe(II)-sensing homologue Fur provides clues as to why these two proteins with similar structures respond to two very different metal ions.

PMID:
11523983
DOI:
10.1021/bi0155448
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center