The zinc metalloregulatory protein Synechococcus PCC7942 SmtB binds a single zinc ion per monomer with high affinity in a tetrahedral coordination geometry.
The Synechococcus PCC7942 SmtB is a zinc-responsive transcriptional repressor and a member of the ArsR superfamily of prokaryotic metalloregulatory transcription factors. The mechanism of negative regulation by Zn(II) and other metals as well as the coordination chemistry (stoichiometry, affinity, and specificity) of SmtB is poorly understood. In contrast to previous results [Kar, S. R., Adams, A. C., Lebowitz, J., Taylor, K. B., and Hall, L. M. (1997) Biochemistry 36, 15343-15348], we find that fully reduced SmtB binds 1 mol equiv of Zn(II) with a very high affinity, K(Zn) in excess of 10(11) M(-1) (pH 7.4, 0.15 M KCl, 22 degrees C). Optical spectroscopic experiments reveal that SmtB binds 1 mol equiv of Co(II) in a tetrahedral or distorted tetrahedral environment with one or two cysteine thiolate ligands in the first coordination shell. Zn(II) and Co(II) EXAFS studies are consistent with the optical spectroscopic data, and further suggest the presence of a mixture of carboxylate and imidazole-containing ligands. K(Co) was determined to be 1.7 (+/-0.1) x 10(9) M(-1) in a chelator (EGTA) competition assay; 1 equiv of Zn(II) results in complete displacement of the bound Co(II). SmtB also binds 1 mol equiv of Ni(II), which, when formed at low Ni(II):SmtB molar ratios, adopts a non-native, six-coordinate complex characterized by at least two histidine and no thiolate ligands. The hierarchy of metal binding affinities is Zn(II) >> Co(II) >> Ni(II).
VanZile
M L
ML
Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843-2128, USA.
Cosper
N J
NJ
Scott
R A
RA
Giedroc
D P
DP
D001426:Bacterial Proteins / Q000737:chemistry*; D001667:Binding, Competitive; D003035:Cobalt / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000737:chemistry; D008668:Metallothionein / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry*; D012118:Resorcinols / Q000737:chemistry; D012996:Solutions; D013054:Spectrophotometry, Atomic; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis; D017186:Titrimetry; D014965:X-Rays; D015032:Zinc / Q000737:chemistry*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D012097:Repressor Proteins; D012118:Resorcinols; D012996:Solutions; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D003035:Cobalt; D009532:Nickel; D008668:Metallothionein; D015032:Zinc; C028398:4-(2-pyridylazo)resorcinol; D016257:Fura-2