Structural Characterization of Distinct α3N and α5 Metal Sites in the Cyanobacterial Zinc Sensor SmtB

• Published in: Biochemistry (Easton) 2002-08, Vol.41 (31), p.9765-9775
• Main Authors: VanZile, Michael L, Chen, Xiaohua, Giedroc, David P
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0201771

SmtB is required for Synechococcus to effect a response to toxic concentrations of Zn(II) and other heavy metals. Direct binding of inducing metal ions to SmtB transcriptionally derepresses the expression of SmtA, a prokaryotic class II metallothionein. Homodimeric SmtB binds one Zn(II) or Co(II) per monomer in a cysteine thiolate-containing site in a tetrahedral coordination geometry [VanZile, M. L., et al. (2000) Biochemistry 39, 11818−11829]. In this report, characterization of a set of cysteine substitution mutants of SmtB reveals that SmtB homodimer binds Zn(II) or Co(II) in one of two mutually exclusive metal binding sites, termed α3N and α5, with very high equilibrium affinities. Both sites are characterized by similar affinities for Co(II) (K Co ≈ 2−5 × 109 M-1), while the Zn(II) affinities are at least 20-fold different (K Zn α3N ≥ 1013 M-1; K Zn α5 ≈ 5 × 1011 M-1). Co(II) bound exclusively at the α5 sites is capable of rapid equilibration between the α3N and α5 sites upon reduction of the mixed disulfides in S-methylated SmtB. These results suggest that the α3N or α5 metal sites might play distinct roles in this Zn(II)-sensing protein, systematically investigated in the following paper [VanZile, M. L., Chen, X., and Giedroc, D. P. (2002) Biochemistry 41, 9776−9786]. Since both the α3N and α5 sites are present in many members of the SmtB/ArsR family of metal sensor proteins, the presence of these two metal binding sites may explain some of the functional diversity in metal responses across this family of proteins.