A CuI-sensing ArsR family metal sensor protein with a relaxed metal selectivity profile

ArsR (or ArsR/SmtB) family metalloregulatory homodimeric repressors collectively respond to a wide range of metal ion inducers in regulating homeostasis and resistance of essential and nonessential metal ions in bacteria. BxmR from the cyanobacterium Osciliatoria brevis is the first characterized ArsR protein that senses both Cu-I/Ag-I and divalent metals Zn-II/Cd-II in cells by regulating the expression of a P-type ATPase efflux pump (Bxa1) and an intracellular metallothionein (BmtA). We show here that both pairs of predicted alpha 3N and alpha 5 sites bind metal ions, but with distinct physicochemical and functional metal specificities. Inactivation of the thiophilic alpha 3N site via mutation (C77S) abolishes regulation by both Cd-II and Cu-I, while Zn-II remains a potent allosteric negative effector of operator/promoter binding (Delta G(c) >= +3.2 kcal mol(-1)). In contrast, alpha(5) site mutant retains regulation by all four metal ions, albeit with a smaller coupling free energy (Delta G(c) approximate to +1.7 (+/- 0.1) kcal mol(-1)). Unlike the other metals ions, the BxmR dimer binds 4 mol equiv of Cu-I to form an alpha 3N binuclear (Cu2S4)-S-I cluster by X-ray absorption spectroscopy. BxmR is thus distinguishable from other closely related ArsR family sensors, in having evolved a metalloregulatory alpha 3N site that can adopt an expanded range of coordination chemistries while maintaining redundancy in the response to Zn-II. The evolutionary implications of these findings for the ArsR metal sensor family are discussed.