Coordination of Zn2+ (and Cd2+) by prokaryotic metallothionein. Involvement of his-imidazole.

In mammalian metallothionein Zn2+ is exclusively coordinated to Cys-thiolate to form clusters in which the metal is thermodynamically stable but also kinetically labile. By contrast, little is known about coordination to prokaryotic metallothionein, SmtA. 3 nmol of Zn2+ nmol-1 SmtA were displaced by 8 nmol of p-(hydroxymercuri)phenylsulfonate implicating eight of the nine Cys in the coordination of three metal ions. None of the Zn2+ associated with SmtA was accessible to 4-(2-pyridylazo)resorcinol prior to the addition of p-(hydroxymercuri)phenylsulfonate. An unusual feature of SmtA is the presence of three His residues, and we have investigated whether these contribute to metal coordination. Less Zn2+ was associated with purified SmtA(H40R/H49R/H55R), in which all three His residues were substituted with Arg, and approximately one equivalent of Zn2+ was immediately accessible to 4-(2-pyridylazo)resorcinol. Following incubation of SmtA with 111Cd, three 111Cd resonances were detected, two in a range expected for CdS4 and the third indicative of either CdNS3 or CdN2S2 coordination. Two-dimensional TOCSY 1H NMR and 111Cd-edited 1H NMR showed two His residues bound to 111Cd, confirming CdN2S2 coordination. The pH of half-dissociation of Zn2+ increased from 4.05 for SmtA to 5.37 for SmtA(H40R/H49R/H55R). Equivalent values for single His mutants SmtA(H40R), SmtA(H49R), and SmtA(H55R) were 4.62, 4.48, and 3.81, respectively, revealing that conversion of His40 or His49 to Arg impairs Zn2+ binding at the CdN2S2 and CdS4 sites. Only approximately two equivalents of Zn2+ were associated with purified SmtA(H49R). The appearance of a fourth 111Cd resonance at lower pH suggests that an alternative CdN2S2 site also exists.