COORDINATION OF ZN2- INVOLVEMENT OF HIS-IMIDAZOLE( (AND CD2+) BY PROKARYOTIC METALLOTHIONEIN )

In mammalian metallothionein Zn2+ is exclusively coordinated to Cys-th iolate to form clusters in which the metal is thermodynamically stable but also kinetically labile, By contrast, little is known about coord ination to prokaryotic metallothionein, SmtA, 3 nmol of Zn2+ nmol(-1) SmtA were displaced by 8 nmol of p-(hydroxymercuri)phenylsulfonate imp licating eight of the nine Cys in the coordination of three metal ions . None of the Zn2+ associated with SmtA was accessible to 4-(2-pyridyl azo)resorcinol prior to the addition of p-(hydroxymercuri)phenylsulfon ate. An unusual feature of SmtA is the presence of three His residues, and we have investigated whether these contribute to metal coordinati on. Less Zn2+ was associated with purified SmtA(H40R/H49R/H55R), in wh ich all three His residues were substituted with Arg, and approximatel y one equivalent of Zn2+ was immediately accessible to 4-(2-pyridylazo )resorcinol. Following incubation of SmtA with Cd-111, three Cd-111 re sonances were detected, two in a range expected for CdS4 and the third indicative of either CdNS3 or CdN2S2 coordination. Two-dimensional TO CSY H-1 NMR and Cd-111-edited H-1 NMR showed two His residues bound to Cd-111, confirming CdN2S2 coordination. The pH of half-dissociation o f 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 S mtA(H55R) were 4.62, 4.48, and 3.81, respectively, revealing that conv ersion of His(40) or His(49) to Arg impairs Zn2+ binding at the CdN2S2 and CdS4 sites. Only approximately two equivalents of Zn2+ were assoc iated with purified SmtA(H49R), The appearance of a fourth Cd-111 reso nance at lower pH suggests that an alternative CdN2S2 site also exists .