Noncooperative Metalation of Metallothionein 1a and Its Isolated Domains with Zinc

Mammalian metallothioneins (MTs) are a family of small cysteine-rich proteins capable of binding 7 Zn2+ or Cd2+ ions into two distinct domains: an N-terminal beta-domain that binds 3 Zn2+ or Cd2+ and a C-terminal alpha-domain that binds 4 Zn2+ or Cd2+. MT has been implicated in a number of physiological functions, including metal ion homeostasis, toxic metal detoxification, and as a protective agent against oxidative stress. Conventionally, MT has been understood to coordinate metal ions in a cooperative fashion. Under this mechanism of metalation, the only species of biological relevance would be the metal-free (apo-) form of the protein and the fully metalated (holo-) form of the protein. However, art increasing body of evidence suggests that metalation occurs in a noncooperative manner. If this latter mechanism is correct, then partially metalated forms of the protein will be stable and able to take part in cellular chemistry. We report in this paper conclusive evidence that shows that biologically essential zinc binds to MT in a noncooperative manner. In addition, we report for the first time the stability of a Zn-5-MT species. The implications of these findings are discussed in terms of the mechanism of metalation.