Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry

Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrome try of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu12MT specie s. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Additi on of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ion s whoses masses were consistent with ape-MT, Cu4MT, Cu6MT, and Cu12MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu- 4 to Cu12MT species. However, formation of the tetracopper MT species remai ned cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the alpha or beta domain, domain peptides of MT3 were used. Addition of Cu(I) to the ap o beta domain resulted in a peak consistent with the formation of a four-Cu (I) cluster. This is consistent with reports that Cu(I) ions bind preferent ially to the beta domain of MTs.