Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: Evidence for metal-binding cooperativity

Electrospray ionization (ESI) mass spectra of both well-characterized and n ovel metallothioneins (MTs) from various species were recorded to explore t heir metal-ion-binding modes and stoichiometries. The ESI mass spectra of t he zinc-and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data ob tained by other methods suggest that these MTs bind zinc or cadmium in a si ngle predominant form and are consistent with the presence of three- and fo ur-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially b ind 12 copper ions. To obtain additional information on the formation and r elative stability of metal-thiolate clusters in MTs, a mass spectrometric t itration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resultin g complexes were measured by ESI mass spectrometry. These experiments revea led that the formation of the four-metal cluster and of the thermodynamical ly less stable three-metal cluster is sequential and largely cooperative fo r both zinc and cadmium. Minor intermediate forms between metal-free MT, Me 4MT, and fully reconstituted Me7MT were also observed. The addition of incr easing amounts of cadmium to metal-free blue crab MT-I resulted in prominen t peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecti ng the known structure of this MT with two Me(3)Cys(9) centers. In a simila r reconstitution experiment performed with Caenorhabditits elegams MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were o bserved.