Supermetalation of the β Domain of Human Metallothionein 1a

Metallothionein has been implicated in a number of functions, including toxic metal detoxification, as a metal chaperone and in metal ion homeostasis. In this payer, we demonstrate that the beta domain of human metallothionein 1a, well-known to bind three Zn2+ or Cd2+ ions with nine cysteinyl sulfurs, is also capable of binding an additional Cd2+ ion, leading to the formation of the supermetalated Cd-4-beta-rhMT 1a. This intermediate, either by itself or in concert with the a. domain of human metallothionein, is a likely model for metal exchange with the apoenzyme, which is one of the key roles of metallothionein. Through electrospray ionization (ESI) mass spectrometry and circular dichroism (CD) and ultraviolet (UV) spectroscopy, we show that the addition of 4.4 molar equiv of CdSO4 to a solution of Cd-3-beta-rhMT 1a leads to the complete conversion to Cd-4-beta-rhMT 1a. ESI mass spectrometry was used to determine the exact speciation of beta-rhMT 1a. While the UV absorption spectrum increased slightly, the CD spectrum of Cd-4-beta-rhMT 1a showed significant changes with the appearance of a sharp monophasic peak at 252 nm in contrast to the derivative-shaped envelope of the Cd-3-beta-rhMT 1a species [peak extrema at (+)262 and (-)236 nm], indicating disruption of the exciton coupling in the metal thiolate cluster. Additionally, both direct and indirect Cd-113 nuclear magnetic resonance (NMR) spectra of the Cd-3-beta-rhMT 1a and Cd-4-beta-rhMT 1a species were recorded. The Cd-113 NMR spectrum of Cd-4-beta-rhMT 1a contained four cadmium peaks in the tetrahedral thiolate region at 688.8, 650.3, 635.9, and 602.5 ppm. This represents the first report of both NMR data for isolated Cd-3-beta-rhMT 1a and supermetalated Cd-4-beta-rhMT 1a.