Kinetic analysis of arsenic-metalation of human metallothionein: significance of the two-domain structure.

Metallothionein (MT) is ubiquitous in Nature, underlying MT's importance in the cellular chemistry of metals. Mammalian MT consists of two metal-binding domains while microorganisms like cyanobacteria consist of a single metal-binding domain MT. The evolution of a two-domain protein has been speculated on for some time; however, no conclusive evidence explaining the evolutionary necessity of the two-domain structure has been reported. The results presented in this report provide the complete kinetic analysis and subsequent mechanism of the As(3+)-metalation of the two-domain beta alpha hMT and the isolated single domain fragments using time- and temperature-resolved electrospray ionization mass spectrometry. The mechanism for beta alpha hMT binding As(3+) is noncooperative and involves six sequential bimolecular reactions in which the alpha domain binds As(3+) first followed by the beta domain. At room temperature (295 K) and pH 3.5, the sequential individual rate constants, k(n) (n = 1-6) for the As(3+)-metalation of beta alpha hMT starting at k(1beta alpha) are 25, 24, 19, 14, 8.7, and 3.7 M(-1)s(-1). The six rate constants follow an almost linear trend directly dependent on the number of unoccupied sites for the incoming metal. Analysis of the temperature-dependent kinetic electrospray ionization mass spectra data allowed determination of the activation energy for the formation of As(1)-H(17)-beta alpha hMT (14 kJ mol(-1)) and As(2-6)-beta alpha hMT (22 kJ mol(-1)). On the basis of the increased rate of metalation for the two-domain protein when compared with the isolated single-domain, we propose that there is an evolutionary advantage for the two-domain MT structures in higher organism, which allows MT to bind metals faster and, therefore, be a more efficient metal scavenger.