The number of reported cases of chronic
arsenic poisoning is on the rise throughout the world, making the study of the long-term effects of
arsenic critical. As(3+) binds readily to
biological thiols, including mammalian
metallothionein (MT), which is an ubiquitous
sulfur-rich
metalloprotein known to coordinate a wide range of metals. The two-domain mammalian
protein binds divalent metals (M) into two
metal-thiolate clusters with stoichiometries of M(3)S(cys9) (beta) and M(4)S(cys11) (alpha). We report that As(3+) binds with stoichiometries of As(3)S(cys9) (beta) and As(3)S(cys11) (alpha) to the recombinant human
metallothionein (rhMT)
isoform 1a
protein. Further, we report the complete kinetic analysis of the saturation reactions of the separate alpha and beta domains of rhMT with As(3+). Speciation in the metalation reactions was determined using time- and temperature-resolved electrospray ionization mass spectrometry. The binding reaction of As(3+) to the alpha and beta MT domains is shown to be noncooperative and involves three sequential, bimolecular metalation steps. The analyses allow for the first time the complete simulation of the experimental data for the stepwise metalation reaction of MT showing the relative concentrations of the
metal-free, apo MT and each of the As-MT intermediate species as a function of time and temperature. At room temperature (298 K) and pH 3.5, the individual rate constants for the first, second, and third As(3+) binding to apo-alphaMT are 5.5, 6.3, and 3.9 M(-)(1) s(-)(1) and for apo-betaMT the constants are 3.6, 2.0, and 0.6 M(-)(1) s(-)(1). The activation energy for formation of As(1)-H(6)-betaMT is 32 kJ mol(-)(1), for As(2)-H(3)-betaMT it
is 35 kJ mol(-)(1), for As(3)-betaMT it is 29 kJ mol(-)(1), for As(1)-H(8)-alphaMT it
is 33 kJ mol(-)(1), for As(2)-H(5)-alphaMT it is 29 kJ mol(-)(1), and for As(3)-H(2)-alphaMT it
is 23 kJ mol(-)(1).