title: Arsenic binding to human metallothionein.
abstract: 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).
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7.
authors.forename: Thanh T
authors.initials: TT
authors.lastname: Ngu
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stillman
mesh_terms: D000595:Amino Acid Sequence; D001151:Arsenic / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics
chemical_list: D008668:Metallothionein; D001151:Arsenic