Properties of the reaction of chromate with metallothionein.

Abstract

The reactivity of chromate or Cr(VI) with rabbit liver metallothionein (MT) was explored in this study. Zn(7)-MT reacts very slowly with Cr(VI) in a process characterized by a second-order rate constant of 3.9 x 10(-)(4) M(-)(1) s(-)(1). During the reaction, Zn(2+) was released from the protein. In contrast, apo-MT reduces chromate quicker and in this reaction is much more effective as a reducing agent, when compared to Cys or GSH. The kinetics are consistent with a reaction pathway involving an initial binding step followed by the reduction of Cr(VI). In the process, MT sulfhydryl groups were oxidized at the same rate that Cr(VI) disappeared. A Cr(V) intermediate was detected by EPR spectroscopy immediately upon mixing apo-MT with Cr(VI). The Cr(V) signal decayed during the reaction but was quite stable and could be observed for hours once the supply of thiols was depleted. The g values for the Cr(V) species were 2.014 and 1.987. The kinetics of the reaction of Cr(VI) and the concentration of the intermediate Cr(V) signal were independent of the oxygen concentration and were unaffected by the presence of superoxide dismutase, catalase, or DMSO. In the presence of oxygen, oxy radicals were generated according to ESR spin-trapping experiments with 5,5'-dimethyl-1-pyrroline-N-oxide. Superoxide dismutase decreased and catalase or DMSO largely inhibited the formation of the spin-trapped adduct. Cr(III), the presumed final species of the Cr(VI) reduction, formed a stable complex with apo-MT in the absence of oxygen with an average stoichiometry of two Cr ions bound per protein molecule. Upon addition of O(2), the complex slowly dissociated.