Long-lived chromium(IV) and chromium(V) metabolites in the chromium(VI)-glutathione reaction: NMR, ESR, HPLC, and kinetic characterization

The reduction of Cr(VI) by glutathione (GSH) in an excess of the oxidized form of glutathione (GSSG) proceeds through long-lived intermediates in the pH range 1.8-3.5. Time domain magnetic susceptibility measurements yielded a molar susceptibility of 3.6 +/- 0.2 x 10(-3) cm3/mol for the intermediates corresponding to a magnetic moment of 2.9 +/- 0.1-mu(B) at 25-degrees-C for the intermediate. The magnetic moment data along with the ESR measurements unequivocally establish that Cr(IV) is the dominant long-lived intermediate in this reaction and that Cr(V) accounts for < 5% of the intermediate. These intermediates are formed by two parallel pathways: an internal electron-transfer process within a Cr(VI)-thioester precursor (k(et) = 7.2 x 10(-3) s-1; K(formation) = 4.0 x 10(2) M-1 at pH 2.70) and a bimolecular reaction between the precursor complex and GSH (k = 0.89 M-1 s-1). Second-order rate constants increase with the increase in [H+], consistent with the established acid-catalyzed substitution mechanism for Cr(VI) centers. Conditional formation constants for the thioester complex (evaluated from this study and earlier reports by others) follow a similar trend. This trend in conditional formation constants indicates that the rate of dissociation for the precursor complex does not increase as much as the rate of its formation with an increase in [H+]. The chromium(IV) intermediate further reacts with GSH to form Cr(III) products. The magnetic susceptibility and magnetic moment for the Cr(III) products were evaluated to be 6.7 +/- 0.2 x 10(-3) cm3/mol and 4.0 +/- 0.1-mu(B). The decomposition of the intermediate is a second-order processes (first with respect to each reactant) (k = 0.13 M-1 s-1 at pH 2.7), and the second-order rate constants exhibit no appreciable change with the change in pH. Chromium(III) products obtained in GSSG or in self-buffered (GSH) show very similar spectral features: two absorption bands near 570 nm (epsilon = 31-33 M-1 cm-1) and 410 nm (epsilon = 26-31 M-1 cm-1). It is proposed that rapid ligand exchange or rearrangement takes place prior to the rate-limiting reduction of Cr(IV) by the tripeptide. Major chromium(III) product(s) are monocationic and contain at least 1 mol of GSH and 0.5 mol of GSSG/mol of Cr(III).