Is vanadate reduced by thiols under biological conditions? Changing the redox potential of V(V)/V(IV) by complexation in aqueous solution

Abstract

Although dogma states that vanadate is readily reduced by glutathione, cysteine, and other thiols, there are several examples documenting that vanadium(V)-sulfur complexes can form and be observed. This conundrum has impacted life scientists for more than two decades. Investigation of this problem requires an understanding of both the complexes that form from vanadium(IV) and (V) and a representative thiol in aqueous solution. The reactions of vanadate and hydrated vanadyl cation with 2-mercaptoethanol have been investigated using multinuclear NMR, electron paramagnetic resonance (EPR), and UV-vis spectroscopy. Vanadate forms a stable complex of 2:2 stoichiometry with 2-mercaptoethanol at neutral and alkaline pH. In contrast, vanadate can oxidize 2-mercaptoethanol; this process is favored at low pH and high solute concentrations. The complex that forms between aqueous vanadium(IV) and 2-mercaptoethanol has a 1:2 stoichiometry and can be observed at high pH and high 2-mercaptoethanol concentration. The solution structures have been deduced based on coordination induced chemical shifts and speciation diagrams prepared. This work demonstrates that both vanadium(IV) and (V)-thiol complexes form and that redox chemistry also takes place. Whether reduction of vanadate takes place is governed by a combination of parameters: pH, solute- and vanadate-concentrations and the presence of other complexing ligands. On the basis of these results it is now possible to understand the distribution of vanadium in oxidation states (IV) and (V) in the presence of glutathione, cysteine, and other thiols and begin to evaluate the forms of the vanadium compounds that exert a particular biological effect including the insulin-enhancing agents, antiamoebic agents, and interactions with vanadium binding proteins.

Figure 1

⁵¹V (top) and ¹H (bottom) NMR spectra of solutions containing vanadate (12.0 mM) and 2-mercaptoethanol (12.0 mM) at pH 8.20±0.05. The spectra were recorded at 300 ± 1 K.

Figure 2

The pH dependence of complex [1] and vanadate monomer [V₁] is shown. Data points were obtained by quantitative ¹H and ⁵¹V NMR spectroscopy on solutions containing vanadate (4.0 mM) and 2-mercaptoethanol (12.0 mM) in the absence of KCl.

Figure 3

Plots of complexes [1] (signal at −362 ppm) and [2] (signal at −385 ppm) as a function of [H₂VO₄⁻]²[H₂L]² (here H₂L = HOCH₂CH₂SH) and [1]², respectively. Data points were obtained by quantitative ¹H and ⁵¹V NMR spectroscopy.

Figure 4

Solution structure for the 2:2 vanadium complex with 2-mercaptoethanol, 1, and two likely structures for the 4:4 vanadium complex with 2-mercaptoethanol, 2. Only one of several possible stereoisomers is shown.

Figure 5

⁵¹V NMR spectra of solutions containing KVO₃ (50 mM) and 2-mercaptoethanol (50 mM) at pH 8.25 ± 0.05. CH₃CN is added up to 87 wt%, and spectra at 0, 68, 75, 81, 84 and 87 wt% are shown.

Figure 6

EPR spectra recorded in solutions containing VOSO₄ (10 mM) and increasing concentrations of 2-mercaptoethanol (from 0 to 200 mM) in 0.60 M borate buffer (pH 9.6).

Figure 7

Normalized EPR spectra recorded in solutions with VOSO₄ (10 mM) and 2-mercaptoethanol (200 mM) at pH 3.2 to 13.4.

Figure 8

The concentration of complex 3 and [VO(OH)₃]⁻ ([VO(OH)₃(H₂O)₂]⁻) are shown as a function of pH. Data points were obtained by quantitative EPR spectroscopy in solutions with VOSO₄ (10 mM) and 2-mercaptoethanol (200 mM). No complex signal is observed outside the pH region 8 – 14. The smooth line combining the experimental points for [3] and [VO(OH)₃]⁻ is calculated using the stability constants and the reaction VO²⁺ + 2 HOCH₂CH₂S⁻ [VO(OCH₂CH₂S)₂]²⁻ + 2 H⁺.

Figure 9

Plot of complex [3] in borate buffer at pH 9.6 as a function of [VO(H₂O)₅²⁺][HL⁻]² (here HL⁻ = HOCH₂CH₂S⁻). The linear relationship indicates a 1:2 complex stoichiometry for 3. Other possibilities result in non-linear plots (Figure S5).

Figure 10

Four possible solution structures for complex 3.

Figure 11

Absorption spectra of the vanadate- and vanadyl-2-mercaptoethanol complexes prepared under anaerobic conditions from vanadate (20 mM) and 2-mercaptoethanol (200 mM) at pH 8.9 at different times after the solution preparation: (a) 5 min; (b) 5 hours; and (c) at pH 3.0 (not time dependent).

Figure 12

The [1] and [2], [HOCH₂CH₂SH] and [(HOCH₂CH₂S)₂] during the first 3 hours of reaction. The initial solution were added vanadate (200 mM), HOCH₂CH₂SH (200 mM), KCl (0.4 M) and borate buffer (0.4 M) at pH 8.95.