Mechanistic Studies on the Reaction of Nitrocobalamin with Glutathione: Kinetic evidence for formation of an aquacobalamin intermediate

Abstract

The essential but also toxic gaseous signaling molecule nitric oxide is scavenged by the reduced vitamin B₁₂ complex cob(II)alamin. The resulting complex, nitroxylcobalamin (NO^--Cbl(III)), is rapidly oxidized to nitrocobalamin (NO₂Cbl) in the presence of oxygen; however it is unlikely that nitrocobalamin is itself stable in biological systems. Kinetic studies on the reaction between NO₂Cbl and the important intracellular antioxidant, glutathione (GSH), are reported. In this study, a reaction pathway is proposed in which the β-axial ligand of NO₂Cbl is first substituted by water to give aquacobalamin (H₂OCbl⁺), which then reacts further with GSH to form glutathionylcobalamin (GSCbl). Independent measurements of the four associated rate constants k₁, k_-1, k₂, and k_-2 support the proposed mechanism. These findings provide insight into the fundamental mechanism of ligand substitution reactions of cob(III)alamins with inorganic ligands at the β-axial site.

Keywords: Bioinorganic chemistry; Cob(III)alamin; Kinetics; Reaction mechanisms; Vitamin B12.

Figure 1

Structure of cobalamins showing the two axial sites (upper = β, lower = α) with respect to the corrin ring.

Figure 2

(a) UV-vis spectra for the reaction of GSH (5.00 × 10⁻² M) with NO₂Cbl (5.0 × 10⁻⁵ M) at pH 4.00 (25.0 °C, 0.020 M NaOAc, I = 1.0 M, NaCF₃SO₃). Selected spectra for the reaction are shown every 1.00 min. (b) Plot of absorbance at 354 nm versus time for the experiment shown in 2(a). Data were fitted to a first-order rate equation, giving k_obs = (1.15 ± 0.07) × 10⁻² s⁻¹.

Figure 3

Plots of k_obs vs [GSH] at pH 4.00 (a) and 7.00 (b) for the reaction between NO₂Cbl (5.00 × 10⁻⁵ M) and glutathione (25.0 °C, 0.020 M NaOAc (a) or 0.020 M KH₂PO₄ (b), I = 1.0 M (NaCF₃SO₃)). Data in (a) were fitted to eq (2) in the text fixing k₋₂ = 7.4 × 10⁻⁴ s⁻¹, giving k₁ = (1.75 ± 0.02) × 10⁻² s⁻¹ and K = 94.5 ± 3.7 M⁻¹ at pH 4.00. Data in (b) were fitted to eq (2) fixing k₋₂ = 0 s⁻¹, giving k₁ = (1.73 ± 0.05) × 10⁻² s⁻¹ and K = 102.1 ± 9.7 M⁻¹ at pH 7.00.

Figure 4

Plot of k_obs vs [GSH] for the reaction between NO₂Cbl (5.0 × 10⁻⁵ M) and GSH in the presence of 5.00 × 10⁻⁴ M NaNO₂ at pH 7.00 (25.0°C, 0.020 M KH₂PO₄, 5.00×10⁻⁴ M NaNO₂, I = 1.0 M, NaCF₃SO₃). Data were fitted to eq (2) in the text fixing k₋₂ = 0, giving k₁ = (1.60 ±0.05) × 10⁻²s⁻¹ and K = 16.2 ± 0.2 M⁻¹.

Scheme 1

Proposed reaction pathway for the reaction of NO₂Cbl with GSH. Note that in aqueous solution H₂OCbl⁺ exists in equilibrium with HOCbl (pK_a(H₂OCbl⁺) = 7.8 ^[17]); however at the pH conditions of our kinetic experiments HOCbl formation is unimportant, since the values of the rate constants k₋₁ and k₂ are the same at pH 4.00 and 7.00.