Biologically relevant mechanism for catalytic superoxide removal by simple manganese compounds

Abstract

Nonenzymatic manganese was first shown to provide protection against superoxide toxicity in vivo in 1981, but the chemical mechanism responsible for this protection subsequently became controversial due to conflicting reports concerning the ability of Mn to catalyze superoxide disproportionation in vitro. In a recent communication, we reported that low concentrations of a simple Mn phosphate salt under physiologically relevant conditions will indeed catalyze superoxide disproportionation in vitro. We report now that two of the four Mn complexes that are expected to be most abundant in vivo, Mn phosphate and Mn carbonate, can catalyze superoxide disproportionation at physiologically relevant concentrations and pH, whereas Mn pyrophosphate and citrate complexes cannot. Additionally, the chemical mechanisms of these reactions have been studied in detail, and the rates of reactions of the catalytic removal of superoxide by Mn phosphate and carbonate have been modeled. Physiologically relevant concentrations of these compounds were found to be sufficient to mimic an effective concentration of enzymatic superoxide dismutase found in vivo. This mechanism provides a likely explanation as to how Mn combats superoxide stress in cellular systems.

Scheme 1.

Reduction of MTS by O₂⁻ to form MF. The reduction can be inhibited by SOD. MTS and MF structures in SI Text.

Fig. 1.

MnHCO₃⁺ catalyzes the disappearance of O₂⁻, but Mn(II) citrate does not as measured by MTS reduction. O₂⁻ generated at 0.45 μM⋅s⁻¹ by ⁶⁰Co. [O₂⁻] was determined by measuring MF formation. The arrows and symbols below the x axis show the point of stoichiometric equivalence between O₂⁻ and Mn(II). Solutions contained 0, 25, 50, or 100 μM Mn, 0.5 M ethanol, 150 μM MTS, and 50 mM citrate, pH 7 (A), or 50 mM carbonate, pH 8 (B).

Fig. 2.

MnHCO₃⁺ and MnHPO₄ do not form an oxidized Mn final product when they react with O₂⁻ but Mn(II) citrate and MnP₂O₇²⁻ do. (A) Mn-citrate forms a Mn(III) product (open arrow), reduction of MTS (filled arrow) occurs only after Mn(III) oxidation. (B) MnHCO₃⁺ did not form a Mn(III), the reduction of MTS (filled arrow). (C) MnP₂O₇²⁻ was similar to Mn-citrate (A). (D) MnHPO₄ was similar to MnHCO₃⁺ (B). Solutions were irradiated with ⁶⁰Co to produce O₂⁻. Solutions contained 50 μM MTS, 50 μM MnSO₄, 0.5 M ethanol, 2 U/mL catalase, and 50 mM citrate (A), pyrophosphate (B), carbonate (C), or phosphate (D). All solutions were adjusted to pH 7 except carbonate (C), which was pH 8.7.

Fig. 3.

Determination of rate constants and intermediate spectra by pulse radiolysis. (A) Spectra of different species in the reaction of O₂⁻ with MnHPO₄. The absorption bands were measured immediately after the pulse (green triangles), 2 ms after the pulse (blue squares), and 20 ms after the pulse (red circles). The pulse radiolysis trace at 280 nm (B) and 230 nm (C) were made by piecing together a 20-ms and a 500-ms time-scale trace. The solution contained 50 mM phosphate, 50 μM MnSO₄, 0.5 M ethanol, and ∼2.4 μM O₂⁻ (pH 7.0).

Fig. 4.

The forward reaction (pseudo-first-order) of Mn(II) phosphate (blue, k₃), MnHCO₃⁺ (green, k₃), and Mn(II) citrate (cyan, k₁) with O₂⁻ is fast, whereas the back reaction is slower, as determined by pulse radiolysis. Rates depend on initial [O₂⁻] (1–10 μM). Solutions contained 0.5 M ethanol and 50 mM phosphate, carbonate, or citrate. All solutions were adjusted to pH 7, except carbonate, which was pH 8.7.

Scheme 2.

Proposed noncatalytic mechanism of O₂⁻ reaction with MnP₂O₇²⁻ or Mn(II) citrate. L, citrate/pyrophosphate.

Scheme 3.

Proposed catalytic mechanism of MnHPO₄ and MnHCO₃⁺ disproportionation of O₂. L, phosphate/carbonate. Anionⁿ⁻ is additional bound L.

Fig. 5.

Kinetics determined by pulse radiolysis. (A and B) Dependence of k₄ and k₅ on [HPO₄²⁻]. (C and D) Dependence of k₄ and k₅ on [HCO₃⁻]. Rates were fitted on initial [O₂⁻] (1–10 μM). All solutions contained 0.5 M ethanol and 100 μM MnSO₄, and phosphate solutions were pH 7 and carbonate pH 8.3.

Fig. 6.

Spectra of MnOO⁺ intermediates are similar. (A) First intermediates and O₂⁻ (black line) spectra. (B–D) Second intermediates spectra. Pulse radiolysis was used to determine the extinction coefficient for each species at the indicated wavelengths for points in B–D. The dashed lines are Mn(III) samples made from Mn(III) acetate and 0.5 M carbonate or phosphate (C and D) or from ⁶⁰Co irradiation (B). Pulse radiolysis: Mn(II) citrate (circles), MnHCO₃⁺ (squares), and MnHPO₄ (triangles), were made by adding 50 μM MnSO₄ (MnHPO₄ and MnHCO₃⁺) or 200 μM MnSO₄ [Mn(II) citrate] to 50 mM of the corresponding anion. All solutions contained 0.5 M ethanol and were pH 7 except MnHCO₃⁺, which was pH 7.7.

Fig. 7.

Computer modeling of the catalytic superoxide dismutase activity of Mn(II) phosphate and carbonate. CuZnSOD (yellow squares) was modeled at 1 μM in all cases. The rate of autodismutation of superoxide at pH 7 was used as a competing reaction to determine noncatalytic reaction of superoxide (purple diamonds). (A and B) Superoxide was modeled as a 25-μM superoxide burst, MnHPO₄ (blue circles) or MnHCO₃ (green triangles) removed an amount of superoxide equal to that removed by 1 μM CuZnSOD. (C and D) Superoxide was modeled as a slow constant flux of 6.0 μM⋅s⁻¹. [Mn] with 5 mM HPO₄²⁻ or 5 mM HCO₃⁻ were chosen to have a steady-state [O₂⁻] identical to that of 1 μM CuZnSOD.