Ascorbate reacts with singlet oxygen to produce hydrogen peroxide

Abstract

Singlet oxygen is a highly reactive electrophilic species that reacts rapidly with electron-rich moieties, such as the double bonds of lipids, thiols, and ascorbate (AscH-). The reaction of ascorbate with singlet oxygen is rapid (k = 3 x 10(8) M(-1) s(-1)). Here we have investigated the stoichiometry of this reaction. Using electrodes to make simultaneous, real-time measurements of oxygen and hydrogen peroxide concentrations, we have investigated the products of this reaction. We have demonstrated that hydrogen peroxide is a product of this reaction. The stoichiometry for the reactants of the reaction (1 1O2 + 1AscH--->1H2O2 + 1dehydroascorbic) is 1:1. The formation of H2O2 results in a very different oxidant that has a longer lifetime and much greater diffusion distance. Thus, locally produced singlet oxygen with a half-life of 1 ns to 1 micros in a biological setting is changed to an oxidant that has a much longer lifetime and thus can diffuse to distant targets to initiate biological oxidations.

Figure 1. Singlet Oxygen reacts with ascorbate to produce H₂O₂ with a 1:1 stoichiometry

These plots show the real-time, simultaneous measurements of oxygen consumption (1) and hydrogen peroxide production (2) with ascorbate (1 mM) and Photofrin® (225 μg mL^-1) in metal-free phosphate buffer, pH 6.5. Visible light (hν; 350 J m^-2 s^-1) initiated oxygen consumption and production of H₂O₂. Little if any oxygen consumption is observed (3) in controls: In the absence of light, or Photofrin, or ascorbate, or light but no Photofrin; of course no H₂O₂ is formed.

Figure 2. Sodium azide suppressed oxygen consumption and H₂O₂ production during photo-oxidation of ascorbate

These plots show the real-time, simultaneous measurements of oxygen consumption and hydrogen peroxide production with ascorbate (1 mM) and Photofrin® (225 μg mL^-1) in metal-free phosphate buffer, pH 6.5. Visible light (hν, 350 J m^-2 s^-1) initiated oxygen consumption (1,2) and production of H₂O₂ (3,4). The addition of NaN₃ (1 mM) is indicated by arrow for the curves 2, 4. As seen, this addition created a disturbance with the H₂O₂-electrode, while no such disturbance was seen with the O₂-electode. However, results are based on the slope of the linear portion of the curves before and after addition of azide. All solutions were air-saturated.

Figure 3. A proposed mechanism for the production of H₂O₂

In a near neutral aqueous solution, singlet oxygen may react with the electron-rich carbon-3 of ascorbate. This intermediate will have high electron density at C-2, leading to a rearrangement of the hydroperoxyl moiety, allowing ketal formation at C-3, resulting in rapid oxidation of ascorbate to form dehydroascorbic (in the ketal form) and H₂O₂ [19]. Kwon and Foote observed both the carbon-2 and carbon-3 adduct when ascorbate was present as the di-acid, with the C-2-OOH adduct in greater abundance. The actual nature of the intermediate maybe more complex, due to the well-known hydrolysis reactions of ascorbate and DHA [24]. Because of the stability of the ascorbate radical, a small fraction of ascorbate may reduce singlet oxygen by one-electron, forming superoxide and the ascorbate free radical [20].