A concerted electron /proton transfer mechanism for the oxidation of ascorbic acid

The reaction between ascorbic acid (AH-) and cytochrome b561 is a good model for redox reactions between metalloproteins and specific organic substrates. Treatment with diethylpyrocarbonate (DEPC) inhibits reduction of cytochrome b561 by AH-, but activity can be restored by treatment with hydroxylamine suggesting the involvement of an essential histidine residue. DEPC inactivates cytochrome bss1 more rapidly at alkaline pH, consistent with histidine modification. DEPC does not affect the absorption spectrum of cytochrome b561 or change the midpoint reduction potential confirming that the heme is unaffected. AH- protects the cytochrome from inactivation by DEPC indicating that the essential histidine is in the AH- binding site. Furthermore, DEPC treatment inhibits oxidation of the cytochrome by semidehydroascorbate but not by ferricyanide. This supports a mechanism in which AH- loses a hydrogen atom by donating a proton to histidine and transferring an electron to the heme.;To test this mechanism, the ability of histidine or imidazole to facilitate AH- oxidation in other reactions was also examined. Ascorbate oxidase is inhibited by DEPC. The DEPC-inactivated enzyme catalyzes oxygen consumption in the presence of AH- and imidazole. Oxygen is reduced to H2O in this reaction, as catalase doesn't affect the rate. This rate is faster at pH 7.5 than at pH 5.5, indicating that the unprotonated imidazole catalyzes the reaction. A high concentration (200 mM) imidazole is required, however, consistent with the weak affinity between ascorbate and free histidine.;Histidine (imidazole) catalyzes slower oxygen consumption in the presence of AH- but in the absence of ascorbate oxidase. Again, a high concentration of histidine or imidazole is required, and the pH must be above 7. Catalase reduces the rate of oxygen consumption by 50%, indicating that O2 is reduced to H2O2. This is consistent with the reduction of O2 by ascorbate in two one-electron steps.;Histidine (imidazole) promotes the oxidation of ascorbate as examined by cyclic voltammetry. The oxidation peak of the cyclic voltammogram of AH- shifts to lower potentials in the presence of high concentrations of histidine or imidazole. The shift is greater above pH 7, confirming the requirement for unprotonated imidazole.;These results are consistent with a concerted proton/electron transfer mechanism in which imidazole acts as a proton acceptor allowing electron transfer from AH- to take place.