| Abstract
| - The 1 equiv reaction between ascorbic acid and cytochrome b561 is a good model for redoxreactions between metalloproteins (electron carriers) and specific organic substrates (hydrogen-atomcarriers). Diethyl pyrocarbonate inhibits the reaction of cytochrome b561 with ascorbate by modifying ahistidine residue in the ascorbate-binding site. Ferri/ferrocyanide can mediate reduction of DEPC-treatedcytochrome b561 by ascorbic acid, indicating that DEPC-inhibited cytochrome b561 cannot accept electronsfrom a hydrogen-atom donor like ascorbate but can still accept electrons from an electron donor likeferrocyanide. Ascorbic acid reduces cytochrome b561 with a Km of 1.0 ± 0.2 mM and a Vmax of 4.1 ± 0.8s-1 at pH 7.0. Vmax/Km decreases at low pH but is approximately constant at pH >7. The rate constant foroxidation of cytochrome b561 by semidehydroascorbate decreases at high pH but is approximately constantat pH <7. This suggests that the active site must be unprotonated to react with ascorbate and protonatedto react with semidehydroascorbate. Molecular modeling calculations show that hydrogen bonding betweenthe 2-hydroxyl of ascorbate and imidazole stabilizes the ascorbate radical relative to the monoanion. Theseresults are consistent with the following mechanism for ascorbate oxidation. (1) The ascorbate monoanionbinds to an unprotonated site (histidine) on cytochrome b561. (2) This complex donates an electron toreduce the heme. (3) The semidehydroascorbate anion dissociates from the cytochrome, leaving a protonassociated with the binding site. (4) The binding site is deprotonated to complete the cycle. In thismechanism, an essential role of the cytochrome is to bind the ascorbate monoanion, which does not reactby outer-sphere electron transfer in solution, and complex it in such a way that the complex acts as anelectron donor. Thermodynamic considerations show that no steps in this process involve large changesin free energy, so the mechanism is reversible and capable of fulfilling the cytochrome's function ofequilibrating ascorbate and semidehydroascorbate.
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