Ascorbic acid reduction of compound I of mammalian catalases proceeds via specific binding to the NADPH binding pocket.

Mammalian (Clade 3) catalases utilize NADPH as a protective cofactor to prevent one-electron reduction of the central reactive intermediate Compound I (Cpd I) to the catalytically inactive Compound II (Cpd II) species by re-reduction of Cpd I to the enzyme's resting state (ferricatalase). It has long been known that ascorbate/ascorbic acid is capable of reducing Cpd I of NADPH-binding catalases to Cpd II, but the mode of this one-electron reduction had hitherto not been explored. We here demonstrate that ascorbate-mediated reduction of Cpd I, generated by addition of peroxoacetic acid to NADPH-free bovine liver catalase (BLC), requires specific binding of the ascorbate anion to the NADPH binding pocket.