Proton Delivery to Ferryl Heme in a Heme Peroxidase: Enzymatic Use of the Grotthuss Mechanism

We test the hypothesized pathway by which protons are passed from the substrate, ascorbate, to the ferryl oxygen in the heme enzyme ascorbate peroxidase (APX). The role of amino acid side chains and bound solvent is demonstrated. We investigated solvent kinetic isotope effects (SKIE) for the wild-type enzyme and several site-directed replacements of the key residues which form the proposed proton path. Kinetic constants for H2O2-dependent enzyme oxidation to Compound I, k1, and subsequent reduction of Compound II, k3, were determined in steady-state assays by variation of both H2O2 and ascorbate concentrations. A high value of the SKIE for wild type APX (Dk3 = 4.9) as well as a clear nonlinear dependence on the deuterium composition of the solvent in proton inventory experiments suggest the simultaneous participation of several protons in the transition state for proton transfer. The full SKIE and the proton inventory data were modeled by applying Gross–Butler–Swain–Kresge theory to a proton path inferred...