Kinetic and mechanistic studies detailing the oxidation of substrates derived from the 20 natural amino acids by the ferryl complex [Fe(IV)(O)(N4Py)](2+) are described. Substrates of the general formula Ac-AA-NHtBu were treated with the ferryl complex under identical conditions ([Ac-AA-NHtBu] = 10 mM, [Fe] = 1 mM, 1:1 H(2)O/CH(3)CN), and pseudo-first-order rate constants were obtained. Relative rate constants calculated from these data illustrated the five most reactive substrates; in order of decreasing reactivity were those derived from Cys, Tyr, Trp, Met, and Gly. Second-order rate constants were determined for these substrates by varying substrate concentration under pseudo-first-order conditions. Substrates derived from the other natural amino acids did not display significant reactivity, accelerating decomposition of the ferryl complex at a rate less than 10 times that of the control reaction with no substrate added. Ferryl decomposition rates changed in D(2)O/CD(3)CN for the Cys, Tyr, and Trp substrates, giving deuterium kinetic isotope effects of 4.3, 29, and 5.2, respectively, consistent with electron-transfer, proton-transfer (Cys and Trp), or hydrogen atom abstraction (Tyr) mechanisms. Decomposition rates for [Fe(IV)(O)(N4Py)](2+) in the presence of the Met and Gly substrates were identical in H(2)O/CH(3)CN versus D(2)O/CD(3)CN solvents. A deuterium kinetic isotope effect of 4.8 was observed with the labeled substrate 2,2-d(2)-Ac-Gly-NHtBu, consistent with [Fe(IV)(O)(N4Py)](2+) abstracting an alpha-hydrogen atom from Ac-Gly-NHtBu and generating a glycyl radical. Abstraction of alpha-hydrogen atoms from amino acid substrates other than Gly and oxidation of side chains contained in the amino acids other than Cys, Tyr, Trp, and Met were slow by comparison.