The nature of O 2 activation by the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylic acid oxidase

Abstract

Ethylene is a plant hormone important in many aspects of plant growth and development such as germination, fruit ripening, and senescence. 1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO), an O₂-activating ascorbate-dependent nonheme iron enzyme, catalyzes the last step in ethylene biosynthesis. The O₂ activation process by ACCO was investigated using steady-state kinetics, solvent isotope effects (SIEs), and competitive oxygen kinetic isotope effects (¹⁸O KIEs) to provide insights into the nature of the activated oxygen species formed at the active-site iron center and its dependence on ascorbic acid. The observed large ¹⁸O KIE of 1.0215 ± 0.0005 strongly supports a rate-determining step formation of an Fe^IV O species, which acts as the reactive intermediate in substrate oxidation. The large SIE on k_cat/K_m(O₂) of 5.0 ± 0.9 suggests that formation of this Fe^IV O species is linked to a rate-limiting proton or hydrogen atom transfer step. Based on the observed decrease in SIE and ¹⁸O KIE values for ACCO at limiting ascorbate concentrations, ascorbate is proposed to bind in a random manner, depending on its concentration. We conclude that ascorbate is not essential for initial O₂ binding and activation but is required for rapid Fe^IV O formation under catalytic turnover. Similar studies can be performed for other nonheme iron enzymes, with the ¹⁸O KIEs providing a kinetic probe into the chemical nature of Fe/O₂ intermediates formed in the first irreversible step of the O₂ activation.