The nature of O2 activation by the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylic acid oxidase

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 O-2-activating ascorbate-dependent nonheme iron enzyme, catalyzes the last step in ethylene biosynthesis. The O-2 activation process by ACCO was investigated using steady-state kinetics, solvent isotope effects (SIEs), and competitive oxygen kinetic isotope effects (O-18 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 O-18 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(O-2) 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 O-18 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 O-2 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 O-18 KlEs providing a kinetic probe into the chemical nature of Fe/O-2 intermediates formed in the first irreversible step of the O-2 activation.