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Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):1814-9. doi: 10.1073/pnas.0711626105. Epub 2008 Jan 31.

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

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1
Department of Chemistry, University of California, Berkeley, CA 94720, USA.

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 kcat/Km(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.

PMID:
18238897
PMCID:
PMC2538845
DOI:
10.1073/pnas.0711626105
[Indexed for MEDLINE]
Free PMC Article
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