The nature of O2 activation by the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylic acid oxidase Liviu M. Mirica and Judith P. Klinman* Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, CA 94720 Contributed by Judith P. Klinman, December 10, 2007 (sent for review November 15, 2007) Ethylene is a plant hormone important in many aspects of plant studied extensively, and it generally is accepted that O2 activation growth and development such as germination, fruit ripening, and at the iron center is linked to oxidative decarboxylation of the senescence. 1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase ␣KG, ultimately forming an FeIVAO species as the reactive (ACCO), an O2-activating ascorbate-dependent nonheme iron en- intermediate (14–17). This high-valent Fe/O2 species functions zyme, catalyzes the last step in ethylene biosynthesis. The O2 as a ‘‘generic’’ oxidant for a wide range of oxidation/oxygenation activation process by ACCO was investigated using steady-state reactions. kinetics, solvent isotope effects (SIEs), and competitive oxygen In contrast to the ␣KG-dependent enzymes, where ␣KG kinetic isotope effects (18O KIEs) to provide insights into the nature rather than substrate binds to the metal center, spectroscopic of the activated oxygen species formed at the active-site iron studies of ACCO have shown that ACC coordinates to the iron center and its dependence on ascorbic acid. The observed large 18O center via both its amino and carboxylate groups (10, 18).† This -KIE of 1.0215 ؎ 0.0005 strongly supports a rate-determining step finding implies a distinctly different mode for reductant inter formation of an FeIVAO species, which acts as the reactive inter- action with ACCO than for most other nonheme iron enzymes. mediate in substrate oxidation. The large SIE on kcat/Km(O2)of Magnetic circular dichroism (MCD) studies indicate that the suggests that formation of this FeIVAO species is linked active-site iron center is six-coordinate in the resting state 0.9 ؎ 5.0 to a rate-limiting proton or hydrogen atom transfer step. Based on Fe(II)-ACCO (19). On addition of ACC and ascorbate, the iron 18 the observed decrease in SIE and O KIE values for ACCO at center becomes five-coordinate, allowing for O2 binding and limiting ascorbate concentrations, ascorbate is proposed to bind in activation. These results are in line with steady-state kinetic a random manner, depending on its concentration. We conclude analyses that suggest an ordered process where ACC binding to that ascorbate is not essential for initial O binding and activation 2 ACCO must precede O2 binding, although it could not be but is required for rapid FeIVAO formation under catalytic turn- distinguished whether ascorbate binds before ACC or after over. Similar studies can be performed for other nonheme iron O2 (12). enzymes, with the 18O KIEs providing a kinetic probe into the Single-turnover experiments showed that a substoichiometric chemical nature of Fe/O2 intermediates formed in the first irre- amount of ethylene was formed in the absence of ascorbate, with versible step of the O activation. 2 the presence of either CO2 or bicarbonate being essential for enzyme turnover (13). Although ascorbate was proposed to act 2 His, 1-Asp proteins ͉ high-valent iron oxo species ͉ mainly as a reductant for restoring the iron to the ferrous state, oxygen-18 kinetic isotope effects ͉ nonheme iron enzymes the rate of ethylene formation under single-turnover conditions was significantly lower than under steady-state conditions, sug- thylene is a major plant hormone important in many aspects gesting that ascorbate is needed for O2 activation under catalytic Eof plant growth and development such as germination, fruit turnover (Fig. 2). ripening, and senescence (1). The ability to control ethylene From these accumulated results, it is clear that the complexity formation in a time-dependent manner would have far-reaching of the ACCO chemistry presents a challenge for mechanistic economic, agricultural, and environmental implications. The last enzymologists and requires further examination. In this study, step in the biosynthesis of ethylene, the two electron oxidation steady-state kinetics, solvent isotope effects (SIEs), and com- of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, petitive oxygen kinetic isotope effects (18O KIEs) are used to CO2, and HCN, is catalyzed by ACC oxidase (ACCO) (2–4). provide insights into the rate-limiting steps contributing to the This reaction also requires the concomitant reduction of O2 to interaction of O2 with ACCO, the nature of the Fe/O2 interme- water and the oxidation of ascorbate to dehydroascorbate (Fig. diates, and the role of ascorbate in O2 activation. Of particular IV 1), whereas CO2 (or bicarbonate) has been shown to act as an interest is the investigation of the Fe AO species formation and activator for ethylene formation (5). its proposed role in substrate oxidation (20). ACCO belongs to the family of O2-activating nonheme iron enzymes. Although the sequence homology among these en- Results zymes is not high, all their active sites contain a single ferrous ion SIEs on Steady-State Parameters. The kinetic parameters for bound in a tridendate ligand arrangement referred to as a ACCO have been measured by monitoring the amount of O2 ‘‘2-His-1-carboxylate facial triad’’ (4). A crystal structure of consumed by using a Clark electrode. The obtained kinetic ACCO has been reported (6), revealing a solvent-exposed active site and confirming the ligation of the iron center (His-177, Asp-179, and His-234) inferred from mutagenesis studies (7), Author contributions: L.M.M. and J.P.K. designed research; L.M.M. performed research; and the general jellyroll motif found in other nonheme iron and L.M.M. and J.P.K. wrote the paper. enzymes (8, 9). The authors declare no conflict of interest. What distinguishes ACCO from the other enzymes of this Freely available online through the PNAS open access option. family is the two-electron donating cosubstrate needed for *To whom correspondence should be addressed. E-mail: [email protected]. † reducing O2 to water: ascorbate in the case of ACCO (10–13) It also was shown that ACC and O2 could bind simultaneously to the iron, with ascorbate and ␣-ketoglutarate (␣KG) in almost all other known examples proposed to bind to the enzyme for efficient electron transfer (10, 13). (4). The mechanism of ␣KG-dependent enzymes has been © 2008 by The National Academy of Sciences of the USA 1814–1819 ͉ PNAS ͉ February 12, 2008 ͉ vol. 105 ͉ no. 6 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711626105 Downloaded by guest on September 23, 2021 high-valent FeIVAO species in the first irreversible step (see below). The measured 18O KIE for the much slower background O2 consumption caused by ascorbate and iron in the absence of enzyme is 1.0111 Ϯ 0.0002 (data not shown), excluding an artificial inflation of the ACCO 18O KIE value from the non- enzymatic reactions. Fig. 1. The reaction catalyzed by ACCO. 18 SIE on O KIE. To test the role of protons during O2 activation, the 18 Ϫ1 O KIE also was measured in D2O. Because of lower activity of parameters [kcat ϭ 36.7 Ϯ 1.1 min , Km(ACC) ϭ 92 Ϯ 10 ␮M, ACCO in D2O, data points for the ACCO reaction were Km(Asc) ϭ 5.5 Ϯ 0.7 mM, Km(O2) ϭ 67 Ϯ 6 ␮M] are similar to our previous results (12). Because both ACC and ascorbate have collected only for fractional conversions between 10% and 30%. The measured 18O KIE for ACCO in D O is 1.0209 Ϯ 0.0004 solvent-exchangeable protons, the only deuterium isotope ef- 2 (Fig. 4A). This value is within experimental error of the 18O KIE fects that can be measured are kinetic SIEs. Comparison of the in H O. The lack of a SIE on the 18O KIE of ACCO indicates kinetic parameters measured in both H O and D O revealed the 2 2 2 that solvent deuteration does not impact the relative activation presence of SIEs (Table 1). The measured values for D2Ok , cat barriers of the steps leading up to and including the first D2Ok /K (ACC), and D2Ok /K (Asc) are Ϸ2.3, identical cat m cat m irreversible step of k /K (O ). within experimental error, suggesting changes in hydrogen bond- cat m 2 ing between the reactants and the transition state. None of the Ascorbate Effect on SIE and 18O KIE. Ascorbate is an essential kinetic parameters has been found to vary between pH 6 and 8 substrate for ACCO and acts as a reductant to fully reduce O2 (data not shown), indicating the lack of a pKa effect as the origin D2O Ϯ to water. Its role in ACCO activity was tested by measuring the of the SIEs. Interestingly, the kcat/Km(O2) value is 5.0 0.9 kinetic parameters at lower ascorbate concentration (2 mM, (Table 1 and Fig. 3), an uncommonly large value for a SIE. Such 35% of its Km value). Because of lower activity of ACCO at this a value suggests that the rate-determining step in O2 activation ascorbate concentration, data points were collected only for involves either a proton-coupled electron transfer (PCET) or fractional conversions between 10% and 30%. Measurement of hydrogen atom transfer (HAT), as discussed below. the 18O KIE for ACCO under these conditions reveals a value of 1.0157 Ϯ 0.0004 (Fig. 4B), lower than the value obtained under 18 18 18 O KIE. The O KIE on kcat/Km(O2), kcat/Km(O2), was measured saturating ascorbate concentration (1.0215 Ϯ 0.0005).