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Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: single turnover reaction

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Abstract

The final step in the biosynthesis of the plant hormone ethylene is catalyzed by the non-heme iron-containing enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). ACC is oxidized at the expense of O2 to yield ethylene, HCN, CO2, and two waters. Continuous turnover of ACCO requires the presence of ascorbate and HCO3 − (or an alternative form), but the roles played by these reagents, the order of substrate addition, and the mechanism of oxygen activation are controversial. Here these issues are addressed by development of the first functional single turnover system for ACCO. It is shown that 0.35 mol ethylene/mol Fe(II)ACCO is produced when the enzyme is combined with ACC and O2 in the presence of HCO3 − but in the absence of ascorbate. Thus, ascorbate is not required for O2 activation or product formation. Little product is observed in the absence of HCO3 −, demonstrating the essential role of this reagent. By monitoring the EPR spectrum of the sample during single turnover, it is shown that the active site Fe(II) oxidizes to Fe(III) during the single turnover. This suggests that the electrons needed for catalysis can be derived from a fraction of the initial Fe(II)ACCO instead of ascorbate. Addition of ascorbate at 10% of its K m value significantly accelerates both iron oxidation and ethylene formation, suggesting a novel high-affinity effector role for this reagent. This role can be partially mimicked by a non-redox-active ascorbate analog. A mechanism is proposed that begins with ACC and O2 binding, iron oxidation, and one-electron reduction to form a peroxy intermediate. Breakdown of this intermediate, perhaps by HCO3 −-mediated proton transfer, is proposed to yield a high-valent iron species, which is the true oxidizing reagent for the bound ACC.

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Notes

  1. The kinetic constant values at 2 µM ACCO concentration were also re-determined for the enzyme used in this study: K m for ascorbate=2.5±0.2 mM; K m for ACC=0.15±0.05 mM

  2. It is not possible to fit these g-values exactly to a particular E/D for the usual case of g 0=2.0 and no perturbations of the spin environment such as spin coupling were observed. We are currently investigating whether these factors are applicable to this system. Another possibility is that there is more than one species present, each with a different E/D value. The g=4.77 and 4.05 fit approximately to the middle doublet of an S=5/2, E/D=0.24 spin system. The E/D=0.24 spin system has expected g-values at 4.8, 4.06, 3.72, the last of which may be broad and not observed. In this case, the anomalous middle resonance observed at g=4.3 would derive from another minor S=5/2 species with an E/D of 0.33. Temperature and microwave power variations failed to further resolve these species

  3. The ethylene assay employed here uses chemical quench with TCA to terminate the reaction. This chemical quench step denatures the protein, which results in release of the reaction products. Thus, this enzyme assay reflects ethylene formation as opposed to ethylene release

Abbreviations

ACC:

1-aminocyclopropane-1-carboxylic acid

ACCO:

ACC oxidase

AEC:

1-amino-2-ethylcyclopropane-1-carboxylic acid

MOPS:

3-(N-morpholino)propanesulfonic acid

SAL:

saccharic acid 1,4-lactone

TCA:

trichloroacetic acid

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Acknowledgements

This work was supported by NIH Grants GM24689 (to J.D.L.) and GM33162 (to L.Q.). A.M.R. was supported in part by Training Grant GM08277

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Author notes

  1. Amy M. Rocklin

    Present address: Foley & Lardner Inc., Washington, DC, USA

  2. Hung-wen Liu

    Present address: Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas, Austin, TX , 78712, USA

  3. Keisuke Kato

    Present address: School of Pharmaceutical Science, Toho University, Miyama 2-2-1, Funabashi-shi, 274, Chiba , Japan

Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology, and Biophysics and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN , 55455, USA

    Amy M. Rocklin & John D. Lipscomb

  2. Department of Chemistry and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN , 55455, USA

    Keisuke Kato, Hung-wen Liu & Lawrence Que Jr.

Authors
  1. Amy M. Rocklin
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  2. Keisuke Kato
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  3. Hung-wen Liu
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  4. Lawrence Que Jr.
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  5. John D. Lipscomb
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Corresponding authors

Correspondence to Lawrence Que Jr. or John D. Lipscomb.

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Rocklin, A.M., Kato, K., Liu, Hw. et al. Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: single turnover reaction. J Biol Inorg Chem 9, 171–182 (2004). https://doi.org/10.1007/s00775-003-0510-3

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  • DOI: https://doi.org/10.1007/s00775-003-0510-3

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