Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases
Abstract
Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O2, or 2) solvent remains bound to iron but changes its equilibrium position to allow O2 binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H2O versus D2O) on two stilbenoid-cleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (kH/kD ~ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (kH/kD ~ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5–8.5 and that the Fe(II)–aquo bond is minimally altered by substrate binding. In this study, we pinpointed themore »
- Authors:
-
[1]
- Case Western Reserve Univ., Cleveland, OH (United States)
- Brookhaven National Lab. (BNL), Upton, NY (United States); Case Western Reserve Univ., Cleveland, OH (United States)
- Publication Date:
- Research Org.:
- Brookhaven National Laboratory (BNL), Upton, NY (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)
- OSTI Identifier:
- 1679969
- Report Number(s):
- BNL-219982-2020-JAAM
Journal ID: ISSN 0021-9258
- Grant/Contract Number:
- SC0012704; GM103403; RR029205; AC02-06CH11357; AC02-76SF00515; P41-GM-103393; KP1605010; KC0401040; EB009998
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Journal of Biological Chemistry
- Additional Journal Information:
- Journal Volume: 294; Journal Issue: 27; Journal ID: ISSN 0021-9258
- Publisher:
- American Society for Biochemistry and Molecular Biology
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; enzyme kinetics; iron; crystallography; carotenoid; dioxygenase; non-heme iron; O2 activation; solvent isotope effect; stilbenoid X-ray; absorption spectroscopy
Citation Formats
Khadka, Nimesh, Farquhar, Erik R., Hill, Hannah E., Shi, Wuxian, von Lintig, Johannes, and Kiser, Philip D. Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases. United States: N. p., 2019.
Web. doi:10.1074/jbc.ra119.007535.
Khadka, Nimesh, Farquhar, Erik R., Hill, Hannah E., Shi, Wuxian, von Lintig, Johannes, & Kiser, Philip D. Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases. United States. https://doi.org/10.1074/jbc.ra119.007535
Khadka, Nimesh, Farquhar, Erik R., Hill, Hannah E., Shi, Wuxian, von Lintig, Johannes, and Kiser, Philip D. Tue .
"Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases". United States. https://doi.org/10.1074/jbc.ra119.007535. https://www.osti.gov/servlets/purl/1679969.
@article{osti_1679969,
title = {Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases},
author = {Khadka, Nimesh and Farquhar, Erik R. and Hill, Hannah E. and Shi, Wuxian and von Lintig, Johannes and Kiser, Philip D.},
abstractNote = {Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O2, or 2) solvent remains bound to iron but changes its equilibrium position to allow O2 binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H2O versus D2O) on two stilbenoid-cleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (kH/kD ~ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (kH/kD ~ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5–8.5 and that the Fe(II)–aquo bond is minimally altered by substrate binding. In this study, we pinpointed the origin of the differential kinetic behaviors of NOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism.},
doi = {10.1074/jbc.ra119.007535},
journal = {Journal of Biological Chemistry},
number = 27,
volume = 294,
place = {United States},
year = {Tue May 28 00:00:00 EDT 2019},
month = {Tue May 28 00:00:00 EDT 2019}
}
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Figures / Tables:
Figure 1: Resting state structure of the CA01 active site. The iron center of CAO1, a representative stilbene-cleaving CCD, is shown in the figure. Four histidine residues (His-197, -248, -313, and-510) coordinate the Fe(II) ion with an exchangeable aquo ligand as the fifth coordinating ligand. The figure was produced usingmore »
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