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. 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.
Highlights
Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates
The divergent kinetic behavior between NOV2 and CAO1 prompted us to examine their solvent kinetic isotope effect (sKIE) dependences in more detail
NOV2 and CAO1 crystallographic and Mossbauer spectroscopy studies have indicated that solvent remains bound to iron in the presence of organic substrate [14], we considered the possibility that substrate binding may weaken the metal–aquo bond in CAO1 more so than in NOV2, altering the rate-limiting step
Summary
Crystal structures of cobalt-substituted CAO1 in complex with stilbenes showed that substrates bind ϳ4 –5 Å away from the metal center, allowing the solvent to remain bound to the metal without inducing major conformational changes in the protein structure [14]. Comparative Mossbauer studies on NOV2, CAO1, and ACO and their respective ES complexes showed only minor differences in the electronic environment of the active-site Fe(II) atom, indicating small changes in iron coordination upon substrate binding to the active site [14]. A normal KIE is expected if proton transfer occurs during a rate-limiting step of the reaction [22] These isotope effect studies were complemented with XAS and X-ray crystallography to further address O2 gating and the proton transfer events associated with CCD catalysis. Our data on NOV2 and CAO1, respectively, indicate that the dissociation of solvent ligand from the Fe(II) ion provides the binding site for O2, and a single proton transfer occurs during a step that is rate-limiting under some conditions. We demonstrate that a single active-site amino acid difference between these two enzymes alters the rate-limiting step of catalysis
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
