6n21

Publication Abstract from PubMed

Carotenoid cleavage dioxygenases (CCDs) use a non-heme 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 the 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 ligand to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism.

Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases.,Khadka N, Farquhar ER, Hill HE, Shi W, von Lintig J, Kiser PD J Biol Chem. 2019 May 28. pii: RA119.007535. doi: 10.1074/jbc.RA119.007535. PMID:31138651^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.