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This content will become publicly available on March 25, 2026

Title: Spectroscopy and crystallography define carotenoid oxygenases as a new subclass of mononuclear non-heme FeII enzymes
Carotenoid cleavage dioxygenases (CCDs) are non-heme FeII enzymes that catalyze the oxidative cleavage of alkene bonds in carotenoids, stilbenoids, and related compounds. How these enzymes control the reaction of O2 with their alkene substrates is unclear. Here, we apply spectroscopy in conjunction with X-ray crystallography to define the iron coordination geometry of a model CCD, CAO1, in its resting state and following substrate binding and coordination sphere substitutions. Resting CAO1 exhibits a five-coordinate (5C), square pyramidal FeII center that undergoes steric distortion towards a trigonal bipyramidal geometry in the presence of piceatannol. Titrations with the O2-analog, nitric oxide (NO), show a >100-fold increase in iron-NO affinity upon substrate binding, defining a crucial role for the substrate in activating the FeII site for O2 reactivity. The importance of the 5C FeII structure for reactivity was probed through mutagenesis of the second-sphere Thr151 residue of CAO1, which occludes ligand binding at the sixth coordination position. A T151G substitution resulted in the conversion of the iron center to a six-coordinate (6C) state and a 135-fold reduction in apparent catalytic efficiency towards piceatannol compared to the wild-type enzyme. Substrate complexation resulted in partial 6C to 5C conversion, indicating solvent dissociation from the iron center. Additional substitutions at this site demonstrated a general functional importance of the occluding residue within the CCD superfamily. Taken together, these data suggest an ordered mechanism of CCD catalysis occurring via substrate-promoted solvent replacement by O2. CCDs thus represent a new class of mononuclear non-heme FeII enzymes.  more » « less
Award ID(s):
2107713
PAR ID:
10579589
Author(s) / Creator(s):
; ; ; ; ;
Corporate Creator(s):
Editor(s):
NA
Publisher / Repository:
Elsevier
Date Published:
Journal Name:
Journal of Biological Chemistry
ISSN:
0021-9258
Page Range / eLocation ID:
108444
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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