Spectroscopy and crystallography define carotenoid oxygenases as a new subclass of mononuclear non-heme FeII enzymes.

Abstract

Carotenoid cleavage dioxygenases (CCDs) are non-heme Fe^II enzymes that catalyze the oxidative cleavage of alkene bonds in carotenoids, stilbenoids, and related compounds. How these enzymes control the reaction of dioxygen (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 (Neurospora crassa carotenoid oxygenase 1), in its resting state and following substrate binding and coordination sphere substitutions. Resting CAO1 exhibits a five-coordinate (5C), square pyramidal Fe^II center that undergoes steric distortion toward a trigonal bipyramidal geometry in the presence of piceatannol. Titrations with the O₂-analog, nitric oxide, show a >100-fold increase in iron-nitric oxide affinity upon substrate binding, defining a crucial role for the substrate in activating the Fe^II site for O₂ reactivity. The importance of the 5C Fe^II 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 state and a 135-fold reduction in apparent catalytic efficiency toward piceatannol compared with the wildtype enzyme. Substrate complexation resulted in partial six-coordinate 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 Fe^II enzymes.