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

Carotenoid cleavage dioxygenases (CCDs) are non-heme Fe enzymes that catalyze the oxidative cleavage of alkene bonds in carotenoids, stilbenoids, and related compounds. How these enzymes control the reaction of dioxygen (O) 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 center that undergoes steric distortion toward a trigonal bipyramidal geometry in the presence of piceatannol. Titrations with the O-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 site for O reactivity. The importance of the 5C Fe 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 O. CCDs thus represent a new class of mononuclear non-heme Fe enzymes.