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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. 

Enzymes canusually be unambiguously assigned to one of seven classes specifying the basic chemistry of their catalyzed reactions. Less frequently, two or more reaction classes are catalyzed by a single enzyme within one active site. Two examples are an isomerohydrolase and an isomero-oxygenase that catalyze isomerization-coupled reactions crucial for production of visionsupporting 11-cis-retinoids. In these enzymes, isomerization is obligately paired and mechanistically intertwined with a second reaction class. A handful of other enzymes carrying out similarly coupled isomerization reactions have been described, some ofwhich havebeensubjectedtodetailedstructure–function analyses. Herein we reviewtheserarefied enzymes,focusingonthe mechanisticand structural basis of their reaction coupling with the goal of revealing catalytic commonalities. 

For sustained vision, photoactivated rhodopsin (Rho*) must undergo hydrolysis and release of all- trans -retinal, producing substrate for the visual cycle and apo-opsin available for regeneration with 11- cis -retinal. The kinetics of this hydrolysis has yet to be described for rhodopsin in its native membrane environment. We developed a method consisting of simultaneous denaturation and chromophore trapping by isopropanol/borohydride, followed by exhaustive protein digestion, complete extraction, and liquid chromatography–mass spectrometry. Using our method, we tracked Rho* hydrolysis, the subsequent formation of N -retinylidene-phosphatidylethanolamine ( N -ret-PE) adducts with the released all- trans -retinal, and the reduction of all- trans -retinal to all- trans -retinol. We found that hydrolysis occurred faster in native membranes than in detergent micelles typically used to study membrane proteins. The activation energy of the hydrolysis in native membranes was determined to be 17.7 ± 2.4 kcal/mol. Our data support the interpretation that metarhodopsin II, the signaling state of rhodopsin, is the primary species undergoing hydrolysis and release of its all- trans -retinal. In the absence of NADPH, free all- trans -retinal reacts with phosphatidylethanolamine (PE), forming a substantial amount of N -ret-PE (∼40% of total all- trans -retinal at physiological pH), at a rate that is an order of magnitude faster than Rho* hydrolysis. However, N -ret-PE formation was highly attenuated by NADPH-dependent reduction of all- trans -retinal to all- trans -retinol. Neither N -ret-PE formation nor all- trans -retinal reduction affected the rate of hydrolysis of Rho*. Our study provides a comprehensive picture of the hydrolysis of Rho* and the release of all- trans -retinal and its reentry into the visual cycle, a process in which alteration can lead to severe retinopathies.