Carotenoid cleavage oxygenases (CCOs) are non-heme iron enzymes that catalyze scission of alkene groups in carotenoids and stilbenoids to form biologically important products. CCOs possess a rare four-His iron center whose resting-state structure and interaction with substrates are incompletely understood. Here， we address this knowledge gap through a comprehensive structural and spectroscopic study of three phyletically diverse CCOs. The crystal structure of a fungal stilbenoid-cleaving CCO， CAO1， reveals strong similarity between its iron center and those of carotenoid-cleaving CCOs， but with a markedly different substrate-binding cleft. These enzymes all possess a five-coordinate high-spin Fe(II) cen... More
Carotenoid cleavage oxygenases (CCOs) are non-heme iron enzymes that catalyze scission of alkene groups in carotenoids and stilbenoids to form biologically important products. CCOs possess a rare four-His iron center whose resting-state structure and interaction with substrates are incompletely understood. Here， we address this knowledge gap through a comprehensive structural and spectroscopic study of three phyletically diverse CCOs. The crystal structure of a fungal stilbenoid-cleaving CCO， CAO1， reveals strong similarity between its iron center and those of carotenoid-cleaving CCOs， but with a markedly different substrate-binding cleft. These enzymes all possess a five-coordinate high-spin Fe(II) center with resting-state Fe-His bond lengths of ∼2.15 Å. This ligand set generates an iron environment more electropositive than those of other non-heme iron dioxygenases as observed by Mössbauer isomer shifts. Dioxygen (O) does not coordinate iron in the absence of substrate. Substrates bind away (∼4.7 Å) from the iron and have little impact on its electronic structure， thus excluding coordination-triggered O binding. However， substrate binding does perturb the spectral properties of CCO Fe-NO derivatives， indicating proximate organic substrate and O-binding sites， which might influence Fe-O interactions. Together， these data provide a robust description of the CCO iron center and its interactions with substrates and substrate mimetics that illuminates commonalities as well as subtle and profound structural differences within the CCO family.
