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 4-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 5-coordinate high-spin Fe(II) center with resti... 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 4-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 5-coordinate high-spin Fe(II) center with resting state Fe-His bond lengths of ~2.15 Å. This ligand set generates a more electropositive iron environment relative to other non-heme iron dioxygenases as observed by Mössbauer isomer shifts. Dioxygen (O2) 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 O2 binding. However, substrate binding does perturb the spectral properties of CCO Fe-NO derivatives indicating proximate organic substrate and O2 binding sites, which might influence Fe-O2 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.
