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© 2021 American Chemical SocietyCysteine dioxygenase (CDO) is a nonheme mononuclear iron enzyme, which catalyzes the oxidation of cysteine to cysteine sulfinic acid. Crystal structure studies of mammalian CDO showed that there is a cross–linked cysteine–tyrosine (Cys–Tyr) cofactor in its active site. Moreover, the formation of the Cys–Tyr cofactor requires the metal cofactor (Fe2+) and O2, and it was previously considered to substantially enhance the catalytic efficiency and half–life of CDO. Recently, a pure human CDO (F2–CDO) without including the Cys–Tyr cofactor was crystalized by the site–directed mutagenesis approach in the anaerobic condition. In this work, to gain insights into the formation mechanism of the Cys–Tyr cofactor and whether it can really promote the catalytic reactivity of CDO, a series of computational models have been constructed, and quantum mechanical/molecular mechanical (QM/MM) calculations have been performed. Our calculation results reveal that WT–CDO and F2–CDO follow different mechanisms for the formation of the Cys–Tyr cofactor. In F2–CDO, the cofactor formation contains the H–abstraction, C–S bond formation, intramolecular F migration, and aromatization of the residue F2Y157, in which the Fe–coordinate dioxygen can be recovered after the formation cofactor; however, in the WT–CDO, the cofactor formation shows some differences. During the reaction, hydrogen peroxide is generated, and the final aromatization requires the assistance of one water molecule. Furthermore, the overall barriers of cofactor formation are always higher than l–cysteine oxidation for both WT–CDO and F2–CDO irrespective of the absence or presence of the cofactor. Thus, we can theoretically confirm that the Cys–Tyr cofactor is not essential for the oxidation activity of CDO, and cofactor formation is just an accompanying reaction but not a prerequisite for the oxidation reaction. These results may provide useful information for understanding the catalysis of CDO.
