An intriguing aspect of the green fluorescent protein (GFP) is the autocatalytic post-translational modification that results in the formation of its chromophore. Numerous experimental and theoretical studies indicate that cyclization is the first and the most important step in the maturation process. In this work, two proposed mechanisms for the cyclization were investigated by using the hybrid density functional theory method B3LYP. Cluster models corresponding to the two mechanisms proposed by Wachter et al. [J. Biol. Chem. 2005, 280, 26248-26255] are constructed on the basis of the X-ray crystal structure (PDB entry 2AWJ) and corresponding reaction path potential energy profiles for the two cyclization mechanisms are presented. Our results suggest that the backbone condensation initiated by deprotonation of the Gly67 amide nitrogen is easier than deprotonation of the Tyr66 α-carbon. Moreover, Arg96 fulfills the role of stabilizing the enolate moiety, and Glu222 plays the role of a general base. The formation of the cyclized product is found to be 16.0 and 18.6 kcal/mol endothermic with respect to the two models, which is in agreement with experimental observation
