An interesting aspect of the green fluorescent protein(GFP)is its autocatalytic chromophore maturation.Numerous experimental studies have indicated that dehydration is the last step in the chromophore maturation process of wild-type GFP.Based on the crystal structure of wild-type GFP,the mechanism of the reverse reaction of dehydration was investigated by using density functional theory(DFT)in this study.The reaction mechanism involves two steps: the first step is hydroxyl anion attacking five-membered heterocyclic of chromophore,and the second step is the proton on guanidinium of Arg96 transferring to the β-carbon anion of Tyr66.Our results proposed that the reverse reaction of dehydration is endothermic,which means that the dehydration is exothermic.In addition,our results show that the rate-limiting step of the mechanism is the proton on guanidinium of Arg96 transferring to the β-carbon anion of Tyr66,which is consistent with the experimental observation.