In green fluorescent protein (GFP), chromophore biosynthesis is initiated by a spontaneous main-chain condensation reaction. Nucleophilic addition of the Gly 67 amide nitrogen to the Ser 65 carbonyl carbon is catalyzed by the protein fold and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg 96 and Glu 222 in enhanced GFP (EGFP). In the R96M variant, the rate of chromophore formation is greatly reduced (time constant ‫؍‬ 7.5 ؋ 10 3 h, pH 7) and exhibits pH dependence. In the E222Q variant, the rate is also attenuated at physiological pH (32 h, pH 7) but is accelerated severalfold beyond that of EGFP at pH 9 -10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of 1 h (pH 7-10). Mass spectrometric results for R96M and E222Q indicate accumulation of the pre-cyclization state, consistent with rate-limiting backbone condensation. The pH-rate profile implies that the Glu 222 carboxylate titrates with an apparent pK a of 6.5 in R96M and that the Gly 67 amide nitrogen titrates with an apparent pK a of 9.2 in E222Q. These data suggest a model for GFP chromophore synthesis in which the carboxylate of Glu 222 plays the role of a general base, facilitating proton abstraction from the Gly 67 amide nitrogen or the Tyr 66 ␣-carbon. Arg 96 fulfills the role of an electrophile by lowering the respective pK a values and stabilizing the ␣-enolate. Modulating the base strength of the proton-abstracting group may aid in the design of fast-maturing GFPs with improved characteristics for real-time monitoring of cellular events.

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