The maturation process of green fluorescent protein (GFP) entails a protein oxidation reaction triggered by spontaneous backbone condensation. The chromophore is generated by full conjugation of the Tyr66 phenolic group with the heterocycle, a process that requires C-H bond scission at the benzylic carbon. We have prepared isotope-enriched protein bearing tyrosine residues deuterated at the beta carbon, and have determined kinetic isotope effects (KIEs) on the GFP self-processing reaction. Progress curves for the production of H 2 O 2 and the mature chromophore were analyzed by global curve fitting to a three-step mechanism describing pre-oxidation, oxidation and post-oxidation events. Although a KIE for protein oxidation could not be discerned (k H /k D = 1.1 ± 0.2), a full primary KIE of 5.9 (± 2.8) was extracted for the post-oxidation step. Therefore, the exocyclic carbon is not involved in the reduction of molecular oxygen. Rather, C-H bond cleavage proceeds from the oxidized cyclic imine form, and is the rate-limiting event of the final step. Substantial pH-dependence of maturation was observed upon substitution of the catalytic glutamate (E222Q), indicating an apparent pK a of 9.4 (± 0.1) for the base catalyst. For this variant, a KIE of 5.8 (± 0.4) was determined for the intrinsic time constant that is thought to describe the final step, as supported by ultra-high resolution mass spectrometric results. The data are consistent with general base catalysis of the postoxidation events yielding green color. Structural arguments suggest a mechanism in which the highly conserved Arg96 serves as catalytic base in proton abstraction from the Tyr66-derived beta carbon. KeywordsProtein maturation; chromophore biosynthesis; fluorescent proteins; KIE; deuterium isotope effect; general base catalysis; arginine as catalyst In recent years, the post-translational modifications yielding for the colorful chromophores of GFP-like proteins have been studied extensively (1,2). Members of the family of fluorescent proteins are evolutionarily related to its founding member avGFP from the jellyfish Aequorea victoria (3), and are generally found in marine organisms such as reef-building corals. Fluorescent proteins (FPs) have attracted considerable interest, due to their ability to synthesize brightly fluorescing entities from intrinsic amino acid residues, such that the intense coloration of the mature protein appears to be an inherent property of the particular genetic sequence. The interior of the eleven-stranded β-barrel of FPs contains a helical peptide that spans residues 60 † This work was supported by a grant from the National Science Foundation (NSF MCB-0615938) and a grant from the National Institutes of Health (NIH RO3-EB006413) to R. M. W. NSF Grant CHE-0131222 provided funds to purchase the MALDI instrument. * Author Contact Information: email rwachter@asu.edu, phone 480-965-8188, fax 480-965-2747. NIH Public Access -71 in avGFP, with position 66 occupied by a conserved tyrosine and 67 by a conserved glyci...