Reaction progress of chromophore biogenesis in green fluorescent protein

The mature form of green fluorescent protein (GFP) is generated by a spontaneous self-modification process that is essentially irreversible. A key step in chromophore biosynthesis involves slow air oxidation of an intermediate species, in which the backbone atoms of residues 65-67 have condensed to form a five-membered heterocycle. We have investigated the kinetics of hydrogen peroxide evolution during in vitro GFP maturation and found that the H ₂O₂ coproduct is generated prior to the acquisition of green fluorescence at a stoichiometry of 1:1 (peroxide/chromophore). The experimental progress curves were computer-fitted to a three-step mechanism, in which the first step proceeds with a time constant of 1.5 (±1.1) min and includes protein folding and peptide cyclization. Kinetic data obtained by HPLC analysis support a rapid cyclization reaction that can be reversed upon acid denaturation. The second step proceeds with a time constant of 34.0 (±1.5) min and entails rate-limiting protein oxidation, as supported by a mass loss of 2 Da observed for tryptic peptides derived from species that accumulate during the reaction. The final step in GFP maturation proceeds with a time constant of 10.6 (±1.2) min, suggesting that this step may contribute to overall rate retardation. We propose that under highly aerobic conditions, the dominant reaction path follows a cyclization-oxidation- dehydration mechanism, in which dehydration of the heterocycle is facilitated by slow proton abstraction from the Tyr66 β-carbon. In combination, the results presented here suggest a role for molecular oxygen in trapping the cyclized form of GFP.