Measurement of Internal Substrate Binding in Dehaloperoxidase-Hemoglobin by Competition with the Heme-Fluoride Binding Equilibrium

The application of fluoride anion as a probe for investigating the internal substratebinding has been developed dila applied to dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata. By applying the fluoride titration strategy using UAT=vis spectroscopy, we have studied series of halogenated phenols, other substituted phenolg-, halogenated indoles, and several natural amino acids that bind internally (and noncovalently) in the distal binding pocket of the hen-ie. This approach has identified 2,4=dibromopheno1 (2,4-DBP). as the tightest binding substrate discovered thug far, with approximately 20-fold tighter binding affinity than that of 4-bromophenol (4-BP), a known internally binding inhibitor, in DHP. Combined with resonante Raman spectroscopy, we have confirmed that competitiveJpincling equilibria exist between fluoride anion and internally, bound molecules.' We have further investigated the hydrogen bonding network of the active site of DHP that Stabilizes the exogenous fluoride ligand. These measurements demonstrate a general method for determination of differences in substrate binding affinity based on detection of a competitive fluoride binding equilibrium. The significance of the binding that 2,4,dibromophenol binds more tightly than any other substrate is evident when the structural and mechanistic data are taken into consideration.