A resonance Raman study of substrate and inhibitor binding to protocatechuate-3,4-dioxygenase

Resonance Raman spectra were obtained for complexes of protocatechuate-3,4-dioxygenase with substrate and hydroxybenzoate inhibitors. The data establish metal coordination by these bound species and demonstrate further that tyrosine ligation, present in the resting enzyme, is not altered in the complexes. For the inhibitors, 3-chloro-4-hydroxybenzoate and 3-fluoro-4-hydroxybenzoate, the data are interpreted as indicating iron ligation by the phenolate functionality. For the substrate, 3,4-dihydroxyphenylproprionate, chelation via the o-dihydroxy grouping is proposed. In all three complexes tyrosine ligands present in the resting enzyme are not displaced. The inhibitor scattering intensity was utilized as an internal standard to estimate that two tyrosines are coordinated to the iron at the active site.