Protocatechuate 3,4-dioxygenase (EC 126.96.36.199) from Pseudomonas aeruginosa catalyzes the cleavage of 3,4-dihydroxybenzoate (protocatechuate) into β-carboxy-cis,cis-muconate. The inhibition constants, Ki, of a series of substrate analogues were measured in order to assess the relative importance of the various functional groups on the substrate. Though important for binding, the carboxylate group is not essential for activity. Compounds with para hydroxy groups are better inhibitors than their meta isomers. Our studies of the enzyme inhibitor complexes indicate that the 4-OH group of the substrate binds to the active-site iron. Taken together, Mössbauer, EPR, and kinetic data suggest a mechanism where substrate reaction with oxygen is preceded by metal activation of substrate.