The influence of various C4/C5 substituents in catechol (1,2-dihydroxybenzene) derivatives on the overall rate of conversion by catechol-1,2-dioxygenase from Pseudomonas putida (arvilla) C1 was investigated. Using catechol, 4-methylcatechol, 4-fluorocatechol, 4-chlorocatechol, 4-bromocatechol, 4,5-difluorocatechol and 4-chloro-5-fluorocatechol, it could be demonstrated that substituents at the C4 and/or C5 position decrease the rate of conversion, from 62% (4-methylcatechol) down to 0.7% (4-chloro-5-fluorocatechol) of the activity with non-substituted catechol. The inhibition was reversible upon addition of excess catechol for all substrates tested. This indicates that the lower activities are neither due to irreversible inactivation of the enzyme nor to product inhibition. Based on the reaction mechanism proposed in the literature [Que, L. & Ho, R. Y. N. (1996) Chem. Rev. 96, 2606-2624], the nucleophilic reactivity of the catecholate was expected to be an essential characteristic for its conversion by catechol-1,2-dioxygenase. Therefore, the rates of conversion were compared with calculated energies of the highest occupied molecular orbital (E(HOMO)) of the substrates. A clear quantitative relationship (R>0.97) between the ln kcat and the calculated electronic parameter E(HOMO) was obtained. This indicates that the rate-limiting step of the reaction cycle is dependent on the nucleophilic reactivity of the substrate and not sterically hindered by the relatively large bromine or methyl substituents used in the present study. Possible steps in the reaction mechanism determining the overall rate at 20 degrees C are discussed.