Abstract

The mechanism of o-semiquinone production was examined in the tyrosinase and peroxidase catalyzed oxidations of a series of catecholic compounds using the electron spin resonance (ESR) spin-stabilization approach and in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH). In the tyrosinase process, the nonenzymatic o-semiquinone formation by inverse disproportion mechanism was clearly confirmed. Mechanistic and kinetic studies of o-semiquinone and o-quinone formation by mushroom tyrosinase were carried out by ESR spin stabilization and optical spectroscopy. Two different types of cyclizable catecholic substrates ( -dopa and dopamine 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid) together with an uncyclizable substrate (3,4-dihydroxybenzoic acid) were examined. The reactive quinones were monitored by measuring the apparent initial rates of the o-quinone-MBTH adducts. The transient behaviour of the o-semiquinone was studied by determining the pseudo first-order formation constants (k values in the range 0.226–0.035 s⁻¹), the relative second-order decay kinetic constants (k = 3.3·10² M⁻¹ s⁻¹ for dopamine o-semiquinone) and the maximum concentrations of the o-semiquinone complexes formed in situ with Mg²⁺ ions. The o-semiquinone formation constants are not directly correlated with their maximum concentrations; in fact, the o-semiquinone maximum concentration of the uncyclizable substrate is comparable with that derived from -dopa. Furthermore, the secondary semiquinone formation is slow and not competitive with the primary semiquinone generation. Then, in our model the limiting factor for the o-semiquinone formation, is not simply the substrate ability to cyclize, and, therefore, the potential toxicity of the secondary semiquinone is questionable.

Abbreviations: -dopa, -3,4-dihydroxyphenylalanine; HRP, horseradish peroxidase; MBTH, 3-methyl-2-benzothiazolinone hydrazone; DMAB, 3-dimethylaminobenzoic acid; TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl

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Corresponding author. Address correspondence to: Professor Rosa Pia Ferrari, Dipartimento di Chimica I.F.M., Università di Torino, Via P. Giuria 7, 10125 , Torino, , Italy

*1 This research was supported by grants from the Ministero Italiano Università Ricerca Scientifica e Tecnologica (MURST) and from the Comitato Biotecnologie, Consiglio Nazionale Italiano delle Ricerche (CNR).

¹ Dr. E. Laurenti received a fellowship from the Fondazione Biotecnologie, Torino.

² Prof. L. Casella thanks the European Community, through the contract ERBCHRXCT920014, for financial support.