Halides inhibition of multicopper oxidases studied by FTIR spectroelectrochemistry using azide as an active infrared probe.

An infrared spectroelectrochemical study of Trametes hirsuta laccase and Magnaporthe oryzae bilirubin oxidase has been performed using azide, an inhibitor of multicopper oxidases, as an active infrared probe incorporated into the T2/T3 copper cluster of the enzymes. The redox potential-controlled measurements indicate that N3- stretching IR bands of azide ion bound to the T2/T3 cluster are only detected for the oxidized enzymes, confirming that azide only binds to Cu2+. Moreover, the process of binding/dissociation of azide ion is shown to be reversible. The interaction of halide anions, which also inhibit multicopper oxidases, with the active site of the enzymes was studied by measuring the changes in the azide FTIR bands. Enzymes inhibited by azide respond differently upon addition of fluoride or chloride ions to the sample solution inhibited by azide. Fluoride ions compete with azide for binding at one of the T2/T3 Cu ions, whereas competition from chloride ions is much less evident.