ACTIVE-SITE STUDIES OF DT-DIAPHORASE EMPLOYING ARTIFICIAL FLAVINS

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is an FAD -containing enzyme that catalyzes the 2-electron reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor. In this study, FAD was removed by dialyzing the holoprotein against 2 M KBr, and synthetic analogs of FAD were substituted in the flavin bindi ng site as structural probes. Spectral analysis indicates that the ben zoquinoid forms of 8-mercapto-FAD and 6-mercapto-FAD are stabilized on binding to the enzyme. This is consistent with the fact that the nati ve flavoprotein forms the anion flavin radical upon photoreduction and suggests the presence of a positive charge near the N(1)C(2)O positio n of the isoalloxazine ring. Reactivity studies using 8-chloro- and 8- mercapto-flavins suggest that the 8 position of the FAD is accessible to the solvent. However, the rates of the reactions were dramatically decreased in the presence of the competitive inhibitor, dicumarol. 6-M ercapto-, 6-thiocyanato-, 6-azido-, and 6-amino-flavins were also used as structural probes. The results indicate that the 6 position is acc essible to solvent. Dicumarol binding increases the pK(a) of the enzym e-bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0. The results suggest that DT-diaphorase shows the same properties as the C -C transhydrogenases, and the binding of dicumarol elicits a conformat ional change or an adjustment in the polarity of the FAD pocket. The e nzyme reconstituted with oxidized 5-deaza-FAD has significant catalyti c activity, confirming that DT-diaphorase is an obligatory 2-electron transfer enzyme and plays a role in the detoxification of quinones and quinoid compounds by reducing them to the relatively stable hydroquin ones.