ELECTRON-TRANSFER REACTIONS BETWEEN AROMATIC AMINE DEHYDROGENASE AND AZURIN

Binding and electron transfer reactions between the tryptophan tryptop hylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH), and the type I copper protein azurin have been characterized. In steady-state kinetic assays using azurin as an electron acceptor, it was observed t hat the apparent K-m for azurin decreased with increasing ionic streng th. These results are the opposite of what was observed for the reacti on between the TTQ enzyme methylamine dehydrogenase (MADH) and amicyan in, despite the fact that in both cases the pairs of redox proteins ar e each acidic proteins. It was further demonstrated that azurin does n ot function as an effective electron acceptor for MADH, and that amicy anin does not function as an effective electron acceptor for AADH. Thu s, while the two TTQ enzymes each use type I copper proteins as physio logic electron accepters, there is a strong specificity for which copp er protein serves as a redox partner. The kinetic parameters for the e lectron transfer reactions from reduced AADH to oxidized azurin were d etermined by stopped-flow spectroscopy. Different results were obtaine d depending upon whether AADH was reduced chemically with dithionite o r with the substrate tyramine. The values for the limiting first-order apparent electron transfer rate constant (k(ET)) at 30 degrees C were 4 and 102 s(-1), respectively. Kinetically determined values of K-d a lso differed by a factor of 2.4. These data suggest that the incorpora tion of the substrate-derived amino group into the reduced TTQ of AADH significantly increases the apparent k(ET). The interaction between A ADH and azurin was also quantitated using an ultrafiltration binding a ssay. This yielded a K-d of 300 (m)u M for the AADH-azurin complex. Th is K-d correlated well with the kinetically determined K-d values obta ined from the stopped-flow kinetic studies. Similarities and differenc es in the reactivities of the AADH-azurin and MADH-amicyanin redox pai rs are discussed.