Redox cycles in trimethylamine dehydrogenase and mechanism of substrate inhibition

The steady-state reaction of trimethylamine dehydrogenase (TMADH) with the artificial electron acceptor ferricenium hexafluorophosphate (Fc(+)) has be en studied by stopped-flow spectroscopy, with :particular reference to the mechanism of inhibition by trimethylamine (TMA). Previous studies have sugg ested that the presence of alternate redox cycles is:responsible for the in hibition of activity seen in the high-substrate regime. Here, we demonstrat e that partitioning between these redox cycles (termed the 0/2 and: 1/3 cyc les on the basis of the number of reducing equivalents present in the oxidi zed/reduced enzyme encountered in each cycle) is dependent on both TMA and electron acceptor concentration. The use of Fc(+) as electron acceptor has enabled a study of the major redox forms of TMADH present during steady-sta te turnover at different concentrations of substrate. Reduction of Fc(+) is found to occur via the 4Fe-4S center of TMADH and not the 6-S-cysteinyl fl avin mononucleotide: the direction of electron flow is thus analogous to th e route of electron transfer to the physiological electron acceptor, an ele ctron-transferring flavoprotein (ETF). In steady-state reactions with Fc(+) as electron acceptor, partitioning between the 0/2 land 1/3 redox cycles i s dependent on the concentration of the electron acceptor. In the high-conc entration regime, inhibition is less pronounced, consistent with the predic ted effects on the proposed branching kinetic scheme. Photodiode array anal ysis of the:absorption spectrum of TMADH during steady-state turnover at hi gh TMA concentrations reveals that one-electron reduced TMADH-possessing th e anionic flavin semiquinone-is the predominant species; Conversely, at low concentrations of TMA, the enzyme is predominantly in the oxidized form du ring steady-state turnover. The data, together with evidence derived from e nzyme-monitored turnover experiments performed, at different concentrations of TMA, establish the operation of the branched kinetic scheme in steady-s t ate reactions. With dimethylbutylamine (DMButA) as substrate, the partiti oning between the 0/2 and 1/3 redox cycles is poised more toward the 0/2 cy cle at all DMButA concentrations studied-an observation that is consistent with the inability of DMButA to act as an effective inhibitor of TMADH.