PubMed1996
The tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase (MADH) is covalently modified by nitrogen during its two-electron reduction by methylamine to form an aminoquinol (N-quinol). It is possible, in vitro, to generate unmodified O-quinol and O-semiquinone forms of MADH with dithionite, as well as an N-semiquinone form which contains a substrate-derived nitrogen. Rapid-scanning stopped-flow spectroscopy and global kinetic analysis are used to demonstrate that N-semiquinone is a true physiologic reaction intermediate which accumulates during the two sequential one-electron oxidations of N-quinol MADH by amicyanin. In contrast, no detectable O-semiquinone accumulates during the two sequential one-electron oxidations of the O-quinol form of MADH by amicyanin. This is because the reaction of N-semiquinone with amicyanin is much slower (70 s-1 at 25 degrees C) than the reaction of O-semiquinone ( > 1000 s-1). These rate constants obtained from global analysis of the overall reaction are the same as those obtained when each semiquinone form was made in vitro and then mixed with oxidized amicyanin. The presence of 200 mM NH4Cl during the reaction of O-quinol MADH with amicyanin does not cause any detectable accumulation of a semiquinone species. Thus, the accumulation of the intermediate in the reactions of the N-quinol is not due to the influence of noncovalently bound ammonia at the active site of the O-semiquinone. These data indicate that the intermediate which accumulates during the complete oxidation of substrate-reduced N-quinol MADH is not the O-semiquinone, but the more slowly reacting N-semiquinone, and that the N-semiquinone is a physiologically relevant reaction intermediate. These results also provide good evidence in favor of an aminotransferase mechanism, as opposed to an imine elimination mechanism, for the reaction of MADH with substrate methylamine.