The role of Tyr-169 of trimethylamine dehydrogenase in substrate oxidationand magnetic interaction between FMN cofactor and the 4Fe/4S center

Tyr-169 in trimethylamine dehydrogenase is one component of a triad also co mprising residues His-172 and Asp-267. Its role in catalysis and in mediati ng the magnetic interaction between FMN cofactor and the 4Fe/4S center have been investigated by stopped-flow and EPR spectroscopy of a Tyr-169 to Phe (Y169F) mutant of the enzyme. Tyr-169 is shown to play an important role i n catalysis (mutation to phenylalanine reduces the limiting rate constant f or bleaching of the active site flavin by about 100-fold) but does not serv e as a general base in the course of catalysis. In addition, we are able to resolve two kinetically influential ionizations involved in both the react ion of free enzyme with free substrate (as reflected in k(lim)/K-d) and in the breakdown of the E-ox.S complex (as reflected in k(lim)). In EPR studie s of the Y169F mutant, it is found that the ability of the Y169F enzyme to form the spin-interacting state between flavin semiquinone and reduced 4Fe/ 4S center characteristic of wild-type enzyme is significantly compromised. The present results are consistent with Tyr-169 representing the ionizable group of pK(a) similar to 9.5, previously identified in pH-jump studies of electron transfer, whose deprotonation must occur for the spin-interacting state to be established.