J Biol Chem, Vol. 274, Issue 19, 13155-13161, May 7, 1999

The Role of Tyr-169 of Trimethylamine Dehydrogenase in Substrate Oxidation and Magnetic Interaction between FMN Cofactor and the 4Fe/4S Center

Jaswir Basran, Mei-Huei Jang^§, Michael J. Sutcliffe^¶, Russ Hille^§, and Nigel S. Scrutton

From the  Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom, the ^¶ Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom, and the ^§ Department of Medical Biochemistry, Ohio State University, Columbus, Ohio 43210

Tyr-169 in trimethylamine dehydrogenase is one component of a triad also comprising residues His-172 and Asp-267. Its role in catalysis and in mediating 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 in catalysis (mutation to phenylalanine reduces the limiting rate constant for bleaching of the active site flavin by about 100-fold) but does not serve as a general base in the course of catalysis. In addition, we are able to resolve two kinetically influential ionizations involved in both the reaction 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 studies 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 ~9.5, previously identified in pH-jump studies of electron transfer, whose deprotonation must occur for the spin-interacting state to be established.