Radiolytic studies of trimethylamine dehydrogenase - Spectral deconvolution of the neutral and anionic flavin semiquinone, and determination of rate constants for electron transfer in the one-electron reduced enzyme

Trimethylamine dehydrogenase from the pseudomonad Methylophilus methylotrop hus has been examined using the technique of pulse radiolysis to rapidly in troduce a single reducing equivalent into the enzyme. Using enzyme that has had its iron-sulfur center rendered redox-inert by prior reaction with fer ricenium hexafluorophosphate, we determined the spectral change associated with formation of both the anionic and neutral forms that were generated at high and low pH, respectively, of the unique 6-cysteinyl-FMN of the enzyme . With native enzyme, electron transfer was observed within the radiolytica lly generated one-electron reduced enzyme but only at low pH (6.0). The kin etics and thermodynamics of this electron transfer in one-electron reduced enzyme may be compared with that studied previously in the two-electron red uced enzyme. In contrast to previous studies with two-electron reduced enzy me in which a pK(a) of similar to 8 was determined for the flavin semiquino ne, in the one-electron reduced enzyme the semiquinone was not substantiall y protonated even at pH 6.0. These results indicate that reduction of the i ron-sulfur center of the enzyme significantly decreases the pK(a) of the fl avin semiquinone of the active site. This provides further evidence, in con junction with the strong magnetic interaction known to exist between the ce nters in the two-electron reduced enzyme, that the two redox-active centers in trimethylamine dehydrogenase are in intimate contact with one another i n the active site of the enzyme.