PROTOTROPIC CONTROL OF INTRAMOLECULAR ELECTRON-TRANSFER IN TRIMETHYLAMINE DEHYDROGENASE

The pH dependence of static optical/EPR spectra of trimethylamine dehy drogenase reduced to the level of two equivalents (TMADH(2eq)) has bee n examined and indicates the existence of three different states for t his iron-sulfur flavoprotein, At pH 6, TMADH(2eq) exists principally i n a form possessing flavin mononucleotide hydroquinone, with its iron- sulfur center oxidized, At pH 8, the enzyme principally contains flavi n mononucleotide semiquinone and reduced iron-sulfur, but despite the proximity of the two centers to one another, their magnetic moments do not interact, At pH 10, TMADH(2eq) exhibits the EPR spectrum that is diagnostic of a previously characterized spin-interacting state in whi ch the magnetic moments of the flavin semiquinone and reduced iron-sul fur center are strongly ferromagnetically coupled, The kinetics of the interconversion of these three states have been investigated using a pH jump technique in both H2O and D2O. The observed kinetics are consi stent with a reaction mechanism involving sequential protonation/depro tonation and intramolecular electron transfer events, All reactions st udied show a normal solvent kinetic isotope effect, Proton inventory a nalysis indicates that at least one proton is involved in the reaction between pH 6 and 8, which principally controls intramolecular electro n transfer, whereas at least two protons are involved between pH 8 and 10, which principally control formation of the spin-interacting state , The results of these and previous studies indicate that for TMADH(2e q) between pH 10 and 6, at least three protonation/deprotonation event s are associated with intramolecular electron transfer and formation o f the spin-interacting state, with estimated pK(alpha) values of 6.0, 8.0, and similar to 9.5, These pK(alpha) values are attributed to the flavin hydroquinone, flavin semiquinone, and an undesignated basic gro up on the protein, respectively.