Prototropic control of intramolecular electron transfer in trimethylamine dehydrogenase.

The pH dependence of static optical/EPR spectra of trimethylamine dehydrogenase reduced to the level of two equivalents (TMADH2eq) has been examined and indicates the existence of three different states for this iron-sulfur flavoprotein. At pH 6, TMADH2eq exists principally in a form possessing flavin mononucleotide hydroquinone, with its iron-sulfur center oxidized. At pH 8, the enzyme principally contains flavin 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, TMADH2eq exhibits the EPR spectrum that is diagnostic of a previously characterized spin-interacting state in which the magnetic moments of the flavin semiquinone and reduced iron-sulfur 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 consistent with a reaction mechanism involving sequential protonation/deprotonation and intramolecular electron transfer events. All reactions studied show a normal solvent kinetic isotope effect. Proton inventory analysis indicates that at least one proton is involved in the reaction between pH 6 and 8, which principally controls intramolecular electron 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 TMADH2eq, between pH 10 and 6, at least three protonation/deprotonation events are associated with intramolecular electron transfer and formation of the spin-interacting state, with estimated pK alpha values of 6.0, 8.0, and approximately 9.5. These pK alpha values are attributed to the flavin hydroquinone, flavin semiquinone, and an undesignated basic group on the protein, respectively.