Prototropic control of intramolecular electron transfer in trimethylamine dehydrogenase

Abstract

The pH dependence of static optical/EPR spectra of trimethylamine dehydrogenase reduced to the level of two equivalents (TMADH(2eq)) has been examined and indicates the existence of three different states for this iron- sulfur flavoprotein. At pH 6, TMADH(2eq) 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, TMADH(2eq) 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 TMADH(2eq), 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(a) values of 6.0, 8.0, and ~9.5. These pK(a) values are attributed to the flavin hydroquinone, flavin semiquinone, and an undesignated basic group on the protein, respectively.