Participation of the iron-sulphur cluster and of the covalently bound coenzyme of trimethylamine dehydrogenase in catalysis

Abstract

Bacterial trimethylamine dehydrogenase contains a novel type of covalently bound FMN and a Fe-S center. The dehydrogenase takes up 1.5 mol of dithionite/mol of enzyme and is thereby converted into the flavin quinol-reduced (4Fe-4S) form, with the expected bleaching of the visible adsorption band of the flavin and the emergence of signals of typical reduced ferredoxin in the EPR spectrum. On reduction with a slight excess of substrate, unusual absorption and EPR spectra appear quite rapidly. The latter is attributed to extensive interaction between the reduced (4Fe-4S) center and the flavin semiquinone. The species of enzyme arising during the catalytic cycle were studied by a combination of rapid-freeze EPR and stopped-flow spectrophotometry. The initial reduction of the flavin to the quinol form is far too rapid to be rate-limiting in catalysis, as is the reoxidation of the substrate-reduced enzyme by phenazine methosulfate. Formation of the spin-spin-interacting species from the dihydroflavin is considerably slower, and it may be the rate-limiting step in the catalytic cycle, since its rate of formation agrees reasonably well with the catalytic-center activity determined in steady-state kinetic assays. Besides the interacting form, a 2nd form of the enzyme was noted during reduction by trimethylamine, differing in absorption spectrum, the structure of which remains to be determined.