Participation of the iron-sulphur cluster and of the covalently bound coenzyme of trimethylamine dehydrogenase in catalysis.
Bacterial trimethylamine dehydrogenase contains a novel type of covalently bound flavin mononucleotide and a tetrameric iron-sulphur centre. The dehydrogenase takes up 1.5mol of dithionite/mol of enzyme and is thereby converted into the flavin quinol-reduced (4Fe-4S) form, with the expected bleaching of the visible absorption band of the flavin and the emergence of signals of typical reduced ferredoxin in the electronparamagnetic-resonance spectrum. On reduction with a slight excess of substrate, however, unusual absorption and electron-paramagnetic-resonance spectra appear quite rapidly. The latter is attributed to extensive interaction between the reduced (4Fe-4S) centre and the flavin semiquinone. The species of enzyme arising during the catalytic cycle were studied by a combination of rapid-freeze e.p.r. and stopped-flow spectophotometry. 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 methosulphate. Formation of the spin-spin-interacting species from the dihydroflavin is considerably slower, however, and it may be the rate-limiting step in the catalytic cycle, since its rate of formation agrees reasonably well with the catalytic-centre activity determined in steady-state kinetic assays. In addition to the interacting form, a second form of the enzyme was noted during reduction by trimethylamine, differing in absorption spectrum, the structure of which remains to be determined.
D002384:Catalysis; D004227:Dithionite / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005486:Flavin Mononucleotide / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism*; D008744:Methylamines / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D010619:Phenazines / Q000378:metabolism; D013057:Spectrum Analysis
D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D008744:Methylamines; D010619:Phenazines; D004227:Dithionite; D005486:Flavin Mononucleotide; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase