The reaction of trimethylamine dehydrogenase with electron transferring flavoprotein.
The kinetics of electron transfer between trimethylamine dehydrogenase (TMADH) and its physiological acceptor, electron transferring flavoprotein (ETF), has been studied by static and stopped-flow absorbance measurements. The results demonstrate that reducing equivalents are transferred from TMADH to ETF solely through the 4Fe/4S center of the former. The intrinsic limiting rate constant (klim) and dissociation constant (Kd) for electron transfer from the reduced 4Fe/4S center of TMADH to ETF are about 172 s-1 and 10 microM, respectively. The reoxidation of fully reduced TMADH with an excess of ETF is markedly biphasic, indicating that partial oxidation of the iron-sulfur center in 1-electron reduced enzyme significantly reduces the rate of electron transfer out of the enzyme in these forms. The interaction of the two unpaired electron spins of flavin semiquinone and reduced 4Fe/4S center in 2-electron reduced TMADH, on the other hand, does not significantly slow down the electron transfer from the 4Fe/4S center to ETF. From a comparison of the limiting rate constants for the oxidative and reductive half-reactions, we conclude that electron transfer from TMADH to ETF is not rate-limiting during steady-state turnover. The overall kinetics of the oxidative half-reaction are not significantly affected by high salt concentrations, indicating that electrostatic forces are not involved in the formation and decay of reduced TMADH-oxidized ETF complex.
Huang
L
L
Department of Medical Biochemistry, Ohio State University, Columbus 43210, USA.
Rohlfs
R J
RJ
Hille
R
R
D004579:Electron Transport; D044927:Electron-Transferring Flavoproteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019605:Euryarchaeota / Q000201:enzymology; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010659:Phenylhydrazines / Q000494:pharmacology; D013053:Spectrophotometry
D044927:Electron-Transferring Flavoproteins; D004791:Enzyme Inhibitors; D005420:Flavoproteins; D010659:Phenylhydrazines; C030299:phenylhydrazine; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase