Publications

• The purification and properties of a bacterial trimethylamine dehydrogenase.

  Colby J., Zatman L.J.

  Biochem J 121:9P-10P(1971) [PubMed] [EuropePMC]

• The reaction of trimethylamine dehydrogenase with electron transferring flavoprotein.

  Huang L., Rohlfs R.J., Hille R.

  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 ... >> More

  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. << Less

  J Biol Chem 270:23958-23965(1995) [PubMed] [EuropePMC]

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

  Steenkamp D.J., Singer T.P.

  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 bleach ... >> More

  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. << Less

  Biochem J 169:361-369(1978) [PubMed] [EuropePMC]

• Electron transfer and conformational change in complexes of trimethylamine dehydrogenase and electron transferring flavoprotein.

  Jones M., Talfournier F., Bobrov A., Grossmann J.G., Vekshin N., Sutcliffe M.J., Scrutton N.S.

  The trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH.ETF) electron transfer complex has been studied by fluorescence and absorption spectroscopies. These studies indicate that a series of conformational changes occur during the assembly of the TMADH.ETF electron transfer comp ... >> More

  The trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH.ETF) electron transfer complex has been studied by fluorescence and absorption spectroscopies. These studies indicate that a series of conformational changes occur during the assembly of the TMADH.ETF electron transfer complex and that the kinetics of assembly observed with mutant TMADH (Y442F/L/G) or ETF (alpha R237A) complexes are much slower than are the corresponding rates of electron transfer in these complexes. This suggests that electron transfer does not occur in the thermodynamically most favorable state (which takes too long to form), but that one or more metastable states (which are formed more rapidly) are competent in transferring electrons from TMADH to ETF. Additionally, fluorescence spectroscopy studies of the TMADH.ETF complex indicate that ETF undergoes a stable conformational change (termed structural imprinting) when it interacts transiently with TMADH to form a second, distinct, structural form. The mutant complexes compromise imprinting of ETF, indicating a dependence on the native interactions present in the wild-type complex. The imprinted form of semiquinone ETF exhibits an enhanced rate of electron transfer to the artificial electron acceptor, ferricenium. Overall molecular conformations as probed by small-angle x-ray scattering studies are indistinguishable for imprinted and non-imprinted ETF, suggesting that changes in structure likely involve confined reorganizations within the vicinity of the FAD. Our results indicate a series of conformational events occur during the assembly of the TMADH.ETF electron transfer complex, and that the properties of electron transfer proteins can be affected lastingly by transient interaction with their physiological redox partners. This may have significant implications for our understanding of biological electron transfer reactions in vivo, because ETF encounters TMADH at all times in the cell. Our studies suggest that caution needs to be exercised in extrapolating the properties of in vitro interprotein electron transfer reactions to those occurring in vivo. << Less

  J Biol Chem 277:8457-8465(2002) [PubMed] [EuropePMC]