scholarly journals Mechanistic studies on the dehydrogenases of methylotrophic bacteria. 2. Kinetic studies on the intramolecular electron transfer in trimethylamine and dimethylamine dehydrogenase

1982 ◽  
Vol 207 (2) ◽  
pp. 241-252 ◽  
Author(s):  
D J Steenkamp ◽  
H Beinert
Keyword(s):  
Electron Transfer ◽  
Triplet State ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Reduced Form ◽  
Intramolecular Electron Transfer ◽  
Methylotrophic Bacteria ◽  
Trimethylamine Dehydrogenase ◽  
Substrate Dependence ◽  
Reduced Forms

E.p.r. spectroscopy of the trimethylamine and dimethylamine dehydrogenases of Hyphomicrobium X indicates that the substrate-reduced forms of these enzymes exist in the triplet state, which arise through interaction of a reduced [4Fe-4S] cluster and flavosemiquinone, with e.p.r. signals which differ in detail from those of the trimethylamine dehydrogenase of bacterium W3A1. Under certain conditions the intramolecular electron transfer between the flavoquinol form of 6-S-cysteinyl-FMN and the [4Fe-4S] cluster in all three dehydrogenases was much slower than the preceding reduction of the flavin to the flavoquinol form. Trimethylamine dehydrogenases from both organisms show a time-dependent broadening of the e.p.r. signals centred around g = 2 after mixing with trimethylamine. The broadening of the e.p.r. signals could be correlated with an unexpected dependence of the rate of formation of the triplet state on substrate concentration. A model which accounts in a qualitative manner for the substrate dependence of the formation of the triplet state in the trimethylamine dehydrogenase of Hyphomicrobium X is proposed. The binding of the substrate to the reduced form of the enzyme seems to result in a conformational change of the enzyme to a form in which the rate of intramolecular electron transfer is decreased. This finding may be correlated with the observation of hyperbolic substrate inhibition for both trimethylamine dehydrogenases. The results indicate the transfer of an electron to the [4Fe-4S] cluster to be an obligatory step in catalysis and suggest that the transfer of electrons from these enzymes to electron acceptors is mediated solely through the [4Fe-4S] cluster.

scholarly journals Mechanistic studies on the dehydrogenases of methylotrophic bacteria. 1. The influence of substrate binding to reduced trimethylamine dehydrogenase on the intramolecular electron transfer between its prosthetic groups

1982 ◽  
Vol 207 (2) ◽  
pp. 233-239 ◽  
Author(s):  
D J Steenkamp ◽  
H Beinert
Keyword(s):  
Triplet State ◽  
Intramolecular Electron Transfer ◽  
Methylotrophic Bacteria ◽  
Mechanistic Studies ◽  
Trimethylamine Dehydrogenase ◽  
Ph Values ◽  
Tetramethylammonium Chloride ◽  
Maximal Yield ◽  
Influence Of Substrate ◽  
Prosthetic Groups

The trimethylamine dehydrogenase of bacterium W3A1 is reduced with the formation of a triplet state in which two electrons, derived from the substrate, are distributed between the [4Fe-4S] cluster and 6-S-cysteinyl-FMN semiquinone. In titration experiments at pH 8.5 about 1.0 mol of dimethylamine or 0.5 mol of trimethylamine per mol of the enzyme is required to titrate the enzyme to an endpoint. At pH values less than 8.0, however, an excess of trimethylamine is required to obtain maximal yield of the g = 4 e.p.r. signal, characteristic of the triplet state, or maximal absorbance at 365 nm which indicates formation of the flavin semiquinone. The binding of 0.86 mol of trimethylamine per mol of the enzyme could be detected by a gel chromatographic method. When the enzyme is titrated with dithionite in the presence of tetramethylammonium chloride, an endpoint is reached after the uptake of two electrons which give rise to the triplet state, whereas three electrons are consumed in the absence of tetramethylammonium chloride to reduce the enzyme completely. The enzyme is inhibited noncompetitively by tetramethylammonium chloride and the slopes of double reciprocal plots are a concave upwards function of inhibitor concentration. The data indicate the presence of a binding site for the substrate and other amines on the reduced enzyme which enhances the proportion of enzyme in the triplet state.

Flavin radicals, conformational sampling and robust design principles in interprotein electron transfer: the trimethylamine dehydrogenase-electron-transferring flavoprotein complex

2004 ◽  
Vol 71 ◽  
pp. 1-14
Author(s):  
David Leys ◽  
Jaswir Basran ◽  
François Talfournier ◽  
Kamaldeep K. Chohan ◽  
Andrew W. Munro ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Kinetic Studies ◽  
Molecular Assembly ◽  
Conformational Sampling ◽  
Trimethylamine Dehydrogenase ◽  
Key Residues ◽  
Electron Transferring Flavoprotein ◽  
Electron Transfer Complex

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.

Kinetic studies on intramolecular electron transfer in solution

1981 ◽  
Vol 84 (1) ◽  
pp. 48-53 ◽  
Author(s):  
Dan Huppert ◽  
Hannah Kanety ◽  
Edward M. Kosower
Keyword(s):  
Electron Transfer ◽  
Kinetic Studies ◽  
Intramolecular Electron Transfer

The reactions of hydrazines with trans-Dichlorobis(ethylenediamine)cobalt(III)chloride, and the isolation and kinetic studies of a Chlorobis(ethylenediamine)methylhydrazinecobalt(III) isomer

1973 ◽  
Vol 26 (9) ◽  
pp. 1923 ◽  
Author(s):  
SC Chan ◽  
CK Lee
Keyword(s):  
Electron Transfer ◽  
Kinetic Studies ◽  
Intramolecular Electron Transfer ◽  
Dilute Solution ◽  
Dilute Solutions ◽  
Spectroscopic Observations ◽  
Acid And Base ◽  
Concentrated Solution ◽  
Kinetics Of ◽  
Hydrolysis Of

The reactions of hydrazine, methylhydrazine, and 1,1-dimethylhydrazine with trans-dichlorobis(ethylenediamine)cobalt(III) chloride in concentrated and dilute solutions are studied. In concentrated solution, there is a reduction by hydrazine to form the insoluble polymeric [CoII(N2H4)2Cl2]n, a substitution by methylhydrazine to form chlorobis(ethylenediamine)methylhydrazinecobalt(III) chloride, and a disproportionation with 1,1-dimethylhydrazine to form tris(ethylenediamine)cobalt(III) chloride. In dilute solution, the reaction observed is a hydroxide substitution by hydrolysis of 1,1- dimethylhydrazine, a reduction to soluble cobalt(II)-ethylenediamine species with hydrazine, and a mixture of both processes for methylhydrazine. The chlorobis(ethylenediamine)methylhydrazinecobalt-(III) chloride obtained is a new complex, and is assigned a cis configuration on the basis of spectroscopic observations. The kinetics of its intramolecular electron-transfer, as well as its acid and base hydrolyses are also studied.

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