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.

scholarly journals The Putidaredoxin Reductase-Putidaredoxin Electron Transfer Complex

2005 ◽  
Vol 280 (16) ◽  
pp. 16135-16142 ◽  
Author(s):  
Vadim Yu Kuznetsov ◽  
Emek Blair ◽  
Patrick J. Farmer ◽  
Thomas L. Poulos ◽  
Amanda Pifferitti ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Tight Binding ◽  
Electron Transfer Reaction ◽  
Wild Type ◽  
Transfer Rates ◽  
Marginal Change ◽  
Theoretical Predictions ◽  
Sulfur Protein ◽  
Key Residues ◽  
Electron Transfer Complex

Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) fromPseudomonas putidawas studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and [2Fe-2S] of 14.6 Å. This mode of interaction allows Pdx to easily adjust and optimize orientation of its cofactor relative to Pdr. The key residues of Pdx located at the center, Asp38and Trp106, and at the edge of the protein-protein interface, Tyr33and Arg66, were mutated to test the Pdr-Pdx computer model. The Y33F, Y33A, D38N, D38A, R66A, R66E, W106F, W106A, and Δ106 mutations did not affect assembly of the [2Fe-2S] cluster and resulted in a marginal change in the redox potential of Pdx. The electron-accepting ability of Δ106 Pdx was similar to that of the wild-type protein, whereas electron transfer rates from Pdr to other mutants were diminished to various degrees with the smallest and largest effects on the kinetic parameters of the Pdr-to-Pdx electron transfer reaction caused by the Trp106and Tyr33/Arg66substitutions, respectively. Compared with wild-type Pdx, the binding affinity of all studied mutants to Pdr was significantly higher. Experimental results were in agreement with theoretical predictions and suggest that: (i) Pdr-Pdx complex formation is mainly driven by steric complementarity, (ii) bulky side chains of Tyr33, Arg66, and Trp106prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr, and (iii) transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp106but are likely to include Asp38and Cys39.

scholarly journals Electron Transfer and Conformational Change in Complexes of Trimethylamine Dehydrogenase and Electron Transferring Flavoprotein

2001 ◽  
Vol 277 (10) ◽  
pp. 8457-8465 ◽  
Author(s):  
Matthew Jones ◽  
Francois Talfournier ◽  
Anton Bobrov ◽  
J. Günter Grossmann ◽  
Nikolai Vekshin ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Conformational Change ◽  
Trimethylamine Dehydrogenase ◽  
Electron Transferring Flavoprotein

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 Structural dissection of the reaction mechanism of cellobiose phosphorylase

2006 ◽  
Vol 398 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Masafumi Hidaka ◽  
Motomitsu Kitaoka ◽  
Kiyoshi Hayashi ◽  
Takayoshi Wakagi ◽  
Hirofumi Shoun ◽  
...  
Keyword(s):  
Reaction Mechanism ◽  
Three Dimensional ◽  
Kinetic Studies ◽  
Structural Features ◽  
Nucleophilic Attack ◽  
Dimensional Structure ◽  
Glycoside Hydrolase Family ◽  
Glucose Phosphate ◽  
Cellobiose Phosphorylase ◽  
Key Residues

Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into α-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 Å (1 Å=0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established.

Extensive conformational sampling in a ternary electron transfer complex

2003 ◽  
Vol 10 (3) ◽  
pp. 219-225 ◽  
Author(s):  
David Leys ◽  
Jaswir Basran ◽  
François Talfournier ◽  
Michael J. Sutcliffe ◽  
Nigel S. Scrutton
Keyword(s):  
Electron Transfer ◽  
Conformational Sampling ◽  
Electron Transfer Complex

Electron transfer in trimethylamine dehydrogenase and electron transferring flavoprotein

1999 ◽  
Vol 27 (1) ◽  
pp. A30-A30
Author(s):  
N. S. Scrutton ◽  
M. J. Sutcliffe ◽  
J. Basran ◽  
R. Hille
Keyword(s):  
Electron Transfer ◽  
Trimethylamine Dehydrogenase ◽  
Electron Transferring Flavoprotein
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