Electron transfer and conformational change in complexes of trimethylamine dehydrogenase and electron transferring flavoprotein.
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.
Jones
Matthew
M
Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.
Talfournier
Francois
F
Bobrov
Anton
A
Grossmann
J Günter
JG
Vekshin
Nikolai
N
Sutcliffe
Michael J
MJ
Scrutton
Nigel S
NS
D042964:Acyl-CoA Dehydrogenase; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport; D044927:Electron-Transferring Flavoproteins; D004583:Electrons; D044943:Fatty Acid Desaturases / Q000737:chemistry; D005296:Ferrous Compounds / Q000494:pharmacology; D005420:Flavoproteins / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014965:X-Rays
D044927:Electron-Transferring Flavoproteins; D005296:Ferrous Compounds; D005420:Flavoproteins; D011994:Recombinant Proteins; C064804:ferrocenium; D044943:Fatty Acid Desaturases; D042964:Acyl-CoA Dehydrogenase; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase