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

Abstract

The trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH⅐ETF) 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 TMADH⅐ETF electron transfer complex and that the kinetics of assembly observed with mutant TMADH (Y442F/L/G) or ETF (␣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 TMADH⅐ETF 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 smallangle 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 TMADH⅐ETF 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.Trimethylamine dehydrogenase (TMADH, 1 EC 1.5.99.7) from the methylotrophic bacterium Methylophilus methylotrophus (sp. W 3 A 1 ) is a homodimeric iron-sulfur flavoprotein that forms an electron transfer complex with electron transferring flavoprotein (ETF (1)). Each subunit of TMADH (molecular mass ϳ 83 kDa) contains a covalently linked 6-S-cysteinyl FMN cofactor, a 4Fe-4S center, and a tightly bound ADP of unknown function (2-8). TMADH catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde (Reaction 1),The mechanism of enzyme reduction by trimethylamine has been addressed by stopped-flow spectroscopy in wild-type (9 -11) and mutant (12-15) TMADH enzymes and has recently been reviewed (16). Following substrate reduction of TMADH, electrons are transferred sequentially from the FMN cofactor to the 4Fe-4S center and, subsequently, in an interprotein electron transfer reaction to the FAD of ...

Mitochondrial β‐oxidation

“…Recently, Parker & Engel showed that functional assemblies consisting of MCAD or sarcosine dehydrogenase (an enzyme of one-carbon metabolism which is also dehydrogenated by ETF) together with ETF, ETFD, coenzyme Q (ubiquinone) and complex III could be isolated from sonicated porcine liver mitochondria [45]. Similarly, Jones et al [46] have characterized electron transfer and conformational changes in a complex of trimethylamine dehydrogenase (EC 1.5.99.7, derived from the methylotroph Methylophilus methylotrophus) and ETF, and demonstrate that electron transfer occurs during metastable states of the complex. Further, they show that ETF undergoes a stable conformational change when it interacts with TAMDH which they term Ôstructural imprintingÕ and that this form of semiquinone ETF has an increased rate of electron transfer to the artificial electron acceptor ferricenium.…”

“…Similarly, Jones et al. [46] have characterized electron transfer and conformational changes in a complex of trimethylamine dehydrogenase (EC 1.5.99.7, derived from the methylotroph Methylophilus methylotrophus ) and ETF, and demonstrate that electron transfer occurs during metastable states of the complex. Further, they show that ETF undergoes a stable conformational change when it interacts with TAMDH which they term ‘structural imprinting’ and that this form of semiquinone ETF has an increased rate of electron transfer to the artificial electron acceptor ferricenium.…”

Mammalian Electron Transferring Flavoprotein·Flavoprotein Dehydrogenase Complexes Observed by Microelectrospray Ionization-Mass Spectrometry and Surface Plasmon Resonance

“…This may imply a similar mechanism for the transfer of electrons from both groups of dehydrogenases to ETF. Conformational changes within the interaction domain that occur when substrate binds, however, could increase the affinity of the reduced dehydrogenase for ETF in vivo, providing an advantage for productive interactions resulting in electron transfer (15,16).…”

Metagenomics analysis of methane metabolisms in manure fertilized paddy soil

“…Our results show that PIG specific KO were found in various pathways towards production of methane. (Harms, 1996), and trimethylamine-corrinoid protein Co-methyltransferase (mttB) and dimethylamine/trimethylamine dehydrogenase (dmd-tmd) catalyze the synthesize of Methyl-CoM, a precursor of methane (Yang et al, 1995;Tallant and Krzycki, 1997;Jones et al, 2002;Pritchett and Metcalf, 2005 …”