Trimethylamine Dehydrogenase and Electron Transferring Flavoprotein

Scrutton, Nigel S.; Sutcliffe, Michael J.

doi:10.1007/0-306-46828-x_5

Cited by 7 publications

(7 citation statements)

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“…TMADH is a 166 kDa homodimeric iron–sulfur flavoprotein which catalyses the oxidative demethylation of trimethylamine (TMA) to form dimethylamine and formaldehyde ([35]. Substrate oxidation is accompanied by the transfer of reducing equivalents, first to the covalently bound cofactor 6‐ S ‐cysteinyl FMN [27], followed by reduction of a ferredoxin‐like [4Fe−4S] 2+/+ located approximately 4–6 Å from the 8‐α‐methyl group of FMN [36].…”

Section: Kinetics Of Electron Transfer Between Etf and Partnersmentioning

confidence: 99%

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Dynamics driving function − new insights from electron transferring flavoproteins and partner complexes

2007

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Electron transferring flavoproteins (ETFs) are soluble heterodimeric FAD-containing proteins that function primarily as soluble electron carriers between various flavoprotein dehydrogenases. ETF is positioned at a key metabolic branch point, responsible for transferring electrons from up to 10 primary dehydrogenases to the membrane-bound respiratory chain. Clinical mutations of ETF result in the often fatal disease glutaric aciduria type II. Structural and biophysical studies of ETF in complex with partner proteins have shown that ETF partitions the functions of partner binding and electron transfer between (a) a 'recognition loop', which acts as a static anchor at the ETF-partner interface, and (b) a highly mobile redox-active FAD domain. Together, this enables the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. This 'conformational sampling' enables ETF to recognize structurally distinct partners, whilst also maintaining a degree of specificity. Complex formation triggers mobility of the FAD domain, an 'induced disorder' mechanism contrasting with the more generally accepted models of protein-protein interaction by induced fit mechanisms. We discuss the implications of the highly dynamic nature of ETFs in biological interprotein electron transfer. ETF complexes point to mechanisms of electron transfer in which 'dynamics drive function', a feature that is probably widespread in biology given the modular assembly and flexible nature of biological electron transfer systems.

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Section: Kinetics Of Electron Transfer Between Etf and Partnersmentioning

confidence: 99%

“…Detailed kinetic and mechanistic analyses of the reductive half‐reaction have been studied extensively, and readers are referred to papers such as Scrutton et al . [40], Scrutton and Sutcliffe [35], Roberts et al. [41], Basran et al .…”

Section: Kinetics Of Electron Transfer Between Etf and Partnersmentioning

confidence: 99%

Dynamics driving function − new insights from electron transferring flavoproteins and partner complexes

2007

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“…The mechanism of enzyme reduction by trimethylamine has been addressed by stopped-flow spectroscopy in wild-type (9 -11) and mutant (12)(13)(14)(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 electron transferring flavoprotein (ETF) (17,18).…”

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

Jones¹,

Talfournier²,

Bobrov³

et al. 2002

Journal of Biological Chemistry

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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 ...

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“…Although H-transfer has been shown to occur by vibrationally assisted tunneling in two flavoprotein amine dehydrogenases (6,7), the chemical mechanism of oxidation remains to be established. The cleavage of the reactive C-H bond in amine substrates by flavoproteins has been reviewed (8,9) and discussed in terms of (i) a proton abstraction mechanism involving an active site base to generate a carbanion species (originally proposed for trimethylamine dehydrogenase (TMADH) 1 (10)), (ii) an aminium radical cation mechanism, originally proposed for monoamine oxidase (11), (iii) a mechanism involving H-atom abstraction by an active site radical species (12), and (iv) a mechanism involving nucleophilic attack by the substrate nitrogen on the flavin C4a atom, followed by proton abstraction by an active site base (13). In a modification of the latter mechanism Edmondson and colleagues (14) have suggested that the flavin N-5 atom in monoamine oxidase A acts as a base during substrate C-H bond cleavage.…”

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Optimizing the Michaelis Complex of Trimethylamine Dehydrogenase

Basran

Sutcliffe

Scrutton

2001

Journal of Biological Chemistry

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Recent evidence from isotope studies supports the view that catalysis by trimethylamine dehydrogenase (TMADH) proceeds from a Michaelis complex involving trimethylamine base and not, as thought previously, trimethylammonium cation. In native TMADH reduction of the flavin by substrate (perdeuterated trimethylamine) is influenced by two ionizations in the Michaelis complex with pK a values of 6.5 and 8.4; maximal activity is realized in the alkaline region. The latter ionization has been attributed to residue His-172 and, more recently, the former to the ionization of substrate itself. In the Michaelis complex, the ionization of substrate (pK a ϳ 6.5 for perdeuterated substrate) is perturbed by ϳ؊3.3 to ؊3.6 pH units compared with that of free trimethylamine (pK a ‫؍‬ 9.8) and free perdeuterated trimethylamine (pK a ‫؍‬ 10.1), respectively, thus stabilizing trimethylamine base by ϳ2 kJ mol ؊1. We show, by targeted mutagenesis and stoppedflow studies that this reduction of the pK a is a consequence of electronic interaction with residues Tyr-60 and His-172, thus these two residues are key for optimizing catalysis in the physiological pH range. We also show that residue Tyr-174, the remaining ionizable group in the active site that we have not targeted previously by mutagenesis, is not implicated in the pH dependence of flavin reduction. Formation of a Michaelis complex with trimethylamine base is consistent with a mechanism of amine oxidation that we advanced in our previous computational and kinetic studies which involves nucleophilic attack by the substrate nitrogen atom on the electrophilic C4a atom of the flavin isoalloxazine ring. Stabilization of trimethylamine base in the Michaelis complex over that in free solution is key to optimizing catalysis at physiological pH in TMADH, and may be of general importance in the mechanism of other amine dehydrogenases that require the unprotonated form of the substrate for catalysis.The oxidation of amines is widespread in biology and a number of enzymes have evolved to catalyze these reactions. The oxidoreductases responsible for these reactions can be grouped broadly into the flavoprotein and quinoprotein families. The mechanism of amine oxidation catalyzed by the quinoprotein amine oxidases is understood reasonably well and occurs through the formation of enzyme-substrate covalent adducts with topaquinone or tryptophan tryptophylquinone redox centers (1, 2). In some cases C-H bond cleavage by these enzymes has been shown to involve quantum tunneling mechanisms for transfer of the H nucleus to a base in the enzyme active site (3-5). By contrast, the mechanism of amine oxidation by flavoproteins is understood less well. Although H-transfer has been shown to occur by vibrationally assisted tunneling in two flavoprotein amine dehydrogenases (6, 7), the chemical mechanism of oxidation remains to be established. The cleavage of the reactive C-H bond in amine substrates by flavoproteins has been reviewed (8, 9) and discussed in terms of (i) a proton abstraction mechanism invo...

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Trimethylamine Dehydrogenase and Electron Transferring Flavoprotein

Cited by 7 publications

References 79 publications

Dynamics driving function − new insights from electron transferring flavoproteins and partner complexes

Dynamics driving function − new insights from electron transferring flavoproteins and partner complexes

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

Optimizing the Michaelis Complex of Trimethylamine Dehydrogenase

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