Subunit structure of electron transfer flavoprotein

The electron transfer flavoprotein from pig liver mitochondria is a 57,000-dalton electron transferase which links several primary flavoprotein dehydrogenases with the mitochondrial electron transport system. The protein was previously reported to be a dimer of apparently identical subunits. There are conflicting estimates in the literature regarding the FAD content of the protein. The results presented here clearly show that the protein contains nonidentical subunits based on polyacrylamide gel electrophoresis in the presence of 8 M urea and sodium dodecyl sulfate. The molecular weights of the subunits are 31,000 and 27,000. Analysis of peptides generated by cleavage of the subunits with cyanogen bromide show that the subunits have different primary structures. This result and amino acid analyses of the protein and the purified subunits show that the heterogeneity cannot be due to proteolysis. Using an experimentally determined molar extinction coefficient for the protein-bound flavin, a minimum Mr = 55,000 was calculated, indicating that the protein contains 1 mol of FAD/mol of protein.     

The electron transfer flavoprotein: ubiquinone oxidoreductases

Electron transfer flavoprotein: ubiqionone oxidoreductase (ETF-QO) is a component of the mitochondrial respiratory chain that together with electron transfer flavoprotein (ETF) forms a short pathway that transfers electrons from 11 different mitochondrial flavoprotein dehydrogenases to the ubiquinone pool. The X-ray structure of the pig liver enzyme has been solved in the presence and absence of a bound ubiquinone. This structure reveals ETF-QO to be a monotopic membrane protein with the cofactors, FAD and a [4Fe-4S](+1+2) cluster, organised to suggests that it is the flavin that serves as the immediate reductant of ubiquinone. ETF-QO is very highly conserved in evolution and the recombinant enzyme from the bacterium Rhodobacter sphaeroides has allowed the mutational analysis of a number of residues that the structure suggested are involved in modulating the reduction potential of the cofactors. These experiments, together with the spectroscopic measurement of the distances between the cofactors in solution have confirmed the intramolecular pathway of electron transfer from ETF to ubiquinone. This approach can be extended as the R. sphaeroides ETF-QO provides a template for investigating the mechanistic consequences of single amino acid substitutions of conserved residues that are associated with a mild and late onset variant of the metabolic disease multiple acyl-CoA dehydrogenase deficiency (MADD).     

The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S] (2+,1+) and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S] (2+,1+) to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S] (+) at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF 1e (-) catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF.     

Stabilization of non-productive conformations underpins rapid electron transfer to electron-transferring flavoprotein

Crystal structures of protein complexes with electron-transferring flavoprotein (ETF) have revealed a dual protein-protein interface with one region serving as anchor while the ETF FAD domain samples available space within the complex. We show that mutation of the conserved Glu-165beta in human ETF leads to drastically modulated rates of interprotein electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase. The crystal structure of free E165betaA ETF is essentially identical to that of wild-type ETF, but the crystal structure of the E165betaA ETF.medium chain acyl-CoA dehydrogenase complex reveals clear electron density for the FAD domain in a position optimal for fast interprotein electron transfer. Based on our observations, we present a dynamic multistate model for conformational sampling that for the wild-type ETF. medium chain acyl-CoA dehydrogenase complex involves random motion between three distinct positions for the ETF FAD domain. ETF Glu-165beta plays a key role in stabilizing positions incompatible with fast interprotein electron transfer, thus ensuring high rates of complex dissociation.     

The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer.

Electron-transfer flavoprotein (ETF) serves as an intermediate electron carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells. The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen bonded to N(1) of the flavin ring. We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond. The binding constants for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol. The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF containing 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein. These data suggest that the 4'-hydoxyl-N(1) hydrogen bond stabilizes the anionic semiquinone in which negative charge is delocalized over the N(1)-C(2)O region. Transfer of the second electron to 4'-deoxy-FAD reconstituted ETF is extremely slow, and it was very difficult to achieve complete reduction of the flavin semiquinone to the hydroquinone. The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions. This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (相似文献   

Molecular cloning of a cDNA for alpha-subunit of rat liver electron transfer flavoprotein

Two cDNA clones for the alpha-subunit of rat liver electron transfer flavoprotein were isolated and their nucleotide sequences were determined. The longer cDNA contained a protein-coding region of 900 nucleotides and 3'-noncoding region of 335 nucleotides. The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the sequence of one of the lysyl endopeptidase-digested peptides from the purified alpha-subunit. The molecular weight of the protein calculated from the protein-coding nucleotides was approx. 3,000 daltons smaller than that of the precursor, suggesting that the cDNA was not of full length. The derived amino acid composition fairly agreed with the chemically determined amino acid composition of the purified alpha-subunit, indicating that the protein-coding region contains most of the mature alpha-subunit.     

Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans.

Glutaryl-coenzyme A (CoA) dehydrogenase and the electron transfer flavoprotein (ETF) of Paracoccus denitrificans were purified to homogeneity from cells grown with glutaric acid as the carbon source. Glutaryl-CoA dehydrogenase had a molecular weight of 180,000 and was made up of four identical subunits with molecular weights of about 43,000 each of which contained one flavin adenine dinucleotide molecule. The enzyme catalyzed an oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, was maximally stable at pH 5.0, and lost activity readily at pH values above 7.0. The enzyme had a pH optimum in the range of 8.0 to 8.5, a catalytic center activity of about 960 min-1, and apparent Michaelis constants for glutaryl-CoA and pig liver ETF of about 1.2 and 2.5 microM, respectively. P. denitrificans ETF had a visible spectrum identical to that of pig liver ETF and was made up of two subunits, only one of which contained a flavin adenine dinucleotide molecule. The isoelectric point of P. denitrificans ETF was 4.45 compared with 6.8 for pig liver ETF. P. denitrificans ETF accepted electrons not only from P. denitrificans glutaryl-CoA dehydrogenase, but also from the pig liver butyryl-CoA and octanoyl-CoA dehydrogenases. The apparent Vmax was of similar magnitude with either pig liver or P. denitrificans ETF as an electron acceptor for these dehydrogenases. P. denitrificans glutaryl-CoA dehydrogenase and ETF were used to assay for the reduction of ubiquinone 1 by ETF-Q oxidoreductase in cholate extracts of P. denitrificans membranes. The ETF-Q oxidoreductase from P. denitrificans could accept electrons from either the bacterial or the pig liver ETF. In either case, the apparent Km for ETF was infinitely high. P. denitrificans ETF-Q oxidoreductase was purified from contaminating paramagnets, and the resultant preparation had electron paramagnetic resonance signals at 2.081, 1.938, and 1.879 G, similar to those of the mitochondrial enzyme.     

Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex

The crystal structure of the human electron transferring flavoprotein (ETF).medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF.MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD.ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu(212) and ETF Arg alpha(249) are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced electron transfer rates between both partners.     

A polymorphic position in electron transfer flavoprotein modulates kinetic stability as evidenced by thermal stress

The electron transfer flavoprotein (ETF) is a hub interacting with at least 11 mitochondrial flavoenzymes and linking them to the respiratory chain. Here we report the effect of the ETFα-T/I171 polymorphism on protein conformation and kinetic stability under thermal stress. Although variants have comparable thermodynamic stabilities, kinetically their behavior is rather distinct as ETFα-T171 displays increased susceptibility to cofactor flavin adenine dinucleotide (FAD) loss and enhanced kinetics of inactivation during thermal stress. Mimicking a fever episode yields substantial activity loss. However, the presence of substoichiometric concentrations of GroEL is sufficient to act as an effective buffer against long-term thermal denaturation. Our investigations are compatible with the notion that the ETFα-T171 variant displays an altered conformational landscape that results in reduced protein function under thermal stress.     

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.     

The mitochondrial electron transfer flavoprotein complex is essential for survival of Arabidopsis in extended darkness

In mammals, the electron transfer flavoprotein (ETF) is a heterodimeric protein composed of two subunits, alpha and beta, that is responsible for the oxidation of at least nine mitochondrial matrix flavoprotein dehydrogenases. Electrons accepted by ETF are further transferred to the main respiratory chain via the ETF ubiquinone oxide reductase (ETFQO). Sequence analysis of the unique Arabidopsis homologues of two subunits of ETF revealed their high similarity to both subunits of the mammalian ETF. Yeast two-hybrid experiments showed that the Arabidopsis ETFalpha and ETFbeta can form a heteromeric protein. Isolation and characterization of two independent T-DNA insertional Arabidopsis mutants of the ETFbeta gene revealed accelerated senescence and early death compared to wild-type during extended darkness. Furthermore in contrast to wild-type, the etfb mutants demonstrated a significant accumulation of several amino acids, isovaleryl CoA and phytanoyl CoA during dark-induced carbohydrate deprivation. These phenotypic characteristics of etfb mutants are broadly similar to those that we observed previously in Arabidopsis etfqo mutants, suggesting functional association between ETF and ETFQO in Arabidopsis, and confirming the essential roles of the ETF/ETFQO electron transfer complex in the catabolism of leucine and involvement in the chlorophyll degradation pathway activated during dark-induced carbohydrate deprivation.     

Mutational hotspots in electron transfer flavoprotein underlie defective folding and function in multiple acyl-CoA dehydrogenase deficiency

We have carried out an extensive in silico analysis on 18 disease associated missense mutations found in electron transfer flavoprotein (ETF), and found that mutations fall essentially in two groups, one in which mutations affect protein folding and assembly, and another one in which mutations impair catalytic activity and disrupt interactions with partner dehydrogenases. We have further experimentally analyzed three of these mutations, ETFβ-p.Cys42Arg, ETFβ-p.Asp128Asn and ETFβ–p.Arg191Cys, which have been found in homozygous form in patients and which typify different scenarios in respect to the clinical phenotypes. The ETFβ-p.Cys42Arg mutation, related to a severe form of multiple acyl-CoA dehydrogenase deficiency (MADD), affects directly the AMP binding site and intersubunit contacts and impairs correct protein folding. The two other variations, ETFβ-p.Asp128Asn and ETFβ–p.Arg191Cys, are both associated with mild MADD, but these mutations have a different impact on ETF. Although none affects the overall α/β fold topology as shown by far-UV CD, analysis of the purified proteins shows that both have substantially decreased enzymatic activity and conformational stability. Altogether, this study combines in silico analysis of mutations with experimental data and has allowed establishing structural hotspots within the ETF fold that are useful to provide a rationale for the prediction of effects of mutations in ETF.     

The effect of tetrahydrofolate on the reduction of electron transfer flavoprotein by sarcosine and dimethylglycine dehydrogenases.

Pig liver electron transfer flavoprotein (ETF) is rapidly reduced by sarcosine and dimethylglycine dehydrogenases to the anionic semiquinone form, the subsequent formation of the flavoquinol form being a much slower process. In the presence of tetrahydrofolate the yield of anionic semiquinone at the end of the rapid phase of reduction of ETF is only about 10% less than without tetrahydrofolate, as judged by e.p.r. spectroscopy. Tetrahydrofolate does not alter the rate of reduction of ETF by either sarcosine or dimethylglycine dehydrogenase. Nevertheless, it was clearly demonstrated that tetrahydrofolate is a substrate for both sarcosine and dimethylglycine dehydrogenases and is converted to N5,10-methylenetetrahydrofolate.     

Differential coupling through Val-344 and Tyr-442 of trimethylamine dehydrogenase in electron transfer reactions with ferricenium ions and electron transferring flavoprotein

Modeling studies of the trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH-ETF) electron transfer complex have suggested potential roles for Val-344 and Tyr-442, found on the surface of TMADH, in electronic coupling between the 4Fe-4S center of TMADH and the FAD of ETF. The importance of these residues in electron transfer, both to ETF and to the artificial electron acceptor, ferricenium (Fc(+)), has been studied by site-directed mutagenesis and stopped-flow spectroscopy. Reduction of the 6-(S)-cysteinyl FMN in TMADH is not affected by mutation of either Tyr-442 or Val-344 to a variety of alternate side chains, although there are modest changes in the rate of internal electron transfer from the 6-(S)-cysteinyl FMN to the 4Fe-4S center. The kinetics of electron transfer from the 4Fe-4S center to Fc(+) are sensitive to mutations at position 344. The introduction of smaller side chains (Ala-344, Cys-344, and Gly-344) leads to enhanced rates of electron transfer, and likely reflects shortened electron transfer "pathways" from the 4Fe-4S center to Fc(+). The introduction of larger side chains (Ile-344 and Tyr-344) reduces substantially the rate of electron transfer to Fc(+). Electron transfer to ETF is not affected, to any large extent, by mutation of Val-344. In contrast, mutation of Tyr-442 to Phe, Leu, Cys, and Gly leads to major reductions in the rate of electron transfer to ETF, but not to Fc(+). The data indicate that electron transfer to Fc(+) is via the shortest pathway from the 4Fe-4S center of TMADH to the surface of the enzyme. Val-344 is located at the end of this pathway at the bottom of a small groove on the surface of TMADH, and Fc(+) can penetrate this groove to facilitate good electronic coupling with the 4Fe-4S center. With ETF as an electron acceptor, the observed rate of electron transfer is substantially reduced on mutation of Tyr-442, but not Val-344. We conclude that the flavin of ETF does not penetrate fully the groove on the surface of TMADH, and that electron transfer from the 4Fe-4S center to ETF may involve a longer pathway involving Tyr-442. Mutation of Tyr-442 likely disrupts electron transfer by perturbing the interaction geometry of TMADH and ETF in the productive electron transfer complex, leading to less efficient coupling between the redox centers.     

Mammalian electron transferring flavoprotein.flavoprotein dehydrogenase complexes observed by microelectrospray ionization-mass spectrometry and surface plasmon resonance

Microelectrospray ionization-mass spectrometry was used to directly observe electron transferring flavoprotein.flavoprotein dehydrogenase interactions. When electron transferring flavoprotein and porcine dimethylglycine dehydrogenase or sarcosine dehydrogenase were incubated together in the absence of substrate, a relative molecular mass corresponding to the flavoprotein.electron transferring flavoprotein complex was observed, providing the first direct observation of these mammalian complexes. When an acyl-CoA dehydrogenase family member, human short chain acyl-CoA dehydrogenase, was incubated with dimethylglycine dehydrogenase and electron transferring flavoprotein, the microelectrospray ionization-mass spectrometry signal for the dimethylglycine dehydrogenase.electron transferring flavoprotein complex decreased, indicating that the acyl-CoA dehydrogenases have the ability to compete with the dimethylglycine dehydrogenase/sarcosine dehydrogenase family for access to electron transferring flavoprotein. Surface plasmon resonance solution competition experiments revealed affinity constants of 2.0 and 5.0 microm for the dimethylglycine dehydrogenase-electron transferring flavoprotein and short chain acyl-CoA dehydrogenase-electron transferring flavoprotein interactions, respectively, suggesting the same or closely overlapping binding motif(s) on electron transferring flavoprotein for dehydrogenase interaction.     

Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin binding domain

The crystal structure of electron transfer flavoprotein (ETF) from Paracoccus denitrificans was determined and refined to an R-factor of 19.3% at 2.6 A resolution. The overall fold is identical to that of the human enzyme, with the exception of a single loop region. Like the human structure, the structure of the P. denitrificans ETF is comprised of three distinct domains, two contributed by the alpha-subunit and the third from the beta-subunit. Close analysis of the structure reveals that the loop containing betaI63 is in part responsible for conferring the high specificity of AMP binding by the ETF protein. Using the sequence and structures of the human and P. denitrificans enzymes as models, a detailed sequence alignment has been constructed for several members of the ETF family, including sequences derived for the putative FixA and FixB proteins. From this alignment, it is evident that in all members of the ETF family the residues located in the immediate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for the human residues alphaT266 and betaY16, respectively. Mapping of ionic differences between the human and P. denitrificans ETF onto the structure identifies a surface that is electrostatically very similar between the two proteins, thus supporting a previous docking model between human ETF and pig medium-chain acyl-CoA dehydrogenase (MCAD). Analysis of the ionic strength dependence of the electron transfer reaction between either human or P. denitrificans ETF and MCAD demonstrates that the human ETF functions optimally at low ( approximately 10 mequiv) ionic strength, while P. denitrificans ETF is a better electron acceptor at higher (>75 mequiv) ionic strength. This suggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points between the proteins. Analysis of the electrostatic potentials of the human and P. denitrificans ETFs reveals that the P. denitrificans ETF is more negatively charged. This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an explanation for the opposing ionic strength dependencies for the reaction of MCAD with the two ETFs. Furthermore, by analysis of a model of the previously described human-P. denitrificans chimeric ETF protein, it is possible to identify one region of ETF that participates in docking with ETF-ubiquinone oxidoreductase, the physiological electron acceptor for ETF.     

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