REVIEW ARTICLE Dynamics driving function ) new insights from electron transferring flavoproteins and partner complexes Helen S. Toogood, David Leys and Nigel S. Scrutton Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, UK Introduction Electron transferring flavoprotein (ETF) is positioned at a key metabolic branch point, and is responsible for transferring electrons from up to 10 primary dehydro- genases to the membrane-bound respiratory chain, the nature and diversity of which vary between organisms [1]. ETFs are highly dynamic and engage in novel mechanisms of interprotein electron transfer, which is dependent on large-scale conformational sampling to explore optimal configurations to maximize electronic coupling. Sampling mechanisms enable efficient com- munication with structurally distinct redox partners [2], but require additional mechanisms for complex assembly to impart specificity in the protein–protein interaction. ETFs are soluble heterodimeric FAD-containing proteins that are found in all kingdoms of life. They contain a second nucleotide-binding site which is usually occupied by an AMP molecule [1]. In bacteria and eukaryotes, ETFs function primarily as solu- ble one- or two-electron carriers between various Keywords acyl-CoA dehydrogenase; conformational sampling; electron transferring flavoprotein; imprinting; trimethylamine dehydrogenase Correspondence N. Scrutton, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK Fax: + 44 1613065201 Tel: + 44 1613065152 E-mail: nigel.scrutton@manchester.ac.uk Website: http://www.mib.manchester.ac.uk (Received 10 July 2007, revised 24 August 2007, accepted 14 September 2007) doi:10.1111/j.1742-4658.2007.06107.x 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 structur- ally distinct partners, whilst also maintaining a degree of specificity. Com- plex formation triggers mobility of the FAD domain, an ‘induced disorder’ mechanism contrasting with the more generally accepted models of pro- tein–protein interaction by induced fit mechanisms. We discuss the implica- tions 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. Abbreviations ACAD, acyl-CoA dehydrogenase; DMButA, n-butyldimethylamine; ETF, electron transferring flavoprotein; ETFQO, electron transferring flavoprotein ubiquinone oxidoreductase; Fc + , ferricenium ion (oxidized); GAII, glutaric acidaemia ⁄ aciduria type II; MCAD, medium-chain acyl- CoA dehydrogenase; SAXS, small-angle X-ray solution scattering; TMA, trimethylamine; TMADH, trimethylamine dehydrogenase. FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS 5481 flavoprotein-containing dehydrogenases. Electrons are accepted or donated to ETF via the formation of transient complexes with their partners [3]. Almost all ETFs are mobile carriers containing a flexible domain essential for function [4]. ETFs need to balance pro- miscuity with specificity in their interactions with pro- tein donors and acceptors, in keeping with their function in respiratory pathways. In this review, we discuss new aspects of the structure and mechanism of ‘typical’ ETFs, and explore the diversity in func- tion and structure of ETFs across kingdoms. Finally, we analyse, in the context of new structural informa- tion, the role of clinical mutations in human ETFs and their partner proteins that give rise to severe metabolic diseases. ETF families ETFs across kingdoms interact with a variety of elec- tron donors ⁄ acceptors that are involved in diverse met- abolic pathways. ETFs belong to the same families of a ⁄ b-heterodimeric FAD-containing proteins [5–7]. Members of these families can be divided roughly into three groups based on sequence homology and func- tional types. Group I ETFs are a well-studied group of electron carriers, typically found in mammals and a few bacte- ria. Mammalian ETFs are physiological electron acceptors for at least nine mitochondrial matrix flavo- protein dehydrogenases [4,8]. These dehydrogenases include the chain length-specific acyl-CoA dehydrogen- ases (e.g. medium-chain acyl-CoA dehydrogenase, MCAD) involved in fatty acid b-oxidation, isovaleryl- CoA dehydrogenase, 2-methyl branched-chain acyl- CoA dehydrogenase, glutaryl-CoA dehydrogenase involved in amino acid oxidation, as well as dimethyl- glycine and sarcosine dehydrogenases involved in cho- line metabolism [4,8]. Electrons are passed from these primary dehydrogenases through ETF to membrane- bound ETF ubiquinone oxidoreductase (ETFQO) [9,10]. Another well-studied group I ETF is from the bacte- rium Paracoccus denitrificans [11–13]. It is capable of accepting electrons from P. denitrificans glutaryl-CoA dehydrogenase, in addition to the butyryl-CoA and octanoyl-CoA dehydrogenases from pig liver. The physiological electron acceptor for ETF has been found to be ETFQO [12]. Group II ETFs are homologous to the proteins FixB and FixA, equivalent to a-ETF and b-ETF, respectively, which are found in nitrogen-fixing and diazotrophic bacteria [14]. These ETFs are often electron donors to enzymes such as butyryl-CoA dehydrogenase, and may also accept electrons from donors such as ferredoxin and NADH [15]. No ETF- dependent activity has been observed with the mem- brane-bound respiratory enzymes in nitrogen-fixing bacteria, and so it is thought that the electron transfer pathway from ETF to dinitrogen is via the enzymes ETF:ferredoxin oxidoreductase, ferredoxin, nitrogenase reductase and nitrogenase [14]. A well-studied group II ETF is from the bacterium Methylophilus methylotrophus strain W3A1, which con- tains only one known dehydrogenase partner, namely trimethylamine dehydrogenase (TMADH) [3,16]. FixB ⁄ FixA proteins have been characterized from the micro- aerobic Azorhizobium caulinodans, which is known to accept electrons from pyruvate dehydrogenase under aerobic conditions [14]. The nitrogen-fixing organism Bradyrhizobium japonicum contains two sets of ETF- like genes: one with high homology to group I ETFs (etfSL), and the other very similar to group II FixB ⁄ FixA proteins [17]. Under aerobic conditions, only the etfSL genes are expressed, whereas the reverse is true for anaerobic growth, as nitrogen fixation only occurs anaerobically [17]. One ETF from the anaerobe Megasphaera elsdenii (formerly Peptostreptococcus elsdenii) is unusual, as it contains two FAD-binding sites per ETF molecule, and so does not bind AMP [6,15,18,19]. This ETF serves as an electron donor to butyryl-CoA dehydro- genase via its NADH dehydrogenase activity [6], and is an electron acceptor for d-lactate dehydrogenase [15]. It has also been shown to contain a low percent- age of the modified flavins 6-OH-FAD and 8-OH- FAD [6]. Group III ETFs include a pair of putative proteins, YaaQ and YaaR, located adjacent to the cai operon, which encodes carnitine-inducible proteins in Escheri- chia coli [7]. Group III members will not be discussed further in this review. An examination of the databases of genomic sequences shows organisms containing multiple ETF- like genes as well as ETFs fused with other proteins (Pedant; http://pedant.gsf.de). The genome of the eubacterium Fusobacterium nucleatum ssp. nucleatum (ATCC 25586) suggests the presence of two complete ETF molecules, each positioned upstream of an acyl- CoA dehydrogenase. The genome also contains a large ORF (GI:19704756; Pedant; http://pedant.gsf.de) con- taining a fusion of three proteins comprising an N-ter- minal short-chain acyl-CoA dehydrogenase, followed by the a-subunit only of ETF and a C-terminal rubre- doxin (Fig. 1). As no functional studies of this enzyme have been published, it is presumed that the absence of the b-ETF subunit is a result of its role as a ‘fixed’ ETF and partners – structure, function and dynamics H. S. Toogood et al. 5482 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS electron carrier, although flexibility within the multi- domain complex may be possible. Another example of an organism with multiple ETF content is the iron-reducing, nitrogen-fixing bacterium Geobacter metallireducens (Pedant; http://pedant. gsf.de). At least three of the sets of ETF genes are unusual (e.g. ORF4) as the N-terminal portion of the a-ETF subunit contains the gene sequence encoding a [4Fe)4S] 2+ ⁄ + ferredoxin domain (Fig. 1). These ETFs are found upstream of genes such as putative Fe–S oxidoreductases (Pedant; http://pedant.gsf.de). At least nine other putative [4Fe)4S] 2+ ⁄ + ferredoxin-contain- ing ETFs have been identified (NCBI blast; http:// www.ncbi.nlm.nih.gov/BLAST). Many archaea contain ETF- or FixB⁄ A-like sequences, such as Archaeoglobus fulgidus DSM 4304, Pyrobaculum aerophilum st. IM2, Aeropyrum pernix and Thermoplasma volcanium st. GSS1, but these are absent in methanogens (Pedant; http://pedant.gsf.de). Several genera, such as Thermoplasma and Sulfolobus, contain multiple ETF genes, including a fusion protein of the two subunits, with the b-subunit at the N-termi- nus (ba-ETF). In Sulfolobus solfataricus, ba-ETF is found in an operon-like cluster of genes containing the primary dehydrogenase 2-oxoacid ferredoxin oxido- reductase, a putative ferredoxin-like protein and a FixC-like protein, homologous to the membrane- bound ETF ferredoxin oxidoreductase in nitrogen- fixing organisms [14]. A blast search of the structurally equivalent N-ter- minal (non-FAD-binding) a-ETF and b-ETF sequences against known ORFs showed homology with a variety of adenosine nucleotide-binding enzymes (NCBI blast; http://www.ncbi.nlm.nih.gov). Such enzymes include members of the adenosine nucleotide a-hydrolase superfamily from Oryza sativa, which con- tains an ATP-binding fold [20]. The thiamine bio- synthesis-like protein from three Leishmania species contains b-ETF and aminotransferase components at the N- and C-termini, respectively [21]. This class of enzyme is known to bind ATP. Other ATP-binding enzymes with homology to b-ETF in the database (NCBI blast; http://www.ncbi.nlm.nih.gov) include adenylyl-sulfate kinase from Anaeromyxobacter sp. Fw109-5 (GI:121539501), the predicted glutamate- dependent NAD(+) synthase from Strongylocentrotus purpuratus (GI:115971088) and the asparagine synthase from Desulfovibrio vulgaris ssp. vulgaris DP9 (GI:120564303). As b-ETF typically binds AMP, homology to domains of other enzymes known to bind adenosine nucleotides is not surprising. Sequence homology of ETFs An alignment of a- and b-ETFs from all kingdoms of life (Fig. 2) shows that, within the a-ETF family, the overall sequence homology is low, although high sequence homology is found in the C-terminal region. By contrast, in the b-ETF family, there is a similar degree of sequence similarity throughout the length of the protein. Group I ETFs align better than group II ETFs, although both groups contain significant sequence similarity in conserved regions. The C-terminal portion of a-ETF contains a highly conserved region, known as the b 1 ab 2 region of FAD enzymes, which binds the adenosine pyrophosphoryl moiety of FAD [22]. Within this region is the a-ETF consensus sequence of PX[L,I,V]Y[L,I,V]AXGIS- GX[L,I,V]QHX 2 G [7], similar to the consensus sequence for FAD-binding dehydrogenases of GXGXXGX 15 [E ⁄ D] [22]. The b-ETF family contains a conserved signature sequence of VXRX 2 [E,D]- X 3 [E,Q]X[L,I,V]X 3 LP[C,A][L,I,V] 2 which is used to identify members of the b-ETF family [7]. Adjacent to this signature sequence, group I b-ETFs also show the highly conserved region of DLRLNEPR- YA[S ⁄ T]LPNIMKAKKK (residues 184–204; human numbering), containing the recognition loop and the highly conserved L195 necessary for partner binding in Fusobacterium nucleatum Butyryl-CoA dehydrogenase α-ETF Rubredoxin β-ETF Fusion protein Probable Fe-S oxidoreductase Geobacter metallireducens Ferredoxin α-ETF Rubredoxin oxidoreductase Fusion protein Fig. 1. Schematic diagram of the ‘operon-like’ arrangement of genes and fusion proteins from Fusobacterium nucleatum ssp. nucleatum (ATCC 25586) and Geobacter metallireducens (ORF4; Pedant; http://pedant.gsf.de). H. S. Toogood et al. ETF and partners – structure, function and dynamics FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS 5483 humans [23]. The group II b-ETF from M. methylotro- phus also contains a recognition loop and the highly conserved L193 partner binding to TMADH [3]. Other group II members appear not to contain a significant group I-like recognition loop, suggesting a different mode of partner binding. ETF and partners – structure, function and dynamics H. S. Toogood et al. 5484 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS Structure of ETF Domains of ETF The three-dimensional structures of group I ETFs have been solved from humans (Fig. 3A) [1] and P. denitrifi- cans [13], and group II ETF from M. methylotrophus (W3A1; Fig. 3B) [3]. The structure of the P. denitrifi- cans ETF is nearly identical to human ETF, with the major difference being a random loop between residues b90–96 which is an a-helix in humans [13]. All three structures can be divided into three distinct domains. Domain I is composed of mostly the a-subunit, whereas domain III is made up entirely of the b-sub- unit [1]. These domains share nearly identical polypep- tide folds related by a pseudo-twofold axis, in spite of a lack of sequence similarity. Both domains I and III are composed of a core of a seven-stranded parallel b-sheet, flanked by solvent-exposed a-helices. These domains also contain a three-stranded antiparallel b-sheet with a fourth strand coming from the opposite domain. Together these two domains form a shallow bowl shape, and make up the ‘rigid’ or more static part of the molecule upon which domain II rests. Domain III contains a deeply buried AMP molecule which plays a purely structural role [1]. Domain II is the FAD-binding domain, and is attached to domains I and III by flexible linker regions (Fig. 3) [1]. Domain II can be subdivided into two domains, II a and IIb, which are composed of the C-terminal portions of the a- and b-subunits, respec- tively. Domain IIa is the larger of the two, folds in a manner similar to bacterial flavodoxins [24] and forms most of the region that binds FAD. This is the region of high sequence similarity within the a-subunit. This fold consists of a core of a five-stranded parallel b-sheet surrounded by alternating a-helices [1]. A sixth strand of the b-sheet is provided by the b-subunit. FAD is bound in an orientation in which the isoallox- azine ring is situated in a crevice between domains II and III, with the xylene portion pointed towards the b-subunit. By contrast, domain IIb does not interact with FAD, but instead wraps around the lower portion of domain IIa near domains I and III [1]. Despite the low sequence similarity between the two groups of ETF, the overall folding of the struc- tures is very similar, with the exception of the orien- tation of the flavin-binding domain. Domain II of W3A1 ETF is rotated by about 40° relative to the human and P. denitrificans flavin domains, with Va190 and Pb235 (W3A1 numbering) serving as hinge points [3]. In human ETF, the conserved Eb165 of domain III interacts with Na259, which is located near the conserved Ra249 (Ra237 in W3A1) and FAD (Fig. 4A). There are also hydrophobic interactions between the C7- and C8-methyl groups II II FAD AB FAD III I III I Human W3A 1 Fig. 3. Overall structures of the ETFs from humans (A) and Methylophilus methylotro- phus W3A1 (B). PDB codes: human, 1EFV [1]; W3A1, 1O96 [3]. a- and b-ETF chains are shown as magenta and blue cartoons. FAD and AMP are shown as yellow and orange sticks, respectively. Conserved Leub195 ⁄ 194 for human and W3A1 ETFs, respectively, are shown as red spheres. Fig. 2. Alignment of a-ETFs (A) and b-ETFs (B) across kingdoms. Organisms: BRADI, Bradyrhizobium japonicum etfSL genes (P53573 ⁄ P53575); BRADII, Bradyrhizobium japonicum FixB ⁄ A genes (P10449 ⁄ P53577); HUMAN, mature human sequence (P13804 ⁄ P38117); METH, Methylophilus methylotrophus (P53571 ⁄ P53570); PARA, Paracoccus denitrificans (P38974 ⁄ P38975); SULF, Sulfol- obus solfataricus (Q97V72 ⁄ Q97V71). Sequences were obtained from the Swiss-Prot database (http://www.expasy.org) with accession num- bers in parentheses. The numbering for W3A1 and P. denitrificans a-ETF residues in the text are for the cloned forms of the protein in which a methionine (in bold typeface) has been inserted at the beginning of each gene. Residue colours: orange, FAD binding; blue, AMP binding; red, interaction with partners; green, interaction between domain III and flexible domain II; violet, b-ETF signature sequence; yellow, hinge points. The dotted red line refers to the recognition loop. H. S. Toogood et al. ETF and partners – structure, function and dynamics FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS 5485 of the isoalloxazine ring of FAD and residues Fb41 and Yb16, respectively, of domain III [1]. These interactions are likely to transiently stabilize the fla- vin domain in this position [25]. Sequence alignments show that Eb165 (human numbering, Fig. 1) is highly conserved amongst mostly group I ETFs, including P. denitrificans ETF (Eb162), which also contains the flavin domain in the same position as humans. This suggests that this may be a common orientation of the flavin domain amongst group I members. As a result of the change in orientation of the flavin domain in W3A1 ETF, Eb163 (equivalent to human Eb165) interacts instead with the conserved Ra237 via a bifurcated salt bridge (Fig. 4B) [3]. This arginine resi- due also forms a single salt bridge with Da241 of domain II. A second interaction between these two domains is seen in the low-resolution W3A1 ETF structure [3], between residues Ra211 and Eb37. In humans, the equivalent arginine residue, Ra223, inter- acts directly with the flavin and is over 8 A ˚ from domain III [3]. R 211 E 37 L 184 D 241 W 38 R 237 FAD E 163 F 41 FAD R 249 E 165 N 259 AB CD 3 structures Multiple positions of the flavin domain Low resolution solution structure II II III IIIII Fig. 4. Interactions between domains II and III in human (A) and Methylophilus methylotrophus W3A1 (B) ETFs. PDB codes: human, 1EFV [1]; W3A1, 1O96 [3]. a- and b-ETF chains are shown as magenta and blue cartoons and sticks. FAD is shown as yellow sticks and a water molecule is shown as a red sphere. Hydrogen bonds and hydrophobic interactions are shown as dotted and broken lines, respectively. (C) Small-angle X-ray scattering solvent envelope of W3A1 ETF, with a superimposition of the crystal structures of free ETF within it [4]. a- and b-ETF chains are shown as blue and magenta cartoons, respectively. Domains are labelled with Roman numerals. Adapted from [3]. (D) Superimposition of three free ETF structures showing the two positions of the flavin domain. Adapted from [4]. a- and b-ETF chains are shown as green and red cartoons, respectively. Domains are labelled with Roman numerals. ETF and partners – structure, function and dynamics H. S. Toogood et al. 5486 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS Solution structure of free ETF Small-angle X-ray solution scattering (SAXS) studies carried out on human, P. denitrificans and W3A1 ETFs have shown that the solvent envelopes of each ETF are almost identical, in spite of the different con- formations of domain II [4]. A superimposition of the solvent envelope of W3A1 ETF onto the structure of its free ETF shows that, although domains I and III fit well, the envelope around domain II shows the exis- tence of multiple conformations in solution (Fig. 4C) [3]. These conformations appear to arise from domain II rotating about 30–50° with respect to domains I and III via two flexible hinge regions. This corresponds to a shift in position of domain II from the W3A1 posi- tion to the human ⁄ P. denitrificans position. The lack of an appropriate shoulder in the intermediate angle range, which can be associated with the static lobed domain structures, suggests that all three ETFs possess similar domain arrangements in solution, with the fla- vin domain sampling a range of conformational states. These states are likely to include multiple discrete, but transient states. A superimposition of W3A1 ETFs with different flavin domain positions, modelled by weighted masses molecular dynamics, has shown that these conformations are consistent with the solvent envelope of ETF [3]. The solvent envelopes of both oxidized and reduced W3A1 ETF are essentially identi- cal, suggesting that no large conformational change occurs as a result of changing the redox state [4]. The conformations seen crystallographically may have arisen from the trapping of a particular discrete state as a result of crystal packing constraints, but may also reflect differences in the proportions of the discrete states between the different ETFs [25]. Cofactor binding The isoalloxazine rings of FAD from human and W3A1 ETFs are sandwiched between several conserved residues that make distinct, but structurally equivalent, interactions (Fig. 5A) [1,3]. A key characteristic of ETF FAD-binding domains is the ‘bent’ conformation of the ribityl chain of FAD as a result of 4¢OH hydro- gen bonding with N1 of the isoalloxazine ring [1]. It is thought that the 4¢OH group helps to stabilize the semiquinone ⁄ dihydroquinone couple, and may be involved in electron transfer to ETFQO. Another char- acteristic feature is the absence of aromatic residues that stack parallel to the ring. One or two aromatic residues (Yb16 and Fb41 in humans) are within hydro- phobic interaction distance, but the rings are not ori- ented towards FAD. In its place the guanidinium portion of the side chain of the conserved Ra249 is perpendicular to the xylene portion of the isoalloxazine ring, which may function by stabilizing the anionic reduced FAD [13], and also by conferring a kinetic block on full reduction to the dihydroquinone [3]. Other key interactions include the N1 residue of Ha268 with O2 of the isoalloxazine ring, which may also function in stabilizing the anionic semiquinone [1]. The hydroxyl group of Ta266 interacts with N5 of FAD, which may aid in modulating the redox poten- tial. The ADP moiety of FAD is solvent exposed, more so in W3A1 ETF [3]. Stabilization of the nega- tive charge imposed by the phosphates is achieved through interactions with residues such as Sa248 and Sa281 [1]. A B Fig. 5. (A) Schematic representation of the FAD-binding region of human ETF. PDB code, 1EFV [1]. FAD residues and water are shown as atom-coloured sticks and red circles, respectively. (B) AMP-binding region of human ETF. Residues and FAD are shown as atom-coloured sticks and water molecules are shown as red spheres. Potential interactions are shown as dotted lines. H. S. Toogood et al. ETF and partners – structure, function and dynamics FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS 5487 The AMP-binding sites of all three ETF structures are very similar, both in terms of the position and types of interaction between AMP and b-ETF. AMP is buried deeply within domain III and is thought to play a purely structural role (Fig. 5B) [1]. These inter- actions are mostly backbone interactions; thus, although there is a high degree of conservation of posi- tion of the interacting residues, there is often a low sequence conservation (Fig. 2; blue residues). The phosphate moiety of AMP from humans forms hydro- gen bonds with the residues Ab126, Db29, Nb32, Qb33 and Tb34, as well as a water molecule. A few hydrogen bonds are found to anchor the rest of the AMP molecule, including backbone interactions with Cb66 and Ab9 and two water molecules [1]. It is thought that AMP binding may be a structural rem- nant of a NADP-binding site, which is a known elec- tron donor of the group II ETF from Megasphaera elsdenii, which does not bind AMP [6]. Structure of ETF–partner complexes Methylophilus methylotrophus TMADH:ETF The first structure of an ETF in complex with its part- ner protein was solved between TMADH and ETF from M. methylotrophus W3A1 [3]. The structure of the free TMADH dimer had been solved previously, and was shown to contain the redox-active cofactors 6-S-cysteinyl FMN and [4Fe)4S] 2+ ⁄ + (electron donor to ETF), as well as a purely structural ADP molecule (Fig. 6A) [26,27]. Two crystal forms were obtained for the wild-type complexes, which were found to be virtu- ally identical, suggesting that the structure is largely independent of crystal packing contacts. The total bur- ied interfacial surface visible in the structures was elon- gated in shape and covered 1750 A ˚ 2 , with 10% and 8% of the surface contributed by ETF and TMADH, respectively [3]. Surprisingly, there was a complete absence of density for the mobile flavin domain of ETF, in spite of SDS-PAGE analysis of the TMADH:ETF crystals showing its presence [3]. The structures showed that there was an interaction site between the two proteins, which was distinct from the predicted location of the flavin-binding domain of ETF [3]. This consists of a hydrophobic interaction between a surface patch in the ADP-binding domain of TMADH and a loop in ETF domain III (residues Pb189–Ib197), termed the ‘recognition loop’ (Fig. 6B). This loop consists of the N-terminal portion of an a-helix and part of the preceding loop. A residue key to this interaction is the ETF residue Lb194 (red sphere in Fig. 3), which is buried within this hydro- phobic patch of TMADH. Other hydrophobic residues of ETF interacting with TMADH are Yb191, Ib197 and Sb193, the latter of which stabilizes the initial turn of the a-helix in the recognition loop. These residues are highly conserved, in particular within group I ETFs (Fig. 1). Several residues preceding Yb191 which do not contact TMADH are also conserved, including Lb186, Nb187, P b189 and Rb190. The recognition loop is stabilized by both the close packing of these residues and a bifurcating salt bridge between Rb190 and residues Eb44 and Eb51. Several other residues involved in complex formation include a salt bridge between the N-terminus of TMADH and Db16 of ETF, and a number of direct or water-mediated hydro- gen bonds. This relatively small number of interactions helps to explain why the dissociation constant ($ 5 lm) of TMADH:ETF is weak [3,28]. In free ETF, the recognition loop is more flexible and is oriented slightly differently, with Pb189 and Pb204 serving as hinge points [3]. Limited trypsin pro- teolysis, which removed the recognition loop, produced an ETF whose structure and redox capabilities with dithionite were virtually identical to native ETF, yet it had lost its ability to accept electrons from TMADH. This shows the pivotal role of the recognition loop in complex formation, and serves as an ‘anchor’ distant to the redox centres [3]. This anchor may serve as a means of recognizing specific redox partners, as all that would be required would be a suitably placed hydrophobic patch to interact with the recognition loop [3]. The absence of density for the flavin domain of ETF occurs after residues Va190 and Pb235, which serve as hinge points [3]. This total lack of density was initially surprising, as the free ETF structure showed clear den- sity for the flavin domain, in spite of the known flexi- bility of the molecule in solution from SAXS studies [4]. This suggests that either the flavin domain has an increased mobility within the complex, or packing con- straints with the free ETF structure lock the domain in one position. This mobility of the flavin domain within the complex lends support to the transient nature of the electron transfer-competent state, as predicted from kinetics and other studies [4,25]. Several mutant TMADH:ETF complexes were designed which altered the interactions between the flavin domain and domain III of ETF, as well as its interaction with TMADH (see ‘Human MCAD:ETF’ section below). At least two of each of the mutant com- plex structures were determined, TMADH WT:ETF Eb37Q and TMADH Y442F:ETF WT, including two structures in a new space group (H. S. Toogood, D. Leys & N. S. Scrutton, unpublished results). All ETF and partners – structure, function and dynamics H. S. Toogood et al. 5488 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS structures were virtually identical to the wild-type complex, including the absence of the flavin domain, highlighting the rapid mobility of this domain. Modelling studies in which the flavin domain of ETF was docked into the TMADH:ETF complex, based on its position in free ETF, showed that the flavin domain had to undergo a significant conforma- tional change to prevent clashes with TMADH [3,4]. This is supported by the detection of structural changes on complex formation by observing spectral changes during difference spectroscopy studies of TMADH:ETF [29]. Shifting the domain into a human- like conformation would allow the domain to fit within the allowable space. The ‘empty volume’ observed gp FMN 9 [4Fe-4S] 2+/+ R 37 L 194 Reco nition loo Y442 AMP 6-S-cysteinyl L14 ADP A BC TMADH (monomer) ETF FAD 2 Y442 V344 FAD G479 A480 S391 L393 T414 Q462 H416 Y478 A464 R 195 S 193 A 192 Y 191 Fig. 6. (A) Structure of the TMADH:ETF complex. Only one TMADH and ETF are shown for clarity. PDB code for all, 1O94 [3]. a- and b-ETF chains and TMADH are shown as magenta, blue and green cartoons, respectively. The TMADH cofactor 6-S-cysteinyl FMN is shown as yel- low sticks, and the [4Fe)4S] 2+ ⁄ + centre is shown as red and yellow spheres. TMADH ADP and ETF AMP are shown as orange sticks. Resi- dues Y442 and V344 are shown as blue sticks. The recognition loop of ETF is shown as a red cartoon with the conserved Lb194 residue shown as red sticks. The dotted circle refers to the approximate position of the missing flavin domain. (B) Structure of the recognition loop in TMADH:ETF. Residues are shown as atom-coloured sticks with green and blue carbons for TMADH and ETF, respectively. (C) Model of ETF domain II in the TMADH:ETF complex. a-ETF and TMADH are shown as magenta and green cartoons, respectively. The two FAD mole- cules are shown as yellow sticks. Highlighted residues are shown as atom-coloured sticks with green and magenta carbons for TMADH and ETF, respectively. H. S. Toogood et al. ETF and partners – structure, function and dynamics FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS 5489 between TMADH and ETF is of sufficient size and shape to allow the flavin domain of ETF to undergo a ‘ball-in-socket’ type of motion [3], suggesting that mul- tiple (> 2) conformations are possible. This suggests an ‘induced fit’ model for partner association, with electron transfer likely to be possible from an ensemble of thermodynamically metastable complexes rather than one discrete species [3]. Kinetics studies have shown that, in the electron transfer-competent state, the flavin of ETF is likely to be close to a surface groove of TMADH close to resi- dues V344 and Y442 [30]. Molecular dynamics calcula- tions were performed on the flavin domain of free ETF superimposed onto the complex to determine potential electron transfer-competent states [3]. A model of one of the putative ‘active’ conformations between the [4Fe)4S] 2+ ⁄ + centre of TMADH and the flavin domain of ETF gives an intercofactor distance of less than 14 A ˚ (Fig. 6C) [3]. In this state, the guanidinium ion of the conserved Ra237 is located close to the aro- matic ring and hydroxyl group of Y442 of TMADH. Cross-linking studies using bismaleimidohexane with TMADH Y442C and ETF Ra237C mutants led to the rapid formation of a cross-linked complex, establishing the close contact of these residues in the complex. Also, difference spectroscopy studies with TMADH and the ETF mutant Ra237A showed that electron transfer was severely compromised as a result of a change in the rate of rearrangement of ETF to form the electron transfer-competent state, rather than a change in the intrinsic rate of electron transfer [29]. However, any interactions between TMADH and the flavin domain of ETF are likely to be fleeting, and simply increase the half-life of the electron transfer-competent states to allow fast electron transfer [3]. Human MCAD:ETF To investigate the way in which ETF can interact with its structurally distinct partners, the structure of human ETF with its partner MCAD was determined [23]. The structure of free MCAD had been solved pre- viously, and was shown to be a homotetramer of 43 kDa monomers (dimer of dimers) containing one FAD per monomer [31]. The first structure of the com- plex between MCAD and ETF was found to contain a tetramer of MCAD with one ETF molecule [23]. The total buried interfacial surface visible in the structures (excluding the ETF flavin domain) was elongated in shape and covered 536 A ˚ 2 , with 3.2% and 4.3% of the surface contributed by ETF and MCAD, respectively. In this structure, the flavin domain of ETF was barely visible in the density [23]. Four mutant MCAD:ETF complexes were designed which altered the interactions between the flavin domain and domain III of ETF (MCAD:ETF Eb165A), as well as its interaction with MCAD (MCA D:ETF Ra249A; MCAD E212A:ETF; MCAD E359A:ETF) [25]. The aim was to alter the ratio of the different conformational states sufficiently to trap discrete flavin domain positions. Kinetic studies of these complexes showed a reduction in electron transfer rates [when using 2,6-dichloroindophenol as the terminal electron acceptor], except for the MCAD: ETF Eb165A complex, which showed both a dramatic increase in rate and decrease in the apparent K m value. Crystal structures of all four mutant complexes were obtained (Fig. 7A; last three: H. Toogood, A. van Thiel, D. Leys & N. S. Scrutton, unpublished work), which showed an increase in density for the flavin domain to about 70% occupancy (except for MCAD: ETF Ra249A), with the flavin domain in the same position as in the wild-type structure. In these struc- tures, ETF is interacting with a dimer of MCAD [25]. As with the TMADH:ETF structures, human ETF contains a recognition loop (Pb190–Ib198), including the highly conserved residue Lb195, which interacts with a hydrophobic pocket on MCAD (Fig. 7B) [23]. The recognition loop interacts with the MCAD surface in such a way that causes an extension of a-helix C of MCAD [31], with a nearly perfect alignment of the axes and corresponding dipoles of both helices [23]. The side chain of Lb195 is buried within a hydropho- bic pocket formed by a-helices A, C and D of MCAD, and is lined by residues such as F23, L61, L73 and I83. ETF residues which also interact with this pocket include Yb192, Pb197, Ib198 and Mb199 [23]. A comparison of the free and complex crystal struc- tures reveals that, although MCAD adopts a nearly identical conformation in both structures, ETF adopts a slightly different backbone conformation with more extensive side chain rearrangements, including Lb195 [23]. The structure of the free ETF mutant Lb195A does not show any significant rearrangements of the recogni- tion loop, yet kinetic studies with both MCAD, isovale- ryl-CoA dehydrogenase and the structurally distinct partner dimethylglycine dehydrogenase show a severe decrease in electron transfer rates (A. van Thiel, H. Toogood, H. L. Messiha, D. Leys & N. S. Scrutton, unpublished work). Mutations of MCAD, such as L61M, L73W and L75Y, which were designed to ‘fill in’ the binding pocket, were all severely impaired in elec- tron transfer rates with ETF [25]. Microelectrospray ionization mass spectrometry and surface plasma reso- nance studies showed competitive binding of ETF to acyl-CoA dehydrogenases and dimethylglycine dehydro- ETF and partners – structure, function and dynamics H. S. Toogood et al. 5490 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... existence of two possible routes of electron transfer from the [4Fe)4S]2+ ⁄ + centre to an external electron acceptor The shortest pathway extends from C345, a ligand on the [4Fe)4S]2+ ⁄ + ETF and partners – structure, function and dynamics centre, to V344, which is located at the bottom of a small groove on the surface of TMADH The second pathway extends from C345 to E439 and finally to Y442, the latter... dehydrogenase In Flavins and Flavoproteins (Edmondson DE & McCormick DB, eds), pp 687–690 Walter de Gruyter, Berlin Jones M, Talfournier F, Bobrov A, Grossmann JG, Vekshin N, Sutcliffe MJ & Scrutton NS (200 2) Electron transfer and conformational change in complexes of trimethylamine dehydrogenase and electron transferring flavoprotein J Biol Chem 277, 8457–8465 Thorpe C (199 1) Electron- transferring flavoproteins. .. 1977–1989 Scott JD & Ludwig RA (200 4) Azorhizobium caulinodans electron- transferring flavoprotein N electrochemi- ETF and partners – structure, function and dynamics 15 16 17 18 19 20 21 22 23 24 25 26 27 cally couples pyruvate dehydrogenase complex activity to N2 fixation Microbiology 150, 117–126 Pace CP & Stankovich MT (198 7) Redox properties of electron- transferring flavoprotein from Megasphaera elsdenii Biochim... [3] and small-scale conformational changes in the formation of electron transfer-competent state(s) A simplified kinetic scheme for such a system, where A is one -electron- reduced TMADH (4Fe)4S +) and B is oxidized ETF, is shown in Scheme 1 [30] In this scheme, kr (and k–r) refer to the reversible rate of reor- A branching kinetic steady-state scheme has been proposed for intra- and interprotein electron. .. MJ (200 0) Trimethylamine dehydrogenase and electron transferring flavoprotein Sub-Cell Biochem 35, 145–181 Scrutton NS (200 4) Chemical aspects of amine oxidation by flavoprotein enzymes Nat Product Rep 21, 722–730 Marcus RA & Sutin N (198 5) Electron transfers in chemistry and biology Biochim Biophys Acta 811, 265– 316 Page CC, Moser CC & Dutton PC (200 3) Mechanism for electron transfer within and between... because, although the substrate can donate two electrons at a 5494 FEBS Journal 274 (200 7) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS H S Toogood et al ETF and partners – structure, function and dynamics ETFsq 11 FMN.S (CH 3)3 N 4Fe-4Sox ETFsq + HCHO 4Fe-4Sred 2 FMN 1 (CH 3)2 NH 3 4Fe-4Sox FMNH2 + HCHO (CH 3)3 N 4Fe-4Sox ETFox 4 6 FMNsq.S 7 9 ETFsq (CH 3)3 N 5 FMNsq 10 4Fe-4Sox FMNsq 4Fe-4Sred 4Fe-4Sred... Acta 1433, 139–152 ETF and partners – structure, function and dynamics 66 Dwyer TM, Mortl S, Kemter K, Bacher A, Fauq A & Frerman FE (199 9) The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer Biochemistry 38, 9735–9745 67 Olsen RK, Andresen BS, Christensen E, Bross P, Skovby F & Gregersen N (200 3) Clear relationship between... Mayhew SG (199 5) Cloning of electron- transferring flavoprotein from Megasphaera elsdenii Biochem Soc Trans 23, 379S Sato K, Nishina Y & Shiga K (200 3) Purification of electron- transferring flavoprotein from Megasphaera elsdenii and binding of additional FAD with an unusual absorption spectrum J Biochem 134, 719–729 Yu J, Wang J, Lin W, Li S, Li H, Zhou J, Ni P, Dong W, Hu S, Zeng C, et al (200 5) The genomes... which are compatible with fast electron transfer Transient stabilization of electron transfer-competent states is achieved through interactions between the two partners, including interactions with the conserved Ra237 (W3A1 numbering) [23] This separation of the partner recognition site (recognition loop) from the electron transfer site (flavin domain) is critical in understanding how ETF can interact... transfer flavoprotein families ETF-alpha and ETF-beta Res Microbiol 146, 397–404 Frerman FE (198 8) Acyl-CoA dehydrogenases, electron transfer flavoprotein and electron transfer flavoprotein dehydrogenase Biochem Soc Trans 16, 416–418 Beckmann JD & Frerman FE (198 5) Electron- transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties Biochemistry . REVIEW ARTICLE Dynamics driving function ) new insights from electron transferring flavoproteins and partner complexes Helen S. Toogood, David Leys and Nigel. green and magenta carbons for TMADH and ETF, respectively. H. S. Toogood et al. ETF and partners – structure, function and dynamics FEBS Journal 274 (2007)