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

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

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

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

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

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-

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

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

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

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

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.

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

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 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.

Q163
FAD
Q 265
E212
W166
N354
E359
R
249
Q
285
FAD
T26
L73
L75
G60
L59
L61
F23
I83
F30
E34
P 196
N
197
T
194
A
193
I

tions between ETF and MCAD include direct hydro-
gen bonds between Qa285 ⁄ N354, Qa265 ⁄ E359 and a
phosphate of ETF FAD ⁄ Q163, respectively [25]. The
smallest distance between the isoalloxazine rings of the
two FAD molecules is 9.7 A
˚
, suggesting that this is an
electron transfer-competent state. The indole group of
MCAD W166 is positioned between the isoalloxazine
rings, and is within van der Waals’ contact with both
the C7 and C8 methyl groups of ETF FAD [25].
The complex structure shows that electrostatic inter-
actions are essentially absent from the interface, yet it
is known that the electron transfer rate decreases with
increasing ionic strength [25]. These observations could
be a result of the destabilization of the protein–protein
interaction between E212 and Arga249. Alternatively,
these results may arise from enhanced hydrophobic
interaction at high ionic strength involving the hydro-
phobic patch ⁄ recognition loop. The concomitant
decrease in the rate of complex dissociation following
electron transfer might lead to the observed reduction
in steady-state turnover [25].
Although there are no structural similarities between
TMADH and MCAD, ETF interacts in a similar man-
ner with both proteins [23]. This is a result of the rec-
ognition loop interacting with distinct, but structurally
equivalent, hydrophobic patches on the partners,
which creates a near-identical volume and shape of the
space occupied by the flavin domain of ETF. The rela-

FMN [27], followed by reduction of a ferredoxin-like
[4Fe)4S]
2+ ⁄ +
located approximately 4–6 A
˚
from the
8-a-methyl group of FMN [36]. The physiological ter-
minal electron acceptor of TMADH from M. methy-
lotrophus is ETF, with electron transfer from the
[4Fe)4S]
2+ ⁄ +
centre occurring via quantum electron
tunnelling [37,38]. Stopped-flow kinetics studies of the
reductive half-reaction shows that it occurs in three
kinetic phases. The fast phase represents the two-elec-
tron reduction of 6-S-cysteinyl FMN, followed by
intermediate and slow phases which reflect the transfer
of one electron from the dihydroquinone of flavin to
the [4Fe)4S]
2+ ⁄ +
centre, and the formation of a spin-
interacting state between the flavin semiquinone and
the reduced [4Fe)4S]
2+ ⁄ +
[39]. This latter state is
formed after the binding of a second substrate mole-
cule, which induces the ionization of Y169 located
close to the pyrimidine ring of 6-S-cysteinyl FMN [36].
This state is distinguished by a complex EPR signal
centred near g $ 2 with an unusually intense half-field

ETF and partners – structure, function and dynamics H. S. Toogood et al.
5492 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS
ETF FAD in two single electron transfer steps. The
midpoint reduction potential of the oxidized flavin ⁄
semiquinone couple of ETF (E
ox ⁄ sq
) is unusually posi-
tive (+ 153 mV) [29], but is consistent with the need
to accept electrons from the [4Fe)4S]
2+ ⁄ +
centre of
TMADH, which has a 4Fe)4S
2+
⁄ 4Fe)4S
+
potential
of + 102 mV [47]. This highly positive redox potential
of ETF suggests exceptional stabilization of the anio-
nic semiquinone, most probably because of the loca-
tion of the guanidinium group of the conserved Ra237
over the si face of the flavin isoalloxazine ring.
Conversion of FAD to the dihydroquinone form is
incomplete, with a midpoint potential of the
semiquinone ⁄ dihydroquinone couple of less than
) 250 mV, as a result of the presence of both kinetic
and thermodynamic blocks on full reduction of FAD
[29].
Recent mutagenic studies have shown the impor-
tance of Sa254 as a hydrogen bond donor to the N(5)
atom in the oxidized state of the flavin [48,49]. Muta-

mation [50].
Steady-state kinetic parameters for the electron
transfer between the [4Fe)4S]
2+ ⁄ +
centre of TMADH
and ETF flavin give k
cat
and K
m
values of
16.8 ± 0.5 s
)1
and 14.8 ± 1.2 l m, respectively, at
25 °C [30]. Modelling studies of the complex between
TMADH and ETF, as well as kinetics studies of
TMADH mutants, revealed the existence of a small
surface groove on TMADH which may accommodate
the isoalloxazine ring of FAD bound to ETF [30].
These studies revealed the existence of two possible
routes of electron transfer from the [4Fe)4S]
2+ ⁄ +
cen-
tre to an external electron acceptor. The shortest path-
way extends from C345, a ligand on the [4Fe)4S]
2+ ⁄ +
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 of which forms one side of the groove
on the surface of the enzyme [30].

increase in k
cat
. The kinetics of the reductive half-
reaction of these mutants showed only small changes
in the rate of intramolecular electron transfer from
6-S-cysteinyl FMN to the [4Fe)4S]
2+ ⁄ +
centre,
which may reflect minor structural changes around
the [4Fe)4S]
2+ ⁄ +
centre [30].
Stopped-flow studies on the kinetics of transfer of
electrons from two-electron-reduced TMADH to oxi-
dized ETF revealed complex multiphasic kinetics [51].
To simplify these studies, the 6-S-cysteinyl FMN co-
factor of TMADH was inactivated by phenylhydr-
azine, rendering it inert to reduction ⁄ oxidation. This
allows TMADH to be reduced anaerobically to the
one-electron state, via titration with dithionite, with
the electron located on the [4Fe)4S]
2+ ⁄ +
centre [52].
Fast reaction studies of this one-electron-reduced mod-
ified TMADH with ETF eliminates the complications
arising from internal electron transfer in TMADH
[30]. Initial studies of the kinetics of the oxidative half-
reaction with ETF were carried out at 25 °C, and
showed a hyperbolic dependence on ETF concen-
tration, which exhibited saturation behaviour [53].

[28]. By contrast, the reactions of the mutants and
native TMADH with Fc
+
all showed a linear depen-
dence on Fc
+
concentration at 25 °C. Val344 substitu-
tions to cysteine, alanine or glycine showed a moderate
increase in second-order rate constants, whereas the
opposite was true for the bulkier substitutions tyrosine
and isoleucine, with the latter showing a nearly 20-fold
reduction. This could be the result of a change in the
length of the electron transfer distance and ⁄ or changes
in packing density. These mutations were found to
have little or no effect on the binding or limiting rate
constant (k
lim
) for oxidation of the substrate. These
stopped-flow and steady-state results suggest that elec-
tron transfer to ETF proceeds via the longer pathway
through Y442, whereas electron transfer to Fc
+
is via
the shorter route through V344, as Fc
+
is likely to
penetrate the groove more fully than the ETF flavin.
Substitutions at Y344 showed that shortening the
length of the side chain increased the electron transfer
rates to Fc

k
a
k
Àa
ðABÞ
1
Ð
k
r
k
Àr
ðABÞ
2
!
k
eT
ðA
þ
B
À
Þ!A
þ
þ B
À
ðScheme 1)
As native and V344 mutants of TMADH do not
display saturation kinetics with ETF at 5 °C, this
model predicts that complex formation is rate limiting
[30]. Thus, both k
eT

3
s
)1
. Such
low predicted k
eT
values for Y442 mutants are not
likely to correspond to intrinsic electron transfer rates
for transfers occurring over 11–12 A
˚
[55], and so rate
limitation is likely to be the result of impaired struc-
tural reorganization during complex assembly. In
native TMADH, Y442 may enhance the rates of reor-
ganization of the electron transfer complex by a direct
interaction with ETF via its phenolic hydroxyl group.
Disruption of favourable interactions by Y442 mutants
could thereby alter the nature of the electron transfer-
competent state, such as a change in the [4Fe)4S]
2+ ⁄ +
to ETF FAD distance, leading to a dramatically
reduced k
eT
value [30].
Kinetic scheme of intra- and interprotein
electron transfer
A branching kinetic steady-state scheme has been pro-
posed for intra- and interprotein electron transfer of
TMADH (Fig. 8) [41]. TMADH is unusual as it shows
substrate inhibition at high TMA concentrations. Fig-

experiments with TMA concentrations of 20 lm to
2mm. At high TMA concentrations, the spectrum
indicates that the predominant species at steady state is
the one-electron-reduced flavin semiquinone ⁄ oxidized
[4Fe)4S]
2+ ⁄ +
, consistent with the species predicted to
accumulate in the 1 ⁄ 3 cycle. At low TMA concentra-
tions, the predominant species is oxidized TMADH,
with only a small quantity of flavin semiquinone. Thus,
at low substrate concentrations, the 0⁄ 2 cycle is
predominant and substrate inhibition does not occur.
Interestingly, at low Fc
+
concentrations (oxidizing
substrate), the switch between the 1 ⁄ 3 and 0 ⁄ 2 cycles
with a decrease in TMA concentration does not occur.
Thus, as the ratio of reducing to oxidizing substrate
increases, the level of steady-state enzyme reduction
increases [41].
It is thought that the 1 ⁄ 3 cycle is slower than the
0 ⁄ 2 cycle, indicating that it would be predominant only
at high TMA concentrations or low ETF ⁄ phenazine
methosulfate concentrations. This is because substrate
binding stabilizes the semiquinone form of the flavin in
the one-electron-reduced enzyme [56]. The binding of
substrate to the one-electron-reduced [4Fe)4S]
2+ ⁄ +
centre of TMADH, which is likely to accumulate
under the high substrate concentrations of the steady-

4Fe-4S
red
FMN
ox
.S
FMN
sq
.S
4Fe-4S
ox
FMN.S
4Fe-4S
ox
FMN
sq
4Fe-4S
ox
FMN
sq
.S
FMNH
2
.S
4Fe-4S
ox
(CH
3
)
2
NH

(CH
3
)
3
N
(CH
3
)
3
N
1
2
3
4
5
6
7
8
9
10
11
0/2 cycle 1/3 cycle
Fig. 8. Kinetic scheme of the proposed branching mechanism of electron transfer for TMADH:ETF. In the 0 ⁄ 2 cycle, the enzyme turns over
between the oxidized and two-electron-reduced state. In the 1 ⁄ 3 cycle, the enzyme turns over between the one- and three-electron-reduced
states. Population of the 1 ⁄ 3 cycle leads to substrate inhibition of TMADH. ox, oxidized; red, reduced; S, substrate; sq, semiquinone.
Adapted from [41].
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 5495
This model also predicts that the partitioning of the
two redox cycles is not dependent on the rate of 6-S-

that we have inferred that both structural imprinting
and conformational sampling are the same process.
This is not the case. The timescales for structural
imprinting are far too slow to be associated with elec-
tron transfer from TMADH to ETF as observed in
stopped-flow studies. Conformational sampling is an
intrinsically rapid motion of the FAD domain in the
complex that allows the FAD domain to search out
electron transfer-competent conformations. The struc-
turally imprinted form of ETF accumulates over an
extended time course (typically hours) when as-puri-
fied ETF is incubated with TMADH. The precise
structural change(s) that occurs during the imprinting
reaction is not known, but both fluorescence emission
and anisotropy analysis indicate that there is a slow
structural change in ETF when incubated with
TMADH over extended time periods (H. Messiha,
S. E. Burgess, D. Leys & N. S. Scrutton, unpublished
results).
Mammalian MCAD:ETF
MCAD is a 172 kDa homotetrameric flavoprotein that
catalyses the a,b-dehydrogenation of acyl-CoA thio-
esters to their corresponding trans-2,3-enoyl-CoA [58].
Substrate oxidation is accompanied by the transfer of
reducing equivalents to the covalently bound cofactor
FAD, followed by electron transfer to its physiological
terminal electron acceptor ETF. The kinetic scheme of
MCAD is very complex and involves several interme-
diates. In the reductive half-reaction, after formation
of the initial Michaelis complex, there are multiple

MCAD:product complex by two single-electron reduc-
tions to two oxidized ETF molecules. Reversible prod-
uct release completes the catalytic cycle [59].
Electron transfer between reduced MCAD and oxi-
dized ETF occurs in the presence of bound product
for several reasons. Firstly, acyl-CoA thioesters are rel-
atively weak thermodynamic reductants, and so the
equilibrium is shifted towards product formation by
preferential binding of enoyl-CoA product to the
reduced enzyme [62]. A consequence of this is that the
product must remain bound until MCAD is reoxidized
ETF and partners – structure, function and dynamics H. S. Toogood et al.
5496 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS
by ETF. The product-bound complex also has a higher
kinetic, but not thermodynamic, reductant ability than
free reduced MCAD, as product binding reduces the
pK value of the reduced flavin species [63]. Finally,
the presence of bound product dramatically reduces
the oxidase activity of the enzyme, which prevents the
loss of reducing equivalents to non-ATP-generating
processes [59].
The oxidative half-reaction between product-bound
pig ACAD and ETF at 3 °C is multiphasic, composed
of two rapid phases (t
1 ⁄ 2
$ 20 and 50 ms) and two
slower phases (t
1 ⁄ 2
$ 1 and 20 s) (inset, Fig. 9) [60].
The first and second phases correspond to the reoxida-

constants and overall two-electron redox potential of
the two FADs are similar, the potential of the
oxidized ⁄ semiquinone couple is 116 mV lower than
with unmodified FAD (+ 37 mV for the unmodified
oxidized ⁄ semiquinone couple [66]). This suggests that
the 4¢-hydroxy-N(1) hydrogen bond stabilizes the anio-
nic semiquinone by delocalizing the electron over the
N(1)–C(2)O region. The turnover rate of ETF with
MCAD is significantly reduced with 2¢-deoxy-FAD, a
reflection of the decreased potential of the oxi-
dized ⁄ semiquinone couple [65].
The role of ACAD:ETF complex formation and
reorganization has not been investigated thoroughly in
kinetics studies of mammalian systems. However, com-
plex formation is known to be transient, as shown by
the K
d
values of 2 and 5 lm with dimethylglycine
dehydrogenase and short-chain acyl-CoA dehydrogen-
ases, respectively [32]. Mutagenesis of the conserved
ETF residue Ra249 to alanine or lysine resulted in less
than 10% of the activity with MCAD remaining, with
a decreased potential of the oxidized ⁄ semiquinone cou-
ple to ) 39 mV [23,65]. These changes are most proba-
bly caused by a decrease in complex formation [23], as
well as a reduction in semiquinone stabilization, as a
result of the change of the delocalized positive charge
of arginine to a point charge in lysine [65]. Mutations
of other residues known to be involved in complex for-
mation, such as MCAD residues E212A and L75Y

[E-FAD
ox
~P]
E-FAD
ox
P
P
many steps
S
1
2
3
4
E-FAD
red
H
-
[E-FAD
red
H
-
~S]
S
6
5
[E-FAD
sq
~P]
E-FAD
ox

dissociation that support rapid interprotein electron
transfer [23]. Rapid complex dissociation can occur by
having no more than a few weak interactions between
the partners, or by having one of the redox cofactors
situated in a mobile domain. Both strategies are
employed in ETF–partner protein complexes.
The structures of complexes between ETF and its
partners show a dual mode of interaction [3,23,25].
The recognition loop binds to a hydrophobic patch on
ETF partners, which provides a weak anchoring site
between the two partners. This creates a suitable inter-
facial cavity for the flavin domain to sample a large
range of conformational interactions, some of which
are compatible with fast electron transfer. Transient
stabilization of electron transfer-competent states is
achieved through interactions between the two part-
ners, including interactions with the conserved Ra237
(W3A1 numbering) [23].
This separation of the partner recognition site (rec-
ognition loop) from the electron transfer site (flavin
domain) is critical in understanding how ETF can
interact with specific, yet structurally distinct, partners
[23]. The observed specificity of ETF to its partners
can be understood by the latter needing only to pro-
vide a suitable hydrophobic patch in the correct posi-
tion to ensure interaction with the recognition loop of
ETF. Once a complex is established, the flavin domain
is promiscuous in searching out suitable transient
electron transfer-competent states that place the redox
cofactors within 14 A

E
E
E
MCAD
MCAD
MCAD
FAD
Free ETF
MCAD:ETF Complexes
ETF
ETF
ETF
ETF
ETF
E 165
E
165
L
185
L
185
R 249
R 249
R
249
R
249
R
249
Fig. 10. Schematic diagram of the dynamic

often fatal disease glutaric acidaemia ⁄ aciduria type II
(GAII), also known as multiple acyl-CoA dehydroge-
nase dysfunctional disease (MADD) [67]. This disorder
differs from glutaric aciduria type I, which arises from
defects in glutaryl-CoA dehydrogenase, as this disease
results in the large excretion of compounds such as
butyric and isovaleric acids [68]. GAII is an autosomal
recessively inherited disorder subdivided into IIA, IIB
and IIC, depending on which of the three respective
genes contains mutations [68]. The mutations can lead
to a range from mild to severe cases, with variable pre-
sentation times, depending on the location and nature
of the mutation. The neonatal-onset forms are usually
fatal and are characterized by symptoms such as severe
nonketotic hypoglycaemia, metabolic acidosis and
excretion of large amounts of fatty acid- and amino
acid-derived metabolites. Late-onset GAII symptoms
include lethargy, vomiting, hypoglycaemia, metabolic
acidosis and hepatomegaly, which tend to be periodic
and often preceded by metabolic stress [68,69].
The types of known clinical mutation of a- and
b-ETF include single amino acid substitutions,
Table 1. Clinical mutations of a- and b-ETF. GA, glutaric acidaemia or glutaric aciduria.
Gene
Missense ⁄ nonsense Deletion ⁄ insertion
c
Splicing
Phenotype Reference
Codon change
a

a
base number at the beginning of the codon or the
b
residue number.
c
Number refers
to the codon number range, with lower case bases the deletions. Additional information was obtained from the Human Gene Mutation Data-
base (archive.uwcm.ac.uk ⁄ uwcm ⁄ mg ⁄ hgmd0.html).
d
This mutation results in the skipping of exon 3 or in the creation of a downstream
cryptic splice site and the insertion of the nine 5
¢
proximal nucleotides of intron 3.
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 5499
deletions or insertions of bases resulting in the loss of
amino acids, early termination or frameshifts, and var-
iable gene products resulting from incorrect splicing
(Table 1) [68,70–73]. In some cases, the mutations lead
to a decrease in the amount of mRNA transcript
and ⁄ or protein levels in the cell. Mutations which
destabilize either subunit could lead to a reduction in
the levels of correctly folded protein in the cell. Some
patients completely lack b-ETF transcript or ETF pro-
tein, presumably as a result of mutation(s) in the regu-
latory sequences of the gene that affect expression
and ⁄ or turnover of the corresponding mRNAs [68].
In some cases, the effects of the mutations on ETF
have been determined, or can be inferred by the struc-
tures of free or complexed human ETF. Expression of

abolished, as indicated by the mild form of the disease
[23].
Conclusions
In recent years, detailed biophysical analysis, coupled
with the determination of the structures of ETF–part-
ner protein complexes, has revealed a novel mode of
interprotein electron transfer. Complex formation trig-
gers mobility of the FAD domain, an ‘induced dis-
order’ mechanism contrasting with the more generally
accepted models of protein–protein interaction by
induced fit mechanisms. The subsequent interfacial
motion of the FAD domain is the basis for the interac-
tion of ETF with structurally diverse protein partners.
This motion seeks out optimal geometries and dis-
tances for interprotein electron transfer, a mechanism
termed ‘conformational sampling’ [3]. Given the modu-
lar nature of redox proteins, this might be a more gen-
eral feature of intra- and interprotein electron transfer
in biological systems. Similar mechanisms have been
proposed for intraprotein electron transfer in the
multidomain nitric oxide synthases [76]. In addition,
crystal structures of the cytochrome b
6
f complex have
identified a similar, yet distinct, motion of the Rieske
iron–sulfur domain compared with that observed for
the cytochrome bc
1
complex [77].
The mechanism of conformational sampling identi-

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