Báo cáo khoa học: Dynamics driving function ) new insights from electron transferring flavoproteins and partner complexes pdf

Structural and biophysical studies of ETF in complex with partnerproteins have shown that ETF partitions the functions of partner bindingand electron transfer between a a ‘recognition lo

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 munication with structurally distinct redox partners[2], but require additional mechanisms for complexassembly to impart specificity in the protein–proteininteraction

com-ETFs are soluble heterodimeric FAD-containingproteins that are found in all kingdoms of life Theycontain a second nucleotide-binding site which isusually occupied by an AMP molecule [1] In bacteriaand 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

FAD-10 primary dehydrogenases to the membrane-bound respiratory chain.Clinical mutations of ETF result in the often fatal disease glutaric aciduriatype II Structural and biophysical studies of ETF in complex with partnerproteins have shown that ETF partitions the functions of partner bindingand electron transfer between (a) a ‘recognition loop’, which acts as a staticanchor at the ETF–partner interface, and (b) a highly mobile redox-activeFAD domain Together, this enables the FAD domain of ETF to sample arange of conformations, some compatible with fast interprotein electrontransfer 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 interproteinelectron 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 biologicalelectron 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.

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

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 fromdonors such as ferredoxin and NADH [15] No ETF-dependent activity has been observed with the mem-brane-bound respiratory enzymes in nitrogen-fixingbacteria, and so it is thought that the electron transferpathway from ETF to dinitrogen is via the enzymesETF:ferredoxin oxidoreductase, ferredoxin, nitrogenasereductase and nitrogenase [14]

A well-studied group II ETF is from the bacteriumMethylophilus methylotrophusstrain W3A1, which con-tains only one known dehydrogenase partner, namelytrimethylamine dehydrogenase (TMADH) [3,16] FixB⁄FixA proteins have been characterized from the micro-aerobic Azorhizobium caulinodans, which is known toaccept electrons from pyruvate dehydrogenase underaerobic conditions [14] The nitrogen-fixing organismBradyrhizobium 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 theetfSL genes are expressed, whereas the reverse is truefor anaerobic growth, as nitrogen fixation only occursanaerobically [17]

One ETF from the anaerobe Megasphaera elsdenii(formerly Peptostreptococcus elsdenii) is unusual, as itcontains two FAD-binding sites per ETF molecule,and so does not bind AMP [6,15,18,19] This ETFserves 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 discussedfurther in this review

An examination of the databases of genomicsequences shows organisms containing multiple ETF-like genes as well as ETFs fused with other proteins(Pedant; http://pedant.gsf.de) The genome of theeubacterium Fusobacterium nucleatum ssp nucleatum(ATCC 25586) suggests the presence of two completeETF molecules, each positioned upstream of an acyl-CoA dehydrogenase The genome also contains a largeORF (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 doxin (Fig 1) As no functional studies of this enzymehave been published, it is presumed that the absence ofthe b-ETF subunit is a result of its role as a ‘fixed’

rubre-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 bound ETF ferredoxin oxidoreductase in nitrogen-fixing organisms [14]

membrane-A blast search of the structurally equivalent minal (non-FAD-binding) a-ETF and b-ETFsequences against known ORFs showed homologywith a variety of adenosine nucleotide-binding enzymes(NCBI blast; http://www.ncbi.nlm.nih.gov) Suchenzymes include members of the adenosine nucleotidea-hydrolase superfamily from Oryza sativa, which con-tains an ATP-binding fold [20] The thiamine bio-synthesis-like protein from three Leishmania speciescontains b-ETF and aminotransferase components atthe N- and C-termini, respectively [21] This class ofenzyme is known to bind ATP Other ATP-bindingenzymes with homology to b-ETF in the database(NCBI blast; http://www.ncbi.nlm.nih.gov) includeadenylyl-sulfate kinase from Anaeromyxobacter sp.Fw109-5 (GI:121539501), the predicted glutamate-dependent NAD(+) synthase from Strongylocentrotuspurpuratus(GI:115971088) and the asparagine synthasefrom Desulfovibrio vulgaris ssp vulgaris DP9(GI:120564303) As b-ETF typically binds AMP,homology to domains of other enzymes known to bindadenosine nucleotides is not surprising

N-ter-Sequence homology of ETFs

An alignment of a- and b-ETFs from all kingdoms oflife (Fig 2) shows that, within the a-ETF family, theoverall sequence homology is low, although highsequence homology is found in the C-terminal region

By contrast, in the b-ETF family, there is a similardegree of sequence similarity throughout the length ofthe protein Group I ETFs align better than group IIETFs, although both groups contain significantsequence similarity in conserved regions

The C-terminal portion of a-ETF contains a highlyconserved region, known as the b1ab2 region of FADenzymes, which binds the adenosine pyrophosphorylmoiety of FAD [22] Within this region is the a-ETFconsensus sequence of PX[L,I,V]Y[L,I,V]AXGIS-GX[L,I,V]QHX2G [7], similar to the consensussequence for FAD-binding dehydrogenases ofGXGXXGX15[E⁄ D] [22] The b-ETF family contains aconserved signature sequence of VXRX2[E,D]-

X3[E,Q]X[L,I,V]X3LP[C,A][L,I,V]2 which is used toidentify members of the b-ETF family [7] Adjacent tothis signature sequence, group I b-ETFs also showthe highly conserved region of DLRLNEPR-YA[S⁄ T]LPNIMKAKKK (residues 184–204; humannumbering), containing the recognition loop and thehighly conserved L195 necessary for partner binding in

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).

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 significantgroup I-like recognition loop, suggesting a differentmode of partner binding

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 twodomains, IIa and IIb, which are composed of theC-terminal portions of the a- and b-subunits, respec-tively Domain IIa is the larger of the two, folds in amanner similar to bacterial flavodoxins [24] and formsmost of the region that binds FAD This is the region

of high sequence similarity within the a-subunit Thisfold consists of a core of a five-stranded parallelb-sheet surrounded by alternating a-helices [1] A sixthstrand 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 IIand III, with the xylene portion pointed towards theb-subunit By contrast, domain IIb does not interactwith FAD, but instead wraps around the lower portion

of domain IIa near domains I and III [1]

Despite the low sequence similarity between thetwo 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 ofW3A1 ETF is rotated by about 40 relative to thehuman and P denitrificans flavin domains, withVa190 and Pb235 (W3A1 numbering) serving ashinge points [3] In human ETF, the conservedEb165 of domain III interacts with Na259, which islocated near the conserved Ra249 (Ra237 in W3A1)and FAD (Fig 4A) There are also hydrophobicinteractions between the C7- and C8-methyl groups

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.

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 flavindomain in W3A1 ETF, Eb163 (equivalent to humanEb165) interacts instead with the conserved Ra237 via

a bifurcated salt bridge (Fig 4B) [3] This arginine due also forms a single salt bridge with Da241 ofdomain II A second interaction between these twodomains is seen in the low-resolution W3A1 ETFstructure [3], between residues Ra211 and Eb37 Inhumans, the equivalent arginine residue, Ra223, inter-acts directly with the flavin and is over 8 A˚ fromdomain III [3]

Low resolution solution

structure

II II

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.

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 isperpendicular to the xylene portion of the isoalloxazinering, which may function by stabilizing the anionicreduced FAD [13], and also by conferring a kineticblock on full reduction to the dihydroquinone [3].Other key interactions include the N1 residue ofHa268 with O2 of the isoalloxazine ring, which mayalso function in stabilizing the anionic semiquinone [1].The hydroxyl group of Ta266 interacts with N5 ofFAD, 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 achievedthrough interactions with residues such as Sa248 andSa281 [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.

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 hydrohydro-phobic residues

of ETF interacting with TMADH are Yb191, Ib197and Sb193, the latter of which stabilizes the initial turn

of the a-helix in the recognition loop These residuesare highly conserved, in particular within group IETFs (Fig 1) Several residues preceding Yb191 which

do not contact TMADH are also conserved, includingLb186, Nb187, Pb189 and Rb190 The recognitionloop is stabilized by both the close packing of theseresidues and a bifurcating salt bridge between Rb190and residues Eb44 and Eb51 Several other residuesinvolved in complex formation include a salt bridgebetween the N-terminus of TMADH and Db16 ofETF, and a number of direct or water-mediated hydro-gen bonds This relatively small number of interactionshelps to explain why the dissociation constant( 5 lm) of TMADH:ETF is weak [3,28]

In free ETF, the recognition loop is more flexibleand is oriented slightly differently, with Pb189 andPb204 serving as hinge points [3] Limited trypsin pro-teolysis, which removed the recognition loop, produced

an ETF whose structure and redox capabilities withdithionite were virtually identical to native ETF, yet ithad lost its ability to accept electrons from TMADH.This shows the pivotal role of the recognition loop incomplex formation, and serves as an ‘anchor’ distant

to the redox centres [3] This anchor may serve as ameans of recognizing specific redox partners, as allthat would be required would be a suitably placedhydrophobic patch to interact with the recognitionloop [3]

The absence of density for the flavin domain of ETFoccurs after residues Va190 and Pb235, which serve ashinge points [3] This total lack of density was initiallysurprising, 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 anincreased mobility within the complex, or packing con-straints with the free ETF structure lock the domain inone position This mobility of the flavin domain withinthe complex lends support to the transient nature ofthe electron transfer-competent state, as predicted fromkinetics and other studies [4,25]

Several mutant TMADH:ETF complexes weredesigned which altered the interactions between theflavin domain and domain III of ETF, as well as itsinteraction with TMADH (see ‘Human MCAD:ETF’section below) At least two of each of the mutant com-plex structures were determined, TMADH WT:ETFEb37Q and TMADH Y442F:ETF WT, includingtwo structures in a new space group (H S Toogood,

D Leys & N S Scrutton, unpublished results) All

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 structuralchanges on complex formation by observing spectralchanges during difference spectroscopy studies ofTMADH:ETF [29] Shifting the domain into a human-like conformation would allow the domain to fit withinthe allowable space The ‘empty volume’ observed

T414

Q462 H416

Y478 A464

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.

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 designedwhich altered the interactions between the flavindomain and domain III of ETF (MCAD:ETFEb165A), as well as its interaction withMCAD (MCA D:ETF Ra249A; MCAD E212A:ETF;MCAD E359A:ETF) [25] The aim was to alter theratio of the different conformational states sufficiently

to trap discrete flavin domain positions Kinetic studies

of these complexes showed a reduction in electrontransfer rates [when using 2,6-dichloroindophenol asthe terminal electron acceptor], except for the MCAD:ETF Eb165A complex, which showed both a dramaticincrease in rate and decrease in the apparent Kmvalue.Crystal structures of all four mutant complexes wereobtained (Fig 7A; last three: H Toogood, A vanThiel, D Leys & N S Scrutton, unpublished work),which showed an increase in density for the flavindomain to about 70% occupancy (except for MCAD:ETF Ra249A), with the flavin domain in the sameposition 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 ETFcontains a recognition loop (Pb190–Ib198), includingthe highly conserved residue Lb195, which interactswith 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 ofMCAD [31], with a nearly perfect alignment of theaxes 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 andI83 ETF residues which also interact with this pocketinclude Yb192, Pb197, Ib198 and Mb199 [23]

A comparison of the free and complex crystal tures reveals that, although MCAD adopts a nearlyidentical conformation in both structures, ETF adopts aslightly different backbone conformation with moreextensive side chain rearrangements, including Lb195[23] The structure of the free ETF mutant Lb195A doesnot show any significant rearrangements of the recogni-tion loop, yet kinetic studies with both MCAD, isovale-ryl-CoA dehydrogenase and the structurally distinctpartner dimethylglycine dehydrogenase show a severedecrease in electron transfer rates (A van Thiel,

struc-H Toogood, struc-H L Messiha, D Leys & N S Scrutton,unpublished work) Mutations of MCAD, such asL61M, L73W and L75Y, which were designed to ‘fill in’the binding pocket, were all severely impaired in elec-tron transfer rates with ETF [25] Microelectrosprayionization mass spectrometry and surface plasma reso-nance studies showed competitive binding of ETF toacyl-CoA dehydrogenases and dimethylglycine dehydro-

genase, suggesting similar or closely overlapping

bind-ing sites for each [32] Cross-linkbind-ing experiments with

ETFQO showed that it preferentially interacts with the

b-subunit of ETF [33] These results suggest a similar

mode of interaction between ETF and its structurally

distinct partners [23]

An alignment of MCAD-like partners shows very

little sequence conservation of the residues interacting

with the recognition loop [23] However, the amino acidsubstitutions tend to retain their hydrophobic or hydro-gen-bonding ability, suggesting that ETF does not have

to recognize an exact binding pocket, but a structurallyequivalent one The high conservation of the recogni-tion loop, particularly in group I ETFs, suggests thatETFs across kingdoms may also interact with their part-ners in a similar manner via a recognition loop [23]

ETF MCAD

G60 L59

L61

F23

I83 F30

as yellow and orange sticks, respectively Highlighted side chains of MCAD and ETF 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 ETF Eb165 is shown as a red sphere (B) Structure of the recognition loop in MCAD:ETF Residues are shown as atom-coloured sticks with green and blue carbons for MCAD and ETF, respectively (C) Structure of the electron transfer interaction site a-ETF and MCAD are shown as magenta and green cartoons, respectively The two FAD molecules are shown as yellow sticks Highlighted residues are shown as atom-coloured sticks with green and magenta carbons for MCAD and ETF, respectively.

The orientation of the flavin domain within the

MCAD:ETF complex is dramatically different from its

position in any of the free ETF structures (Fig 7C)

[25] The contact surface between MCAD and the

fla-vin domain is about 330 A˚2, with a shape

complemen-tarity value of 0.56, suggesting that the interaction is

weak and of a transient nature [25] Within this

inter-face, Ra249 of the flavin domain forms a salt bridge

with E212 of MCAD, as well as interacting with E359

via a bridging water molecule This is in agreement

with chemical modification studies, which show that an

arginine residue in ETF and carboxylates on MCAD

are involved in complex formation [34] Other

interac-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-tive positions of the docking sites for the leucine

anchoring residue within the recognition loop between

the two complexes are very similar However, the two

partner proteins interact with ETF via different redox

cofactors, with the electron-donating cofactors in

dif-ferent relative positions within the two complex

struc-tures This highlights the need for the flavin domain to

sample the available conformational space to find an

electron transfer-competent state, as seen by the lack

of density for the flavin domain in both wild-type

structures These conformations are transiently

stabi-lized through key interactions between conserved

resi-dues specific to each dehydrogenase type [23] As boththe [4Fe)4S]2+ ⁄ + and FAD cofactors of TMADHand MCAD, respectively, are located within a 10 A˚radius of the ETF FAD, this suggests that a similarconformation of ETF in both complexes is possible forfast interprotein electron transfer

Kinetics of electron transfer between ETF and partners

Methylophilus methylotrophus TMADH:ETFTMADH is a 166 kDa homodimeric iron–sulfur flavo-protein which catalyses the oxidative demethylation oftrimethylamine (TMA) to form dimethylamine andformaldehyde (Eqn 1) [35] Substrate oxidation isaccompanied by the transfer of reducing equivalents,first to the covalently bound cofactor 6-S-cysteinylFMN [27], followed by reduction of a ferredoxin-like[4Fe)4S]2+ ⁄ +

located approximately 4–6 A˚ from the8-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 electrontunnelling [37,38] Stopped-flow kinetics studies of thereductive half-reaction shows that it occurs in threekinetic phases The fast phase represents the two-elec-tron reduction of 6-S-cysteinyl FMN, followed byintermediate and slow phases which reflect the transfer

of one electron from the dihydroquinone of flavin tothe [4Fe)4S]2+ ⁄ +centre, and the formation of a spin-interacting state between the flavin semiquinone andthe reduced [4Fe)4S]2+ ⁄ +

[39] This latter state isformed after the binding of a second substrate mole-cule, which induces the ionization of Y169 locatedclose to the pyrimidine ring of 6-S-cysteinyl FMN [36].This state is distinguished by a complex EPR signalcentred near g  2 with an unusually intense half-field

g 4 signal [39] However, the kinetics are furthercomplicated as the extent of the biphasic naturechanges with both substrate concentration and pH[39] Detailed kinetic and mechanistic analyses of thereductive half-reaction have been studied extensively,and readers are referred to papers such as Scrutton

et al [40], Scrutton and Sutcliffe [35], Roberts et al.[41], Basran et al [42–46], and references cited therein

ðCH3Þ3N + H2O! ðCH3Þ2NHþ CH2Oþ 2Hþþ 2e ð1Þ

TMADH:ETF oxidative half-reactionThe oxidative half-reaction of TMADH involves thetransfer of two electrons through [4Fe)4S]2+ ⁄ +to the