Structure of electron transfer flavoprotein-ubiquinoneoxidoreductase and electron transfer to themitochondrial ubiquinone poolJian Zhang*, Frank E. Frerman†, and Jung-Ja P. Kim*‡

*Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226; and †Department of Pediatrics, University of Colorado Health SciencesCenter, Denver, CO 80262

Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved September 11, 2006 (received for review June 2, 2006)

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a 4Fe4S flavoprotein located in the inner mitochondrialmembrane. It catalyzes ubiquinone (UQ) reduction by ETF, linkingoxidation of fatty acids and some amino acids to the mitochondrialrespiratory chain. Deficiencies in ETF or ETF-QO result in multipleacyl-CoA dehydrogenase deficiency, a human metabolic disease.Crystal structures of ETF-QO with and without bound UQ weredetermined, and they are essentially identical. The molecule formsa single structural domain. Three functional regions bind FAD, the4Fe4S cluster, and UQ and are closely packed and share structuralelements, resulting in no discrete structural domains. The UQ-binding pocket consists mainly of hydrophobic residues, and UQbinding differs from that of other UQ-binding proteins. ETF-QO isa monotopic integral membrane protein. The putative membrane-binding surface contains an �-helix and a �-hairpin, forming ahydrophobic plateau. The UQOflavin distance (8.5 Å) is shorterthan the UQOcluster distance (18.8 Å), and the very similar redoxpotentials of FAD and the cluster strongly suggest that the flavin,not the cluster, transfers electrons to UQ. Two possible electrontransfer paths can be envisioned. First, electrons from the ETFflavin semiquinone may enter the ETF-QO flavin one by one,followed by rapid equilibration with the cluster. Alternatively,electrons may enter via the cluster, followed by equilibrationbetween centers. In both cases, when ETF-QO is reduced to atwo-electron reduced state (one electron at each redox center), theenzyme is primed to reduce UQ to ubiquinol via FAD.

fatty acid oxidation � iron-sulfur flavoprotein � mitochondrial respiratorychain � membrane protein � acyl-CoA dehydrogenases

E lectron transfer f lavoprotein-ubiquinone oxidoreductase(ETF-QO) is an intrinsic membrane protein located in the

inner mitochondrial membrane. It contains single equivalents ofFAD and a [4Fe4S]2�,1� cluster (1). The protein is the singleinput site to the main respiratory chain for electrons from nineflavoprotein acyl-CoA dehydrogenases and two N-methyl dehy-drogenases (2, 3). The electron acceptor for the dehydrogenasesis the ETF, which is the reductant of ETF-QO. ETF-QO isoxidized by the diffusible ubiquinone (UQ) pool that also isaccessed by NADH-UQ oxidoreductase (Complex I), succi-nate-UQ oxidoreductase (Complex II), the flavin-linked glyc-erol-3-phosphate dehydrogenase, and dihydroorotate dehydro-genase, another flavin-linked UQ oxidoreductase (4). Theubiquinol product of these oxidoreductases transfers electrons tothe bc1 complex (Complex III). Thus, ETF and ETF-QO link theoxidation of fatty acids and some amino acids to the mitochon-drial respiratory system, and the overall electron flow can besummarized as follows: Acyl-CoA3 Acyl-CoA dehydrogenases3 ETF3 ETF-QO3 UQ3 Complex III. Inherited deficien-cies of ETF-QO or ETF cause a metabolic disease, multipleacyl-CoA dehydrogenase deficiency, also known as glutaricacidemia type II (5). This metabolic disease is characterized inits most severe form by delayed neuronal migration, an energy-intensive process, and polycystic kidneys (6).

Reductive titration of ETF-QO by octanoyl-CoA in the pres-ence of catalytic medium-chain acyl-CoA dehydrogenase andETF proceeds to the two-electron reduced state, [4Fe4S]1�, andan anionic flavin semiquinone. Electron transfer in this pathwayis firmly established only for the initial transfer from the primarydehydrogenases to ETF: the reactions proceed as two one-electron transfer steps from the dehydrogenase dihydroquinoneto two equivalents of ETF (7, 8). However, the electron transferpathway is less clear at this point. ETF-QO catalyzes thedisproportionation of ETF semiquinone generated by the pri-mary dehydrogenases at a rate that is catalytically competent toparticipate in the overall transfer of electrons from an acyl-CoAsubstrate to UQ (9). This overall reaction in vitro was establishedonly in a soluble uncompartmentalized system, with a short-chain, water-soluble UQ homolog (9).

ETF-QO, along with the other mitochondrial UQ oxidoreduc-tases, plays a central role in the bioenergetics of aerobic organ-isms and some anaerobic organisms. Three-dimensional struc-tures have been determined for several UQ oxidoreducatases,including succinate-UQ oxidoreductase (10–12), the relatedquinol-fumarate oxidoreductase (13, 14), dihydroorotate dehy-drogenase (15), and the bc1 complex (16–18). These structureshave contributed to an understanding of the distance-dependence of electron transfer (19) and some generalizationsregarding the UQ-binding motifs (20). However, no detailedstructural information has been available for ETF-QO.

We undertook a structural investigation of porcine ETF-QOby using x-ray crystallography to obtain insight into the inter- andintramolecular electron transfers of the protein, the 2e��2H�

reduction of UQ, and the possible mode of binding of ETF-QOto the membrane.

Results and DiscussionThe Overall Structure. In the final structure of UQ containingETF-QO, the entire polypeptide chain was visible except the firstthree residues in one of the two molecules in the asymmetric unitand the first six residues in the other molecule. The residuenumbering system used hereafter corresponds to the matureprotein sequence and can be related to the complete humansequence by addition of 33 residues, the human mitochondrialsignal peptide (21). Each molecule in the asymmetric unit

Author contributions: J.Z. and J.-J.P.K. designed research; J.Z. and J.-J.P.K. performedresearch; J.Z. and J.-J.P.K. analyzed data; F.E.F. contributed new reagents�analytic tools;and J.Z., F.E.F., and J.-J.P.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: ETF, electron transfer flavoprotein; ETF-QO, ETF-ubiquinone oxidoreduc-tase; UQ, ubiquinone; ETF1e�, ETF semiquinone.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org [PDB ID codes 2GMH (UQ-bound structure) and 2GMJ(UQ-free structure)].

‡To whom correspondence should be addressed. E-mail: jjkim@mcw.edu.

© 2006 by The National Academy of Sciences of the USA

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contains one FAD, one 4Fe4S cluster, and one UQ molecule.However, only 5 of the presumed 10 isoprene units could be seenin both of the UQ molecules. The final Rwork and Rfree of thestructure were 21.9% and 25.2%, respectively, for all of the databetween 30.0-Å and 2.5-Å resolutions. The ETF-QO structurewithout bound UQ was determined to 2.7 Å, and the Rwork andRfree of the final model were 22.8% and 25.5%, respectively. Thefolding of porcine ETF-QO is essentially the same, with orwithout UQ, with an rms deviation of 0.26 Å between the twostructures (a detailed comparison is given in Supporting Results,which is published as supporting information on the PNAS website). A ribbon diagram of the UQ-bound structure is shown inFig. 1, and a sequence alignment of ETF-QO from severalspecies along with the secondary structural elements is shown inFig. 5, which is published as supporting information on the PNASweb site. The structure contains 11 �-helices and 19 �-strands,and the molecule forms a single structural domain having threefunctional domains: a FAD domain, a 4Fe4S cluster domain, anda UQ-binding domain. The three domains are closely packed andshare structural elements, and there are no isolated, discretedomains that would be capable of local segmental motion as seenin the Fe-S protein of the bc1 complex (17). The 4Fe4S clusterdomain in ETF-QO consists of N-terminal residues C4–Y16 andC-terminal residues D484–M584. The FAD domain comprisesresidues P17–N106, V141–P235, and S340–V418 and is primarilyan ��� structure. There are two �-sheets in the FAD domain.�-Sheet 1 is a mixed parallel�antiparallel sheet made of strands1, 6, 7, and 8 and is located at the surface of the molecule cappingthe 4Fe4S domain. �-Sheet 2 is composed of strands 1, 2, 5, 8,14, and 15, sandwiched between �-sheet 1 and helices 1, 3, 6, and7. The UQ domain comprises residues T107–V140, Q236–Q339,and S417–F483. It is dominated by �-sheet 3, which is a twisted,mixed parallel�antiparallel sheet comprising strands 3 (3a), 4, 9,

10, 11, 12, and 13. A structural similarity search using DALI (22)indicates that the fold of the core of the 4Fe4S domain is mostsimilar to that of Clostridium acidurici ferredoxin (23). The C�rms difference for the 53 equivalent residues for the twomolecules is 2.5 A. The overall fold of the combined FAD andUQ domains is similar to the p-hydroxylbenzoate hydroxylasefold (24). The C� rms differences for the FAD and UQ�substrate-binding domains of ETF-QO and the hydroxylase are1.9 A (145 equivalent residues) and 2.2 A (75 residues), respec-tively. The fold of the FAD domain of ETF-QO also is similarto that of flavocytochrome c3-fumarate reductase (rms deviation�2.0 A for 127 C� atoms) (25–27) and quinol-fumarate reduc-tase flavin subunit (1.9 A for 145 residues) (28). These domain-or subdomain-level structural similarities imply divergent evo-lution and gene fusions among these functionally related pro-teins. The FAD and 4Fe4S are buried completely in the ETF-QOstructure, as is the benzoquinone ring of UQ. The Fo � Fcomit-map and the relative positions of the three redox centers,FAD, 4Fe4S, and UQ, are shown in Fig. 2.

The FAD Environment. FAD has an extended conformation and isburied completely in the protein (Fig. 1). It is positioned at thecarboxyl side of the parallel �-sheet 2 and the C termini of �1and �6 helices (Fig. 3A). The C7 and C8 methyl groups of theisoalloxazine ring make van der Waal’s contacts with the main-chain N atom of R331 in the plane of �-sheet 3. As in otherflavoproteins, the pyrimidine side of the isoalloxazine ring ishydrogen-bonded to the polypeptide; O2 forms hydrogen bondswith the main-chain nitrogens of G366 and T367 and thehydroxyl of T367, continuing the hydrogen-bonding pattern ofthe �6-helix (Fig. 3A). The interaction of the isoalloxazine ringand the �6-helix is strengthened further by hydrogen bondsbetween the hydroxyl of T367 and the O2 and N1 atoms of theisoalloxazine ring. Other hydrogen-bonding interactions occurbetween the O4 and C85 N atoms, N3 and C85 carbonyl oxygenatoms. In addition to these hydrogen bonds, the positive dipoleof the helix could modulate the redox potential of FAD andstabilize the anionic semiquinone. This helix dipole–flavin in-teraction is present in the p-hydroxybenzoate hydroxylase (24)and glutathione reductase class of flavoproteins (29) and quinol-fumarate reductase (13, 14). A water molecule is located on thesame plane as the flavin ring and is hydrogen-bonded to both theN5 and O2 atoms; however, its functional role is not clear atpresent.

Strands �1, �2 of the �-sheet 2, and helix �1 form the ���dinucleotide-binding motif. Residues G42–G47 contain theADP-binding sequence motif (GXGXXG), form the N terminusof helix �1, and hydrogen-bond to the pyrophosphate moiety ofFAD (Fig. 3A). Like other FAD-containing proteins, the pyro-phosphate moiety is neutralized by R331. The hydroxyl of S82makes a hydrogen bond to 2�-OH of the ribityl chain of FAD andis located beneath the center of the isoalloxazine ring on the

Fig. 1. Ribbon diagram of ETF-QO. The structure comprises three domains:FAD domain (blue), 4Fe4S cluster domain (red), and UQ-binding domain(green). Three redox centers are shown in sticks: FAD (golden yellow), 4Fe4S(magenta), and UQ (dark red). �-Helices and �-strands are numbered sequen-tially from the N terminus to the C terminus. The putative membrane-associated surface regions are shown in cyan. Mitochondrial membrane isdepicted as blue shaded area.

Fig. 2. Electron densities in Fo � Fc omit maps for FAD (3.0�), 4Fe4S (4.0�),and UQ (2.5�). The relative positions and distances (in angstroms) among thethree redox centers are shown.

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si-side with an atom-plane distance of 3.0 A. Thus, it is possiblethat S82 acts as a hydrogen-bond donor and the flavin ring as theacceptor, as observed in some aromatic ring compounds (30). Asimilar interaction has been observed in cholesterol oxidase, inwhich the amide nitrogen of N485 makes a N-H�� interactionwith the pyrimidine side of the FAD ring and modulates theredox potential of the oxidase (31). Thus, the hydrogen bondbetween S82 and the flavin ring in ETF-QO also may influencethe redox potential of the bound FAD. Three residues, R331,T367 and S82, are highly conserved in the ETF-QO sequences(see the supporting information).

The 4Fe4S Cluster Environment. The iron-sulfur cluster in ETF-QOis embedded in two loops that contain residues C528–Y533 andC553–D560. As predicted from the sequence analysis, C528,C553, C556, and C559 from the two loops coordinate the four Featoms in the cluster (21). Residues H503, L504, and W570complete the binding pocket formed by the two loops (Fig. 3B).The cluster is supported further through hydrogen bonds be-tween the S� atoms of the four cysteines and the polypeptidechain; C553 makes hydrogen bonds with H503, C556 bonds withthe hydroxyl of T558, C559 bonds with the phenolic oxygen ofY533 and main-chain N of D560, and C528 bonds with thebackbone nitrogen of A530. Two of the four cluster sulfur atomsmake weak hydrogen bonds, each with the main-chain N atomof K557 or C559 (both distances, 3.4 Å). Such hydrogen bondscan modulate the redox potential of iron-sulfur clusters (32, 33).The relatively positive potential of the ETF-QO cluster, �47

mV, reflects the extensive hydrogen bonding of the cysteinylsulfur and sulfur atoms in the cluster.

UQ Binding. UQ and the flavin isoalloxazine ring are located onthe same side of �-sheet 3. The UQ-binding pocket is mademainly of hydrophobic residues (Fig. 3C). Only one of the twocarbonyl oxygen atoms in the benzoquinone ring is hydrogen-bonded to the polypeptide chain. The O4 atom of UQ makeshydrogen bonds to the backbone nitrogen of G273 and carbonyloxygen of G272. The rest of the molecule is surrounded by mostlyhydrophobic residues making van der Waal’s contacts (Fig. 3C).The phenolic ring of Y271, followed by residues in �11 (G272,G273, and S274), wraps around the C5 methyl, O4 carbonyl, andC3 methoxy groups; F114, H260, and V262 contacts the O1carbonyl and C2 methoxy groups. The isoprene tail is bent suchthat the second isoprene unit contacts the O1 carbonyl group.There is a water molecule that hydrogen-bonds the hydroxyl ofY271 and O4 of the benzoquinone ring. This water may act as theproton donor�acceptor during the UQ redox cycle. ResiduesY271, G272, G273, S274, and H260 are highly conserved amongETF-QOs from different species (Fig. 5). In particular, G273 isabsolutely conserved in all ETF-QO sequences. The absence ofa bulky side chain at position G273 eliminates steric hindranceso that the UQ molecule penetrates deep into its hydrophobicbinding pocket. If electron transfer to UQ involves ubisemiqui-none as a transient intermediate, the semiquinone is protectedfrom reaction with molecular oxygen. Only 5 isoprene units ofthe UQ tail (10 isoprene units for mammalian UQ) are visible.There is a hint of disorder even at the third isoprene unit in one

Fig. 3. Residues in the vicinity of the redox centers. (A) Stereo diagram of the FAD-binding site. The isoalloxazine ring is located at the N terminus of helix �6.The phosphate moiety is located at the N terminus of �1-helix of the ��� Rossmann fold of the protein. Color codes for atoms are oxygen (red), nitrogen (blue),sulfur (purple), phosphorus (brown), protein carbon (yellow), and FAD carbon (green). Hydrogen bonds are shown as dotted lines. (B) The 4Fe4S cluster-bindingresidues. Four cysteine residues (528, 553, 556, and 559) coordinate Fe atoms in the cluster (large dotted lines). They also make hydrogen bonds to the polypeptidechain (dotted lines). Color codes for atoms are the same as in A, except that sulfur is in green and iron is in purple. (C) The UQ-binding site. The O4 atom of theUQ ring is hydrogen-bonded to the main-chain atoms of G272 and G273. A water molecule makes hydrogen bonds to O4 and the hydroxyl of Y271. All otherinteractions involving UQ are of hydrophobic contacts. Color codes are the same as in B.

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of the two ETF-QO molecules in the asymmetric unit, indicatingthat the tail is f lexible (Fig. 2). The second through the fifthisoprene units of the tail are surrounded by mostly hydrophobicresidues, including F114, V125, G434, M435, T438, and G439(Fig. 3C). Thus, the binding mode of UQ observed in ETF-QOis different from those observed in other UQ-binding proteins,e.g., succinate-UQ oxidoreductase (34) and ubiquinol oxidase(35). The binding motif found in other proteins of the respiratoryand photosynthetic systems has semiconserved sequences con-taining a Tyr�Trp or His that make direct hydrogen bonds to O1and�or O4 of the benzoquinone group (20).

Membrane-Binding Surface. By the usual criteria, porcine ETF-QOis classified as an integral membrane protein (36), requiringdetergent to solubilize the protein. In contrast to UQ-bindingproteins of the main respiratory and photosynthetic systems, theETF-QO polypeptide does not traverse the entire membrane. InETF-QO, two highly hydrophobic peptide segments, F114–L131(�3a–�4) and G427–W451 (�9-helix), are located at the surfaceof the molecule and surround the UQ polyisoprene chain (Fig.1). These segments form the entrance of the UQ-binding pocketand likely form the membrane-binding surface (Figs. 1 and 4). Inaddition, there are three detergent molecules surrounding mol-ecule A and two surrounding molecule B, with one moleculeshared between A and B. Furthermore, these two polypeptidesegments are located near the local twofold axis and are sur-rounded by the same hydrophobic segments of the neighboringmolecule. The electrostatic potential map generated by GRASP(37) clearly shows the hydrophobic surface around the UQisoprene tail (Fig. 4). The �9-helix interacts with the membranewith its helical axis approximately parallel to the membranesurface; thus, together with the �-hairpin (�3a–�4), forming a‘‘hydrophobic plateau’’ with an approximate size 25 Å � 30 Å,similar to the ones observed in other monotopic membraneproteins (36), including prostaglandin-H synthase (38) andsqualene-hopene cyclase (39). The positively charged basic

residues near the membrane-associated residues probably play arole in interacting with the phospholipid head groups. Theseresidues include R113, R268, H269, H313, R423, and K454.Assuming these residues interact with the phospholipid headgroups, the benzoquinone head group is penetrating theETF-QO molecule �8 Å into the matrix side of the mitochondria(Fig. 1).

Electron Transfer Pathway. It is clear that ETF-QO catalyzes thereduction of UQ by ETF (1), but details of the reaction remainuncertain. ETF semiquinone (ETF1e�) is the product of theoxidative half-reaction of the acyl-CoA dehydrogenases; how-ever, it is not possible to monitor reduction of UQ by ETF1e� invitro because ETF1e� directly reduces the water-soluble UQ, Q1,with a second-order rate constant of �1,300 M�1 s�1 (9). UQ iscompartmentalized in the membrane phase, which precludes theETF-QO-independent reduction of UQ. ETF1e� can serve asthe direct reductant of ETF-QO, because ETF-QO catalyzes theintermolecular oxidation-reduction of ETF1e� in a novel dis-proportionation of ETF1e� that is kinetically competent toparticipate in the overall electron transfer from an acyl-CoAdehydrogenase to a water-soluble UQ analog, Q1 (9). Physio-logical disproportionation of ETF1e� would effectively increasethe driving force for the reduction of ETF-QO, because thepotential of the hydroqinone�semiquinone couple is �50 mVmore negative than that of the semiquinone�oxidized couple(40). On the other hand, when the reaction is run in the oppositedirection (i.e., when ETF is reduced anaerobically by NADH viaETF-QO in submitochondrial particles in the presence of aninhibitor of the bc1 complex), the anionic ETF1e� is generated(41). The principle of microscopic reversibility suggests thatETF1e� reduces ETF-QO and that the disproportionation reac-tion may be an artifact of the soluble system.

The redox potentials of ETF-QO flavin are �28 mV fortransfer of the first electron and �6 mV for the second electron.The potential of the 4Fe4S cluster is �47 mV (42). The redoxpotentials of the ETF f lavin are �4 mV for oxidized�semiquinone and �50 mV for semiquinone�hydroquinone (40).Thermodynamic considerations and the fact that ETF in vivoutilizes only the oxidized�semiquinone couple promptedPaulsen et al. (42) to propose a model for electron transfer fromETF to ETF-QO to UQ: (i) the ETF1e� reduces ETF-QO oneelectron at a time, first to the FAD of ETF-QO, then from FADto the cluster, forming the two-electron reduced state, i.e., FADsemiquinone and reduced iron-sulfur cluster, and (ii) both ofthese electrons transfer to UQ through the cluster in one-electron transfer steps to form ubiquinol (FAD 3 4Fe4S 3UQ). Because the cluster is an obligatory one electron donor�acceptor, the ubisemiquinone molecule must be formed, at leasttransiently, during catalysis. Another line of evidence suggestingthat electrons enter ETF-QO from ETF at the flavin site andthat the cluster is the electron donor to UQ comes frommutagenesis of C528 in human ETF-QO. Substitution of analanine abolishes quinone reductase activity but retains thedisproportionation activity (6). However, the mutant protein wasexpressed poorly in Saccharomyces cerevisiae, and the effects ofthe mutation on the cluster were not determined other thanthose inferred from the specific activities of ETF-QO in theassays using crude detergent-extracts of yeast mitochondria. Amore quantitative experiment with purified protein is requiredto confirm the results of the mutation studies.

The structure of ETF-QO is not consistent with this model.The structure strongly suggests that the reductant of UQ is theflavin, not the 4Fe4S cluster. The Fe3 atom of the cluster is11.5-Å from C8 of the isoalloxazine ring (Fig. 2). The two redoxcenters are separated by the backbone atoms of R331 and C556,the latter of which coordinates the cluster (Fig. 3 A and B). Theshortest distance from C8 of FAD to the S� of C556 is �9.4 Å.

Fig. 4. Electrostatic potential surface of ETF-QO viewed from the membraneside. Entrance to the UQ-binding site (dashed circle) and the UQ polyisoprenetail (green sticks) are shown. The surrounding positively charged groups (bluepatches) probably are involved in interacting with the negatively chargedmembrane phospholipid heads. The size of the entrance (dashed circle) is �10Å � 6 Å and that of the hydrophobic plateau (blue parallelogram) is �24 Å �30 Å. Color codes are blue for positive (�8 kT), white for neutral, and red fornegative (�8 kT).

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The carbonyl oxygen of C556 is within hydrogen-bonding dis-tance of the backbone nitrogen atom of R331, and this hydrogenbond may electronically couple the flavin and cluster (43). Thedistance between the cluster and UQ, measured between O2 ofUQ and Fe3 of the cluster, is 18.8 Å (Fig. 2), and it is �16.7 Åto the cluster S� of C556. These clusterOUQ distances aresignificantly longer than the 14-Å distance over which efficientelectron transfer by electron tunneling occurs (19). The longdistance from the cluster to UQ makes intraprotein electrontransfer from the flavin to the cluster and then to UQ problem-atic. Three possibilities could explain this apparent problem. (i)A conformational change could bring the cluster closer to theUQ ring. However, the three functional domains of ETF-QO areclosely packed, and individual helices and strands are sharedamong the three domains. Thus, it is unlikely that conforma-tional changes can occur that will significantly decrease theclusterOUQ distance. (ii) The protein could form a dimer asseen in Complex II and related enzymes, such that the 4Fe4Scluster of one monomer could be closer to UQ of the othermonomer for an efficient electron transfer. However, bothcurrent crystal structure analysis and the electrophoretic studiesof ETF-QO (44) suggest that the protein is monomeric. (iii) Itis possible that there is a second UQ site. However, the followingobservations argue against this assertion. First, additional UQcannot be soaked into the crystals, and cocrystallization withadditional UQ did not reveal a second site. Second, when theUQ-free protein was titrated with bromodecyl-UQ or otherquinone analogs, only a single site was detected (45, 46).Therefore, it is unlikely that the electron transfer to the quinoneis from the cluster. On the other hand, the flavin ring and thebenzoquinone ring are in close proximity. The distance betweenC6 of FAD and O3 of UQ is 8.5 Å (C8 of flavin to O2 of UQis 9.9 Å, still much shorter than the distance from the cluster toUQ) (Fig. 3). Furthermore, the two redox centers (FAD andUQ) are at the same side of �-sheet 3, supporting the assumptionthat the flavin is the reductant of UQ, not the cluster. Then, twoelectron transfer pathways can be envisioned (Scheme 1).

The first possible pathway is that the flavin acts both as theelectron acceptor from ETF1e� and the donor to UQ (steps 1 and3 in Scheme 1). Then what is the role of the cluster? WhenETF-QO is reduced by ETF, the ETF-QO flavin is reduced tosemiquinone, and the cluster also is reduced (1, 8), indicatingthat the cluster is not only structural but also is involved in theredox reaction. This finding indicates that an incoming electronfrom ETF rapidly equilibrates between the two redox centers,FAD and the cluster (step 2). When the second electron isintroduced, both the flavin and the cluster are in a one-electronreduced state, i.e., f lavin semiquinone and [4Fe4S]1�. Theenzyme is now primed for two-electron reduction of UQ toubiquinol, one from the flavin and the other from the cluster viaflavin (step 3). Thus, the cluster in ETF-QO has a redox-poisingeffect on the flavin (or electron storage), as in NADH-UQoxidoreductase (Complex I). In the structure of the solubledomain of Complex I from Thermus thermophilus, the FeS

cluster, N1a, is situated away from the main cluster chain andfunctions as an electron storage for FMN (47).

A second possibility is that the cluster is the entry point ofelectrons from ETF (step 1a in Scheme 1). The ETF-QO flavinpotential is only 19 mV lower than that of the cluster, and thereis no reason to exclude the possibility that ETF binding toETF-QO could lower the potential of the cluster to make the twocenters near isopotential, as seen in the heterodimeric periplas-mic nitrate reductase. In the reductase, the potentials of the4Fe4S cluster and heme II of the NapA subunit increase by 180mV and 40 mV, respectively, upon binding the NapB subunit,making the heme and cluster almost isopotential (48). In addi-tion, the ETF-QO cluster is located closer to the surface of theETF-QO molecule than the flavin is (�8 Å vs. �14 Å),suggesting that electron transfer from ETF flavin to theETF-QO cluster would be more favorable. Once an electronenters the cluster, it can shuttle rapidly to the flavin, because thetwo redox centers are almost isopotential. The rest of thepathway is the same as the above (steps 2 and 3 in Scheme 1).If we consider other UQ-binding proteins, such as respiratoryComplexes I and II, in which flavin is the electron acceptor fromNADH and succinate, respectively, we may think that in ETF-QO, FAD is the entrance for electrons from ETF. However, theelectron donor of ETF-QO is another protein (i.e., ETF) and isa one-electron donor as opposed to the two-electron donors,NADH and succinate in Complexes I and II, respectively. Thus,the cluster, an obligatory one-electron acceptor, could well bethe electron entrance point to ETF-QO. Further experimentsare required to distinguish between these two possibilities.

The relative simplicity of the ETF-QO structure permits a newviewpoint for considering the interaction of mitochondrial UQoxidoreductases with the mitochondrial UQ pool. ETF-QOcatalyzes the transfer of electrons from redox systems in themitochondrial matrix to UQ, the mobile electron carrier in themembrane phase, and apparently does so without transmem-brane segments. Thus, the structure of ETF-QO could be aparadigm for understanding how other similar proteins, such asglycerol-3-phosphate dehydrogenase and dihydroorotate dehy-drogenase, which access the UQ pool from the cytosolic andmatrix sides of the inner mitochondrial membrane, respectively,interact with the UQ pool. Also, ETF-QO provides yet anotherattractive model for understanding how lipid substrates accessthe active sites of monotopic membrane proteins (36).

MethodsCrystallization and Data Collection. ETF-QO was purified by theprocedure of Watmough et al. (45) which involves Triton X-100extraction of porcine liver submitochondrial particles. Alterna-tively, the protein was extracted with 40 mM N,N-dimethyl-amine-lauryl N-oxide (LDAO) in the same buffer. In the latterprotocol, all other steps were identical to those described byWatmough et al. There was no difference in activity of theprotein prepared by the two methods; however, the LDAO-solubilized protein fortuitously contained UQ.

Purified ETF-QO solubilized with N,N-dimethylamine-laurylN-oxide (LDAO) was concentrated to 15 mg�ml in 20 mMTris�HCl (pH 8.5) and was crystallized by the hanging drop-vapor diffusion method at 4°C. Hanging drops were made bymixing 2.0 �l each of the protein and reservoir solution [14.5%polyethylene glycol 2000 monomethyl ether (PEG2KMME)�0.5M NaCl�0.1 M Tris, pH 8.0�10% ethylene glycol] and 0.35 �l of6.6 mM �-hexyl-D-glucopyranoside (HBG). All data sets werecollected at 100 K, by using a solution containing 17%PEG2KMME and 20% ethylene glycol as cryoprotectant. Crys-tals belong to the space group P4212 (a � b � 154.3 Å, c � 128.5Å, with two molecules per asymmetric unit). A 2.5-Å native dataset and heavy-atom derivative data sets were collected at 1.000Å by using the BioCars BM-14C beamline at the Advanced

Scheme 1. Possible electron transfer paths between ETF and ETF-QO andwithin ETF-QO. Electrons from ETF1e enter ETF-QO via step 1 (path 1) or 1a(path 2) and are rapidly equilibrated between the two cofactors (step 2). WhenETF-QO is in two-electron reduced state (one electron at each cofactor), bothelectrons are transferred to UQ via FAD (step 3).

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Photon Source (Argonne National Laboratory, Argonne, IL).Native multiple-wavelength anomalous dispersion (MAD) datasets were collected at BM-14D at Advanced Photon Source at1.7389 Å (peak), 1.7426 Å (edge), and 1.6000 Å (remote). Datasets were processed with DENZO�SCALEPACK (49).

The UQ-free protein crystals were obtained in a similarmanner by mixing a 1:1 ratio of the protein solution (17 mg/mlin 20 mM Hepes, pH 7.5�0.2% �-octyl-D-glucopyranoside) andreservoir solution [12% (vol/vol) tertiary butanol�3.5% PEG400�0.1 M CaCl2�0.1 M Hepes, pH 7.5]. Data were collected at4°C with a Rigaku RU200 and an R-AXIS IIc image plate(Rigaku MSC, Woodland, TX). The crystals diffracted to 2.7 Å.The UQ-free ETF-QO crystals also belong to the P4212 spacegroup with cell dimensions a � b � 154.8 Å and c � 130.2 Å.

Structure Determination and Refinement. The structure of ETF-QOcontaining UQ was solved by MIRAS (multiple isomorphousreplacement with anomalous scattering) methods combined withmultiple-wavelength anomalous dispersion phasing (MAD) (50).The native anomalous difference Patterson maps were generatedby Xtalview (51) by using data collected at the peak wavelength(1.7389 Å). The strong peaks at the Harker sections clearlyshowed two iron-sulfur clusters per asymmetric unit and alsoconfirmed the space group to be P4212. All heavy-atom refine-

ments, phasing, electron density modification by solvent flatten-ing, and noncrystallographic twofold averaging were done withPHASES (52). The initial experimental map permitted buildinga polyalanine model with TURBO-FRODO (53). The Sigma-Aweighted map (54) calculated by using the MIRAS�MAD phasescombined with the phases calculated from the initial modelshowed significant improvement and allowed the assignment ofamino acid residues, FAD, and UQ. The structure of UQ-freeETF-QO was solved by difference Fourier techniques using theUQ-containing structure as the starting model. The structurerefinements were done by using CNS (55) alternating withmanual adjustments using TURBO-FRODO. Data collectionand phasing statistics are given in Tables 1 and 2 and therefinement statistics are shown in Table 3, which are publishedas supporting information on the PNAS web site.

This article is dedicated to Helmut Beinert and Frank Ruzicka for theirseminal work on this important enzyme (1) and for Beinert’s continuingcontributions to the field of Fe-S chemistry. We thank the staff of theBioCARS at the Advanced Photon Source for assistance with datacollection. This work was supported by National Institutes of HealthGrants GM29076 (to J.-J.P.K.) and HD08315 (to F.E.F.). Use of theAdvanced Photon Source is supported by the U.S. Department ofEnergy.

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