Structure of electron transfer -ubiquinone and electron transfer to the mitochondrial ubiquinone pool

Jian 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 Sciences Center, 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- Reductive titration of ETF-QO by octanoyl-CoA in the pres- QO) is a 4Fe4S flavoprotein located in the inner mitochondrial ence of catalytic medium-chain acyl-CoA dehydrogenase and membrane. It catalyzes ubiquinone (UQ) reduction by ETF, linking ETF proceeds to the two-electron reduced state, [4Fe4S]1ϩ, and oxidation of fatty acids and some amino acids to the mitochondrial an anionic flavin semiquinone. Electron transfer in this pathway respiratory chain. Deficiencies in ETF or ETF-QO result in multiple is firmly established only for the initial transfer from the primary acyl-CoA dehydrogenase deficiency, a human metabolic disease. dehydrogenases to ETF: the reactions proceed as two one- Crystal structures of ETF-QO with and without bound UQ were electron transfer steps from the dehydrogenase dihydroquinone determined, and they are essentially identical. The molecule forms to two equivalents of ETF (7, 8). However, the electron transfer a single structural domain. Three functional regions bind FAD, the pathway is less clear at this point. ETF-QO catalyzes the 4Fe4S cluster, and UQ and are closely packed and share structural disproportionation of ETF semiquinone generated by the pri- elements, resulting in no discrete structural domains. The UQ- mary dehydrogenases at a rate that is catalytically competent to binding pocket consists mainly of hydrophobic residues, and UQ participate in the overall transfer of electrons from an acyl-CoA binding differs from that of other UQ-binding . ETF-QO is substrate to UQ (9). This overall reaction in vitro was established a monotopic integral membrane . The putative membrane- only in a soluble uncompartmentalized system, with a short- binding surface contains an ␣-helix and a ␤-hairpin, forming a chain, water-soluble UQ homolog (9). hydrophobic plateau. The UQOflavin distance (8.5 Å) is shorter ETF-QO, along with the other mitochondrial UQ oxidoreduc- than the UQOcluster distance (18.8 Å), and the very similar tases, plays a central role in the bioenergetics of aerobic organ- potentials of FAD and the cluster strongly suggest that the flavin, isms and some anaerobic organisms. Three-dimensional struc- not the cluster, transfers electrons to UQ. Two possible electron tures have been determined for several UQ oxidoreducatases, transfer paths can be envisioned. First, electrons from the ETF including succinate-UQ oxidoreductase (10–12), the related flavin semiquinone may enter the ETF-QO flavin one by one, quinol-fumarate oxidoreductase (13, 14), dihydroorotate dehy- followed by rapid equilibration with the cluster. Alternatively, drogenase (15), and the bc1 complex (16–18). These structures electrons may enter via the cluster, followed by equilibration have contributed to an understanding of the distance- between centers. In both cases, when ETF-QO is reduced to a dependence of electron transfer (19) and some generalizations two-electron reduced state (one electron at each redox center), the regarding the UQ-binding motifs (20). However, no detailed is primed to reduce UQ to ubiquinol via FAD. structural information has been available for ETF-QO. We undertook a structural investigation of porcine ETF-QO fatty acid oxidation ͉ iron-sulfur flavoprotein ͉ mitochondrial respiratory by using x-ray crystallography to obtain insight into the inter- and Ϫ ϩ chain ͉ membrane protein ͉ acyl-CoA dehydrogenases intramolecular electron transfers of the protein, the 2e ͞2H reduction of UQ, and the possible mode of binding of ETF-QO lectron transfer flavoprotein-ubiquinone oxidoreductase to the membrane. (ETF-QO) is an intrinsic membrane protein located in the E Results and Discussion inner mitochondrial membrane. It contains single equivalents of FAD and a [4Fe4S]2ϩ,1ϩ cluster (1). The protein is the single The Overall Structure. In the final structure of UQ containing input site to the main respiratory chain for electrons from nine ETF-QO, the entire polypeptide chain was visible except the first flavoprotein acyl-CoA dehydrogenases and two N-methyl dehy- three residues in one of the two molecules in the asymmetric unit drogenases (2, 3). The electron acceptor for the dehydrogenases and the first six residues in the other molecule. The residue is the ETF, which is the reductant of ETF-QO. ETF-QO is numbering system used hereafter corresponds to the mature oxidized by the diffusible ubiquinone (UQ) pool that also is protein sequence and can be related to the complete human accessed by NADH-UQ oxidoreductase (Complex I), succi- sequence by addition of 33 residues, the human mitochondrial nate-UQ oxidoreductase (Complex II), the flavin-linked glyc- signal peptide (21). Each molecule in the asymmetric unit erol-3-phosphate dehydrogenase, and dihydroorotate dehydro- genase, another flavin-linked UQ oxidoreductase (4). The Author contributions: J.Z. and J.-J.P.K. designed research; J.Z. and J.-J.P.K. performed ubiquinol product of these transfers electrons to research; J.Z. and J.-J.P.K. analyzed data; F.E.F. contributed new reagents͞analytic tools; the bc1 complex (Complex III). Thus, ETF and ETF-QO link the and J.Z., F.E.F., and J.-J.P.K. wrote the paper. oxidation of fatty acids and some amino acids to the mitochon- The authors declare no conflict of interest. drial respiratory system, and the overall electron flow can be This article is a PNAS direct submission. summarized as follows: Acyl-CoA 3 Acyl-CoA dehydrogenases 3 3 3 3 Abbreviations: ETF, electron transfer flavoprotein; ETF-QO, ETF-ubiquinone oxidoreduc- ETF ETF-QO UQ Complex III. Inherited deficien- tase; UQ, ubiquinone; ETF1eϪ, ETF semiquinone. cies of ETF-QO or ETF cause a metabolic disease, multiple Data deposition: The atomic coordinates and structure factors have been deposited in the acyl-CoA dehydrogenase deficiency, also known as glutaric , www.pdb.org [PDB ID codes 2GMH (UQ-bound structure) and 2GMJ acidemia type II (5). This metabolic disease is characterized in (UQ-free structure)]. its most severe form by delayed neuronal migration, an energy- ‡To whom correspondence should be addressed. E-mail: [email protected]. intensive process, and polycystic kidneys (6). © 2006 by The National Academy of Sciences of the USA

16212–16217 ͉ PNAS ͉ October 31, 2006 ͉ vol. 103 ͉ no. 44 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604567103 Downloaded by guest on September 26, 2021 Fig. 2. Electron densities in Fo Ϫ Fc omit maps for FAD (3.0␴), 4Fe4S (4.0␴), and UQ (2.5␴). The relative positions and distances (in angstroms) among the three redox centers are shown.

10, 11, 12, and 13. A structural similarity search using DALI (22) indicates that the fold of the core of the 4Fe4S domain is most similar to that of Clostridium acidurici ferredoxin (23). The C␣ rms difference for the 53 equivalent residues for the two molecules is 2.5 A˚ . The overall fold of the combined FAD and UQ domains is similar to the p-hydroxylbenzoate hydroxylase fold (24). The C␣ rms differences for the FAD and UQ͞ substrate-binding domains of ETF-QO and the hydroxylase are 1.9 A˚ (145 equivalent residues) and 2.2 A˚ (75 residues), respec- tively. The fold of the FAD domain of ETF-QO also is similar to that of flavocytochrome c3-fumarate reductase (rms deviation

Fig. 1. Ribbon diagram of ETF-QO. The structure comprises three domains: Ͻ2.0 A˚ for 127 C␣ atoms) (25–27) and quinol-fumarate reduc- BIOPHYSICS FAD domain (blue), 4Fe4S cluster domain (red), and UQ-binding domain tase flavin subunit (1.9 A˚ for 145 residues) (28). These domain- (green). Three redox centers are shown in sticks: FAD (golden yellow), 4Fe4S or subdomain-level structural similarities imply divergent evo- (magenta), and UQ (dark red). ␣-Helices and ␤-strands are numbered sequen- lution and gene fusions among these functionally related pro- tially from the N terminus to the C terminus. The putative membrane- teins. The FAD and 4Fe4S are buried completely in the ETF-QO associated surface regions are shown in cyan. Mitochondrial membrane is Ϫ depicted as blue shaded area. structure, as is the benzoquinone ring of UQ. The Fo Fc omit-map and the relative positions of the three redox centers, FAD, 4Fe4S, and UQ, are shown in Fig. 2. contains one FAD, one 4Fe4S cluster, and one UQ molecule. However, only 5 of the presumed 10 isoprene units could be seen The FAD Environment. FAD has an extended conformation and is in both of the UQ molecules. The final Rwork and Rfree of the buried completely in the protein (Fig. 1). It is positioned at the structure were 21.9% and 25.2%, respectively, for all of the data carboxyl side of the parallel ␤-sheet 2 and the C termini of ␣1 between 30.0-Å and 2.5-Å resolutions. The ETF-QO structure and ␣6 helices (Fig. 3A). The C7 and C8 methyl groups of the without bound UQ was determined to 2.7 Å, and the Rwork and isoalloxazine ring make van der Waal’s contacts with the main- ␤ Rfree of the final model were 22.8% and 25.5%, respectively. The chain N atom of R331 in the plane of -sheet 3. As in other folding of porcine ETF-QO is essentially the same, with or , the pyrimidine side of the isoalloxazine ring is without UQ, with an rms deviation of 0.26 Å between the two hydrogen-bonded to the polypeptide; O2 forms hydrogen bonds structures (a detailed comparison is given in Supporting Results, with the main-chain nitrogens of G366 and T367 and the which is published as supporting information on the PNAS web hydroxyl of T367, continuing the hydrogen-bonding pattern of site). A ribbon diagram of the UQ-bound structure is shown in the ␣6-helix (Fig. 3A). The interaction of the isoalloxazine ring Fig. 1, and a sequence alignment of ETF-QO from several and the ␣6-helix is strengthened further by hydrogen bonds species along with the secondary structural elements is shown in between the hydroxyl of T367 and the O2 and N1 atoms of the Fig. 5, which is published as supporting information on the PNAS isoalloxazine ring. Other hydrogen-bonding interactions occur web site. The structure contains 11 ␣-helices and 19 ␤-strands, between the O4 and C85 N atoms, N3 and C85 carbonyl oxygen and the molecule forms a single structural domain having three atoms. In addition to these hydrogen bonds, the positive dipole functional domains: a FAD domain, a 4Fe4S cluster domain, and of the helix could modulate the redox potential of FAD and a UQ-binding domain. The three domains are closely packed and stabilize the anionic semiquinone. This helix dipole–flavin in- share structural elements, and there are no isolated, discrete teraction is present in the p-hydroxybenzoate hydroxylase (24) domains that would be capable of local segmental motion as seen and glutathione reductase class of flavoproteins (29) and quinol- in the Fe-S protein of the bc1 complex (17). The 4Fe4S cluster fumarate reductase (13, 14). A water molecule is located on the domain in ETF-QO consists of N-terminal residues C4–Y16 and same plane as the flavin ring and is hydrogen-bonded to both the C-terminal residues D484–M584. The FAD domain comprises N5 and O2 atoms; however, its functional role is not clear at residues P17–N106, V141–P235, and S340–V418 and is primarily present. an ␣͞␤ structure. There are two ␤-sheets in the FAD domain. Strands ␤1, ␤2ofthe␤-sheet 2, and helix ␣1 form the ␤␣␤ ␤-Sheet 1 is a mixed parallel͞antiparallel sheet made of strands dinucleotide-binding motif. Residues G42–G47 contain the 1, 6, 7, and 8 and is located at the surface of the molecule capping ADP-binding sequence motif (GXGXXG), form the N terminus the 4Fe4S domain. ␤-Sheet 2 is composed of strands 1, 2, 5, 8, of helix ␣1, and hydrogen-bond to the pyrophosphate moiety of 14, and 15, sandwiched between ␤-sheet 1 and helices 1, 3, 6, and FAD (Fig. 3A). Like other FAD-containing proteins, the pyro- 7. The UQ domain comprises residues T107–V140, Q236–Q339, phosphate moiety is neutralized by R331. The hydroxyl of S82 and S417–F483. It is dominated by ␤-sheet 3, which is a twisted, makes a hydrogen bond to 2Ј-OH of the ribityl chain of FAD and mixed parallel͞antiparallel sheet comprising strands 3 (3a), 4, 9, is located beneath the center of the isoalloxazine ring on the

Zhang et al. PNAS ͉ October 31, 2006 ͉ vol. 103 ͉ no. 44 ͉ 16213 Downloaded by guest on September 26, 2021 Fig. 3. Residues in the vicinity of the redox centers. (A) Stereo diagram of the FAD-. 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-binding residues. Four cysteine residues (528, 553, 556, and 559) coordinate Fe atoms in the cluster (large dotted lines). They also make hydrogen bonds to the polypeptide chain (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 the UQ 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 other interactions involving UQ are of hydrophobic contacts. Color codes are the same as in B.

si-side with an atom-plane distance of 3.0 A˚ . Thus, it is possible mV, reflects the extensive hydrogen bonding of the cysteinyl that S82 acts as a hydrogen-bond donor and the flavin ring as the sulfur and sulfur atoms in the cluster. acceptor, as observed in some aromatic ring compounds (30). A similar interaction has been observed in cholesterol oxidase, in UQ Binding. UQ and the flavin isoalloxazine ring are located on which the amide nitrogen of N485 makes a N-Hѧ␲ interaction the same side of ␤-sheet 3. The UQ-binding pocket is made with the pyrimidine side of the FAD ring and modulates the mainly of hydrophobic residues (Fig. 3C). Only one of the two redox potential of the oxidase (31). Thus, the hydrogen bond carbonyl oxygen atoms in the benzoquinone ring is hydrogen- between S82 and the flavin ring in ETF-QO also may influence bonded to the polypeptide chain. The O4 atom of UQ makes the redox potential of the bound FAD. Three residues, R331, hydrogen bonds to the backbone nitrogen of G273 and carbonyl T367 and S82, are highly conserved in the ETF-QO sequences oxygen of G272. The rest of the molecule is surrounded by mostly (see the supporting information). hydrophobic 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, and The 4Fe4S Cluster Environment. The iron-sulfur cluster in ETF-QO C3 methoxy groups; F114, H260, and V262 contacts the O1 is embedded in two loops that contain residues C528–Y533 and carbonyl and C2 methoxy groups. The isoprene tail is bent such C553–D560. As predicted from the sequence analysis, C528, that the second isoprene unit contacts the O1 carbonyl group. C553, C556, and C559 from the two loops coordinate the four Fe There is a water molecule that hydrogen-bonds the hydroxyl of atoms in the cluster (21). Residues H503, L504, and W570 Y271 and O4 of the benzoquinone ring. This water may act as the complete the binding pocket formed by the two loops (Fig. 3B). proton donor͞acceptor during the UQ redox cycle. Residues The cluster is supported further through hydrogen bonds be- Y271, G272, G273, S274, and H260 are highly conserved among tween the S␥ atoms of the four cysteines and the polypeptide ETF-QOs from different species (Fig. 5). In particular, G273 is chain; C553 makes hydrogen bonds with H503, C556 bonds with absolutely conserved in all ETF-QO sequences. The absence of the hydroxyl of T558, C559 bonds with the phenolic oxygen of a bulky side chain at position G273 eliminates steric hindrance Y533 and main-chain N of D560, and C528 bonds with the so that the UQ molecule penetrates deep into its hydrophobic backbone nitrogen of A530. Two of the four cluster sulfur atoms binding pocket. If electron transfer to UQ involves ubisemiqui- make weak hydrogen bonds, each with the main-chain N atom none as a transient intermediate, the semiquinone is protected of K557 or C559 (both distances, 3.4 Å). Such hydrogen bonds from reaction with molecular oxygen. Only 5 isoprene units of can modulate the redox potential of iron-sulfur clusters (32, 33). the UQ tail (10 isoprene units for mammalian UQ) are visible. The relatively positive potential of the ETF-QO cluster, ϩ47 There is a hint of disorder even at the third isoprene unit in one

16214 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604567103 Zhang et al. Downloaded by guest on September 26, 2021 residues near the membrane-associated residues probably play a role in interacting with the phospholipid head groups. These residues include R113, R268, H269, H313, R423, and K454. Assuming these residues interact with the phospholipid head groups, the benzoquinone head group is penetrating the ETF-QO molecule Ϸ8 Å into the matrix side of the mitochondria (Fig. 1).

Electron Transfer Pathway. It is clear that ETF-QO catalyzes the reduction of UQ by ETF (1), but details of the reaction remain uncertain. ETF semiquinone (ETF1eϪ) is the product of the oxidative half-reaction of the acyl-CoA dehydrogenases; how- ever, it is not possible to monitor reduction of UQ by ETF1eϪ in vitro because ETF1eϪ directly reduces the water-soluble UQ, Q1, with a second-order rate constant of Ϸ1,300 MϪ1 sϪ1 (9). UQ is compartmentalized in the membrane phase, which precludes the ETF-QO-independent reduction of UQ. ETF1eϪ can serve as the direct reductant of ETF-QO, because ETF-QO catalyzes the intermolecular oxidation-reduction of ETF1eϪ in a novel dis- proportionation of ETF1eϪ that is kinetically competent to participate in the overall electron transfer from an acyl-CoA dehydrogenase to a water-soluble UQ analog, Q1 (9). Physio- Fig. 4. Electrostatic potential surface of ETF-QO viewed from the membrane logical disproportionation of ETF1eϪ would effectively increase side. Entrance to the UQ-binding site (dashed circle) and the UQ polyisoprene the driving force for the reduction of ETF-QO, because the tail (green sticks) are shown. The surrounding positively charged groups (blue potential of the hydroqinone͞semiquinone couple is Ϸ50 mV patches) probably are involved in interacting with the negatively charged more negative than that of the semiquinone͞oxidized couple membrane phospholipid heads. The size of the entrance (dashed circle) is Ϸ10 (40). On the other hand, when the reaction is run in the opposite Å ϫ 6 Å and that of the hydrophobic plateau (blue parallelogram) is Ϸ24 Å ϫ BIOPHYSICS ϩ direction (i.e., when ETF is reduced anaerobically by NADH via 30 Å. Color codes are blue for positive ( 8 kT), white for neutral, and red for ETF-QO in submitochondrial particles in the presence of an negative (Ϫ8 kT). inhibitor of the bc1 complex), the anionic ETF1eϪ is generated (41). The principle of microscopic reversibility suggests that of the two ETF-QO molecules in the asymmetric unit, indicating ETF1eϪ reduces ETF-QO and that the disproportionation reac- that the tail is flexible (Fig. 2). The second through the fifth tion may be an artifact of the soluble system. ϩ isoprene units of the tail are surrounded by mostly hydrophobic The redox potentials of ETF-QO flavin are 28 mV for Ϫ residues, including F114, V125, G434, M435, T438, and G439 transfer of the first electron and 6 mV for the second electron. ϩ (Fig. 3C). Thus, the binding mode of UQ observed in ETF-QO The potential of the 4Fe4S cluster is 47 mV (42). The redox potentials of the ETF flavin are ϩ4 mV for oxidized͞ is different from those observed in other UQ-binding proteins, Ϫ ͞ e.g., succinate-UQ oxidoreductase (34) and ubiquinol oxidase semiquinone and 50 mV for semiquinone hydroquinone (40). Thermodynamic considerations and the fact that ETF in vivo (35). The binding motif found in other proteins of the respiratory ͞ and photosynthetic systems has semiconserved sequences con- utilizes only the oxidized semiquinone couple prompted Paulsen et al. (42) to propose a model for electron transfer from taining a Tyr͞Trp or His that make direct hydrogen bonds to O1 ETF to ETF-QO to UQ: (i) the ETF Ϫ reduces ETF-QO one and͞or O4 of the benzoquinone group (20). 1e electron at a time, first to the FAD of ETF-QO, then from FAD to the cluster, forming the two-electron reduced state, i.e., FAD Membrane-Binding Surface. By the usual criteria, porcine ETF-QO semiquinone and reduced iron-sulfur cluster, and (ii) both of is classified as an integral membrane protein (36), requiring these electrons transfer to UQ through the cluster in one- detergent to solubilize the protein. In contrast to UQ-binding electron transfer steps to form ubiquinol (FAD 3 4Fe4S 3 proteins of the main respiratory and photosynthetic systems, the UQ). Because the cluster is an obligatory one electron donor͞ ETF-QO polypeptide does not traverse the entire membrane. In acceptor, the ubisemiquinone molecule must be formed, at least ETF-QO, two highly hydrophobic peptide segments, F114–L131 ␤ ␤ ␣ transiently, during . Another line of evidence suggesting ( 3a– 4) and G427–W451 ( 9-helix), are located at the surface that electrons enter ETF-QO from ETF at the flavin site and of the molecule and surround the UQ polyisoprene chain (Fig. that the cluster is the electron donor to UQ comes from 1). These segments form the entrance of the UQ-binding pocket mutagenesis of C528 in human ETF-QO. Substitution of an and likely form the membrane-binding surface (Figs. 1 and 4). In alanine abolishes quinone reductase activity but retains the addition, there are three detergent molecules surrounding mol- disproportionation activity (6). However, the mutant protein was ecule A and two surrounding molecule B, with one molecule expressed poorly in Saccharomyces cerevisiae, and the effects of shared between A and B. Furthermore, these two polypeptide the mutation on the cluster were not determined other than segments are located near the local twofold axis and are sur- those inferred from the specific activities of ETF-QO in the rounded by the same hydrophobic segments of the neighboring assays using crude detergent-extracts of mitochondria. A molecule. The electrostatic potential map generated by GRASP more quantitative experiment with purified protein is required (37) clearly shows the hydrophobic surface around the UQ to confirm the results of the mutation studies. isoprene tail (Fig. 4). The ␣9-helix interacts with the membrane The structure of ETF-QO is not consistent with this model. with its helical axis approximately parallel to the membrane The structure strongly suggests that the reductant of UQ is the surface; thus, together with the ␤-hairpin (␤3a–␤4), forming a flavin, not the 4Fe4S cluster. The Fe3 atom of the cluster is ‘‘hydrophobic plateau’’ with an approximate size 25 Å ϫ 30 Å, 11.5-Å from C8 of the isoalloxazine ring (Fig. 2). The two redox similar to the ones observed in other monotopic membrane centers are separated by the backbone atoms of R331 and C556, proteins (36), including prostaglandin-H (38) and the latter of which coordinates the cluster (Fig. 3 A and B). The squalene-hopene cyclase (39). The positively charged basic shortest distance from C8 of FAD to the S␥ of C556 is Ϸ9.4 Å.

Zhang et al. PNAS ͉ October 31, 2006 ͉ vol. 103 ͉ no. 44 ͉ 16215 Downloaded by guest on September 26, 2021 cluster, N1a, is situated away from the main cluster chain and functions as an electron storage for FMN (47). A second possibility is that the cluster is the entry point of electrons from ETF (step 1a in Scheme 1). The ETF-QO flavin potential is only 19 mV lower than that of the cluster, and there is no reason to exclude the possibility that ETF binding to Scheme 1. Possible electron transfer paths between ETF and ETF-QO and ETF-QO could lower the potential of the cluster to make the two within ETF-QO. Electrons from ETF1e enter ETF-QO via step 1 (path 1) or 1a centers near isopotential, as seen in the heterodimeric periplas- (path 2) and are rapidly equilibrated between the two cofactors (step 2). When ETF-QO is in two-electron reduced state (one electron at each ), both mic . In the reductase, the potentials of the electrons are transferred to UQ via FAD (step 3). 4Fe4S cluster and heme II of the NapA subunit increase by 180 mV and 40 mV, respectively, upon binding the NapB subunit, making the heme and cluster almost isopotential (48). In addi- The carbonyl oxygen of C556 is within hydrogen-bonding dis- tion, the ETF-QO cluster is located closer to the surface of the tance of the backbone nitrogen atom of R331, and this hydrogen ETF-QO molecule than the flavin is (Ϸ8 Å vs. Ͼ14 Å), bond may electronically couple the flavin and cluster (43). The suggesting that electron transfer from ETF flavin to the distance between the cluster and UQ, measured between O2 of ETF-QO cluster would be more favorable. Once an electron UQ and Fe3 of the cluster, is 18.8 Å (Fig. 2), and it is Ϸ16.7 Å enters the cluster, it can shuttle rapidly to the flavin, because the to the cluster S␥ of C556. These clusterOUQ distances are two redox centers are almost isopotential. The rest of the significantly longer than the 14-Å distance over which efficient pathway is the same as the above (steps 2 and 3 in Scheme 1). electron transfer by electron tunneling occurs (19). The long If we consider other UQ-binding proteins, such as respiratory distance from the cluster to UQ makes intraprotein electron Complexes I and II, in which flavin is the electron acceptor from transfer from the flavin to the cluster and then to UQ problem- NADH and succinate, respectively, we may think that in ETF- QO, FAD is the entrance for electrons from ETF. However, the atic. Three possibilities could explain this apparent problem. (i) electron donor of ETF-QO is another protein (i.e., ETF) and is A conformational change could bring the cluster closer to the a one-electron donor as opposed to the two-electron donors, UQ ring. However, the three functional domains of ETF-QO are NADH and succinate in Complexes I and II, respectively. Thus, closely packed, and individual helices and strands are shared the cluster, an obligatory one-electron acceptor, could well be among the three domains. Thus, it is unlikely that conforma- the electron entrance point to ETF-QO. Further experiments tional changes can occur that will significantly decrease the are required to distinguish between these two possibilities. O cluster UQ distance. (ii) The protein could form a dimer as The relative simplicity of the ETF-QO structure permits a new seen in Complex II and related , such that the 4Fe4S viewpoint for considering the interaction of mitochondrial UQ cluster of one monomer could be closer to UQ of the other oxidoreductases with the mitochondrial UQ pool. ETF-QO monomer for an efficient electron transfer. However, both catalyzes the transfer of electrons from redox systems in the current crystal structure analysis and the electrophoretic studies mitochondrial matrix to UQ, the mobile electron carrier in the of ETF-QO (44) suggest that the protein is monomeric. (iii)It membrane phase, and apparently does so without transmem- is possible that there is a second UQ site. However, the following brane segments. Thus, the structure of ETF-QO could be a observations argue against this assertion. First, additional UQ paradigm for understanding how other similar proteins, such as cannot be soaked into the crystals, and cocrystallization with glycerol-3-phosphate dehydrogenase and dihydroorotate dehy- additional UQ did not reveal a second site. Second, when the drogenase, which access the UQ pool from the cytosolic and UQ-free protein was titrated with bromodecyl-UQ or other matrix sides of the inner mitochondrial membrane, respectively, quinone analogs, only a single site was detected (45, 46). interact with the UQ pool. Also, ETF-QO provides yet another Therefore, it is unlikely that the electron transfer to the quinone attractive model for understanding how lipid substrates access is from the cluster. On the other hand, the flavin ring and the the active sites of monotopic membrane proteins (36). benzoquinone ring are in close proximity. The distance between C6 of FAD and O3 of UQ is 8.5 Å (C8 of flavin to O2 of UQ Methods is 9.9 Å, still much shorter than the distance from the cluster to Crystallization and Data Collection. ETF-QO was purified by the UQ) (Fig. 3). Furthermore, the two redox centers (FAD and procedure of Watmough et al. (45) which involves Triton X-100 UQ) are at the same side of ␤-sheet 3, supporting the assumption extraction of porcine liver submitochondrial particles. Alterna- that the flavin is the reductant of UQ, not the cluster. Then, two tively, the protein was extracted with 40 mM N,N-dimethyl- electron transfer pathways can be envisioned (Scheme 1). amine-lauryl N-oxide (LDAO) in the same buffer. In the latter The first possible pathway is that the flavin acts both as the protocol, all other steps were identical to those described by Watmough et al. There was no difference in activity of the electron acceptor from ETF Ϫ and the donor to UQ (steps 1 and 1e protein prepared by the two methods; however, the LDAO- 3 in Scheme 1). Then what is the role of the cluster? When solubilized protein fortuitously contained UQ. ETF-QO is reduced by ETF, the ETF-QO flavin is reduced to Purified ETF-QO solubilized with N,N-dimethylamine-lauryl semiquinone, and the cluster also is reduced (1, 8), indicating N-oxide (LDAO) was concentrated to 15 mg͞ml in 20 mM that the cluster is not only structural but also is involved in the Tris⅐HCl (pH 8.5) and was crystallized by the hanging drop- redox reaction. This finding indicates that an incoming electron vapor diffusion method at 4°C. Hanging drops were made by from ETF rapidly equilibrates between the two redox centers, mixing 2.0 ␮l each of the protein and reservoir solution [14.5% FAD and the cluster (step 2). When the second electron is polyethylene glycol 2000 monomethyl ether (PEG2KMME)͞0.5 introduced, both the flavin and the cluster are in a one-electron M NaCl͞0.1 M Tris, pH 8.0͞10% ethylene glycol] and 0.35 ␮lof 1ϩ reduced state, i.e., flavin semiquinone and [4Fe4S] . The 6.6 mM ␤-hexyl-D-glucopyranoside (HBG). All data sets were enzyme is now primed for two-electron reduction of UQ to collected at 100 K, by using a solution containing 17% ubiquinol, one from the flavin and the other from the cluster via PEG2KMME and 20% ethylene glycol as cryoprotectant. Crys- flavin (step 3). Thus, the cluster in ETF-QO has a redox-poising tals belong to the space group P4212(a ϭ b ϭ 154.3 Å, c ϭ 128.5 effect on the flavin (or electron storage), as in NADH-UQ Å, with two molecules per asymmetric unit). A 2.5-Å native data oxidoreductase (Complex I). In the structure of the soluble set and heavy-atom derivative data sets were collected at 1.000 domain of Complex I from Thermus thermophilus, the FeS Å by using the BioCars BM-14C beamline at the Advanced

16216 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604567103 Zhang et al. Downloaded by guest on September 26, 2021 Photon Source (Argonne National Laboratory, Argonne, IL). ments, phasing, electron density modification by solvent flatten- Native multiple-wavelength anomalous dispersion (MAD) data ing, and noncrystallographic twofold averaging were done with sets were collected at BM-14D at Advanced Photon Source at PHASES (52). The initial experimental map permitted building 1.7389 Å (peak), 1.7426 Å (edge), and 1.6000 Å (remote). Data a polyalanine model with TURBO-FRODO (53). The Sigma-A sets were processed with DENZO͞SCALEPACK (49). weighted map (54) calculated by using the MIRAS͞MAD phases The UQ-free protein crystals were obtained in a similar combined with the phases calculated from the initial model manner by mixing a 1:1 ratio of the protein solution (17 mg/ml showed significant improvement and allowed the assignment of ͞ ␤ in 20 mM Hepes, pH 7.5 0.2% -octyl-D-glucopyranoside) and residues, FAD, and UQ. The structure of UQ-free ͞ reservoir solution [12% (vol/vol) tertiary butanol 3.5% PEG ETF-QO was solved by difference Fourier techniques using the ͞ ͞ 400 0.1 M CaCl2 0.1 M Hepes, pH 7.5]. Data were collected at UQ-containing structure as the starting model. The structure 4°C with a Rigaku RU200 and an R-AXIS IIc image plate refinements were done by using CNS (55) alternating with (Rigaku MSC, Woodland, TX). The crystals diffracted to 2.7 Å. manual adjustments using TURBO-FRODO. Data collection The UQ-free ETF-QO crystals also belong to the P4212 space ϭ ϭ ϭ and phasing statistics are given in Tables 1 and 2 and the group with cell dimensions a b 154.8 Å and c 130.2 Å. refinement statistics are shown in Table 3, which are published as supporting information on the PNAS web site. Structure Determination and Refinement. The structure of ETF-QO containing UQ was solved by MIRAS (multiple isomorphous This article is dedicated to Helmut Beinert and Frank Ruzicka for their replacement with anomalous scattering) methods combined with seminal work on this important enzyme (1) and for Beinert’s continuing multiple-wavelength anomalous dispersion phasing (MAD) (50). contributions to the field of Fe-S chemistry. We thank the staff of the The native anomalous difference Patterson maps were generated BioCARS at the Advanced Photon Source for assistance with data by Xtalview (51) by using data collected at the peak wavelength collection. This work was supported by National Institutes of Health (1.7389 Å). The strong peaks at the Harker sections clearly Grants GM29076 (to J.-J.P.K.) and HD08315 (to F.E.F.). Use of the showed two iron-sulfur clusters per asymmetric unit and also Advanced Photon Source is supported by the U.S. Department of confirmed the space group to be P4212. All heavy-atom refine- Energy.

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