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Structure of Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase and Electron Transfer to the Mitochondrial Ubiquinone Pool

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Structure of Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase and Electron Transfer to the Mitochondrial Ubiquinone Pool Structure of electron transfer flavoprotein-ubiquinone oxidoreductase 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 proteins. ETF-QO is substrate to UQ (9). This overall reaction in vitro was established a monotopic integral membrane protein. 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 redox 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 enzyme 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 oxidoreductases 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 Protein Data Bank, 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 flavoproteins, 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).
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