Structure of Escherichia coli Succinate:Quinone Oxidoreductase with an Occupied and Empty Quinone-binding Site

Three new structures of Escherichia coli succinate-quinone oxidoreductase (SQR) have been solved. One with the specific quinone-binding site (Q-site) inhibitor carboxin present has been solved at 2.4 Å resolution and reveals how carboxin inhibits the Q-site. The other new structures are with the Q-site inhibitor pentachlorophenol and with an empty Q-site. These structures reveal important details unresolved in earlier structures. Comparison of the new SQR structures shows how subtle rearrangements of the quinone-binding site accommodate the different inhibitors. The position of conserved water molecules near the quinone binding pocket leads to a reassessment of possible water-mediated proton uptake networks that complete reduction of ubiquinone. The dicarboxylate-binding site in the soluble domain of SQR is highly similar to that seen in high resolution structures of avian SQR (PDB 2H88) and soluble flavocytochrome c (PDB 1QJD) showing mechanistically significant structural features conserved across prokaryotic and eukaryotic SQRs.Succinate:quinone oxidoreductase (SQR,⁴ succinate dehydrogenase) and menaquinol:fumarate oxidoreductase (QFR, fumarate reductase), members of the Complex II family, are homologous integral membrane proteins which couple the interconversion of succinate and fumarate with quinone and quinol (1–4). SQR is a key enzyme in the Krebs cycle, oxidizing succinate to fumarate during aerobic growth and reducing quinone to quinol and, thus, acts as a direct link between the Krebs cycle and the respiratory chain. QFR is found in anaerobic or facultative bacteria and lower eukaryotes, where it couples the oxidation of reduced quinones to the reduction of fumarate (1, 4). Escherichia coli SQR has four subunits, two hydrophilic subunits exposed to the cytoplasm (SdhA and SdhB), which interact with two hydrophobic membrane-intrinsic subunits (SdhC and SdhD) (5). SdhA contains the dicarboxylate-binding site and a covalently bound FAD cofactor which cycles between FAD and FADH₂ redox states during succinate oxidation (6). The electrons from succinate oxidation are sequentially transferred via a [2Fe-2S], a [4Fe-4S], and a [3Fe-4S] iron-sulfur cluster relay system in SdhB to a quinone-binding site (Q_P) located at the interface of the SdhB, SdhC, and SdhD subunits. SdhC and SdhD are both composed of three transmembrane helices and coordinate a low spin b-type heme via His residues contributed by each subunit (7, 8).The first structural information about members of the Complex II family came from x-ray structures of the QFR enzymes from E. coli at 3.3 Å resolution (9) and Wolinella succinogenes at 2.2 Å resolution (10). These structures revealed details of the overall architecture of the subunits, the position of key redox cofactors, the electron transfer pathway, and the quinone-binding sites. At around the same time, the structures of soluble fumarate reductases found in anaerobic and microaerophilic bacteria and structurally homologous to the flavoprotein subunit of Complex II were solved by x-ray crystallography (1). Analysis of these soluble fumarate reductases has proven particularly informative in describing the mechanism of fumarate reduction and succinate oxidation at the dicarboxylate-binding site (11–14).Structures of SQRs lagged behind those of the QFRs until the structure of the E. coli enzyme was solved at 2.6 Å (15). This structure, solved in space group R32, revealed that the E. coli enzyme is packed as a trimer. The structures of the SdhA and SdhB subunits were highly similar to those of E. coli and W. succinogenes QFRs, but the transmembrane SdhC and SdhD subunits showed differences compared with their QFR counterparts. The structure revealed the position of the redox sites and the dicarboxylate- and quinone-binding (Q) sites. The heme b molecule was shown to lie away from the electron transfer pathway, suggesting electrons are preferentially transferred from the [3Fe-4S] cluster to ubiquinone, on the grounds of the edge-to-edge distances and redox potentials of the relevant groups. The structure revealed density in the Q-site that was interpreted as ubiquinone, and the position of the binding site was confirmed by the structure of the E. coli enzyme co-crystallized with the Q-site inhibitor 2-(1-methyl-hexyl)-4,6-dinitrophenol (DNP-17, PDB code 1NEN (15)). The E. coli enzyme was subsequently co-crystallized with the Q-site inhibitor Atpenin A5 (AA5) (PDB code 2ACZ (16)). This inhibitor was bound deeper into the quinone-binding site than ubiquinone or DNP-17, suggesting that there are two binding positions for ubiquinone in its binding site. The structure also identified a water-mediated proton pathway, proposed to deliver protons to the quinone-binding site. The first structure of a mitochondrial SQR was from porcine heart at 2.4 Å resolution (PDB code 1ZOY (17). This structure revealed a monomer in the asymmetric unit, suggesting that mitochondrial SQRs were likely to function as monomers. Superposition of the porcine and E. coli SQR structures revealed the high structural similarity of the SdhA and SdhB subunits and the conservation in position of the redox cofactors. Larger divergences were observed in the transmembrane subunits.Further structural information about SQRs was obtained by analysis of structures of avian SQR crystallized with oxaloacetate (2.2 Å resolution, PDB code 1YQ3), with 3-nitropropionate (2.4 Å resolution, PDB code 1YQ4), and with the Q-site inhibitor carboxin (2.1 Å resolution, PDB code 2FBW) (18). These structures revealed important differences in the position of key residues in the dicarboxylate-binding site compared with the E. coli and porcine structures. Arg-297 (equivalent to Arg-298 in porcine and Arg-286 in E. coli SQRs) was ideally located to act as a general base catalyst, accepting a proton during dehydrogenation of succinate, as in the soluble Shewanella flavocytochrome c3 (PDB code 1QJD) (11), suggesting conservation of mechanism between these distantly related enzymes. An unusual cis-serine peptide bond was proposed to position another arginine residue for binding dicarboxylates. Density for the dicarboxylate in 1YQ3 and 2FBW was shown to be distinctly non-planar and could be modeled by the “malate-like intermediate” seen in 1QJD. The nature of the ligand in the dicarboxylate site was further analyzed in a 1.74 Å resolution structure of avian SQR (PDB code 2H88), confirming the high structural similarity of the ligand and binding site residues in the SQR and flavocytochrome c3 structures (11, 12, 14).Despite the structural information described above, there are still unresolved issues regarding the structure and function of SQRs and QFRs. These include the location of conserved waters, which may form a channel involved in protonation of quinone, and the ability of the Q-site to accommodate different quinones and inhibitors. To further address these issues, we pursued structure-function studies of E. coli SQR. We developed alternative crystallization conditions that provided crystals more reproducibly and diffracting to higher resolution. By exchanging the enzyme into decyl-β-d-maltoside (DM) during purification, it was possible to crystallize the enzyme in the orthorhombic P2₁2₁2₁ space group. These crystals routinely diffracted in the 3–3.5 Å resolution range. Co-crystallization with the biochemically well characterized Q-site inhibitor carboxin improved diffraction to 2.1–2.8 Å. This structure shows new features related to the dicarboxylate-binding site of E. coli SQR including a rare cis-peptide bond in SdhA, as found in avian SQR (14), which helps shape the geometry of the active site. Comparisons of the structure with those of SQR binding PCP and SQR with an empty Q-site show how subtle rearrangements of the Q-site accommodate the different inhibitors. The orientation of carboxin in the Q-site differs with computational predictions (16) and with that seen in avian SQR (2FBW). The position of conserved water molecules around the Q-site suggests a new water-mediated proton uptake pathway consistent with recent mutational and biophysical studies (19).