3-Nitropropionic Acid Is a Suicide Inhibitor of Mitochondrial Respiration That, upon Oxidation by Complex II, Forms a Covalent Adduct with a Catalytic Base Arginine in the Active Site of the Enzyme

We report three new structures of mitochondrial respiratory Complex II (succinate ubiquinone oxidoreductase, E.C. 1.3.5.1) at up to 2.1 Å resolution, with various inhibitors. The structures define the conformation of the bound inhibitors and suggest the residues involved in substrate binding and catalysis at the dicarboxylate site. In particular they support the role of Arg297 as a general base catalyst accepting a proton in the dehydrogenation of succinate. The dicarboxylate ligand in oxaloacetate-containing crystals appears to be the same as that reported for Shewanella flavocytochrome c treated with fumarate. The plant and fungal toxin 3-nitropropionic acid, an irreversible inactivator of succinate dehydrogenase, forms a covalent adduct with the side chain of Arg297. The modification eliminates a trypsin cleavage site in the flavoprotein, and tandem mass spectroscopic analysis of the new fragment shows the mass of Arg297 to be increased by 83 Da and to have the potential of losing 44 Da, consistent with decarboxylation, during fragmentation. We report three new structures of mitochondrial respiratory Complex II (succinate ubiquinone oxidoreductase, E.C. 1.3.5.1) at up to 2.1 Å resolution, with various inhibitors. The structures define the conformation of the bound inhibitors and suggest the residues involved in substrate binding and catalysis at the dicarboxylate site. In particular they support the role of Arg297 as a general base catalyst accepting a proton in the dehydrogenation of succinate. The dicarboxylate ligand in oxaloacetate-containing crystals appears to be the same as that reported for Shewanella flavocytochrome c treated with fumarate. The plant and fungal toxin 3-nitropropionic acid, an irreversible inactivator of succinate dehydrogenase, forms a covalent adduct with the side chain of Arg297. The modification eliminates a trypsin cleavage site in the flavoprotein, and tandem mass spectroscopic analysis of the new fragment shows the mass of Arg297 to be increased by 83 Da and to have the potential of losing 44 Da, consistent with decarboxylation, during fragmentation. The toxin 3-nitropropionic acid (3-NP) 5The abbreviations used are: 3-NP, 3-nitropropionic acid; OAA, oxaloacetatic acid; carboxin, 2-methyl-1,4-oxathiin-3-carboxanilide; TTFA, thenoyl trifluoroacetone; SQR, succinate:quinone oxidoreductase; FRD, fumarate reductase; FCc, flavocytochrome c FRD; MS mass spectroscopy. 5The abbreviations used are: 3-NP, 3-nitropropionic acid; OAA, oxaloacetatic acid; carboxin, 2-methyl-1,4-oxathiin-3-carboxanilide; TTFA, thenoyl trifluoroacetone; SQR, succinate:quinone oxidoreductase; FRD, fumarate reductase; FCc, flavocytochrome c FRD; MS mass spectroscopy. is produced by certain plants and fungi. It is a specific inhibitor of mitochondrial respiratory complex II. Fatalities after eating moldy sugarcane have been linked to 3-NP toxicity (1Ming L. J. Toxicol. Clin. Toxicol. 1995; 33: 363-367Crossref PubMed Scopus (76) Google Scholar, 2Liu X. Luo X. Hu W. Biomed. Environ. Sci. 1992; 5: 161-177PubMed Google Scholar). Ruminants grazing in regions with 3-NP-producing plants acquire resistance because of reduction of the nitro group to an amine by ruminal bacteria (3Anderson R.C. Rasmussen M.A. Allison M.J. Appl. Environ. Microbiol. 1993; 59: 3056-3061Crossref PubMed Google Scholar). The effectiveness of 3-NP in vivo after injection or oral administration has made it useful in studies involving tissues or whole animals. Ingestion of 3-NP results in neurodegeneration with symptoms resembling those of Huntington disease (4Beal M.F. Brouillet E. Jenkins B.G. Ferrante R.J. Kowall N.W. Miller J.M. Storey E. Srivastava R. Rosen B.R. Hyman B.T. J. Neurosci. 1993; 13: 4181-4192Crossref PubMed Google Scholar), and conversely Huntington disease results in a loss of complex II activity (5Browne S.E. Bowling A.C. MacGarvey U. Baik M.J. Berger S.C. Muqit M.M. Bird E.D. Beal M.F. Ann. Neurol. 1997; 41: 646-653Crossref PubMed Scopus (741) Google Scholar); thus 3-NP has been used to produce an animal model for the study of Huntington disease (6Borlongan C.V. Koutouzis T.K. Sanberg P.R. Neurosci. Biobehav. Rev. 1997; 21: 289-293Crossref PubMed Scopus (146) Google Scholar, 7Borlongan C.V. Koutouzis T.K. Freeman T.B. Hauser R.A. Cahill D.W. Sanberg P.R. Brain Res. Brain Res. Protoc. 1997; 1: 253-257Crossref PubMed Scopus (51) Google Scholar). Symptoms also include convulsions, and 3-NP is being looked at for inducing a model of epilepsy (8Urbanska E.M. Pol. J. Pharmacol. 2000; 52: 55-57PubMed Google Scholar). Prior subacute 3-NP poisoning seems to provide resistance to ischemic damage to nervous tissue by a preconditioning effect (9Brambrink A.M. Noga H. Astheimer A. Heimann A. Kempski O. Acta Neurochir. Suppl. 2004; 89: 63-66Crossref PubMed Scopus (10) Google Scholar) similar to that resulting from mild ischemia. The target of 3-NP is Complex II, which is both a member of the Krebs tricarboxylic acid cycle (oxidizing succinate to fumarate) and an entry point for electrons into the respiratory chain at the level of ubiquinol. It consists of a large flavoprotein subunit containing covalently bound FAD, an iron-sulfur protein (IP) with three different iron-sulfur clusters, and two small membrane anchor subunits (chains C and D) ligating a single low spin heme of type B. Human genetic defects in the IP subunits or chains C or D lead to development of paragangliomas (10Niemann S. Muller U. Nat. Genet. 2000; 26: 268-270Crossref PubMed Scopus (761) Google Scholar, 11Baysal B.E. Willett-Brozick J.E. Lawrence E.C. Drovdlic C.M. Savul S.A. McLeod D.R. Yee H.A. Brackmann D.E. Slattery III, W.H. Myers E.N. Ferrell R.E. Rubinstein W.S. J. Med. Genet. 2002; 39: 178-183Crossref PubMed Scopus (282) Google Scholar). A mutation in chain C leads to premature aging in nematodes, presumably through excessive production of free radicals (12Senoo-Matsuda N. Hartman P.S. Akatsuka A. Yoshimura S. Ishii N. J. Biol. Chem. 2003; 278: 22031-22036Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Bacterial homologs succinate:quinone oxidoreductase (SQR) and menaquinol: fumarate oxidoreductase (FRD) in Escherichia coli have been studied as genetically manipulable models for the mitochondrial protein. Recent reviews cover this family of enzymes (13Ackrell B.A. FEBS Lett. 2000; 466: 1-5Crossref PubMed Scopus (104) Google Scholar, 14Ackrell B.A. Mol. Aspects Med. 2002; 23: 369-384Crossref PubMed Scopus (75) Google Scholar, 15Cecchini G. Annu. Rev. Biochem. 2003; 72: 77-109Crossref PubMed Scopus (351) Google Scholar, 16Hagerhall C. Biochim. Biophys. Acta. 1997; 1320: 107-141Crossref PubMed Scopus (373) Google Scholar, 17Lancaster C.R. FEBS Lett. 2003; 555: 21-28Crossref PubMed Scopus (32) Google Scholar, 18Ohnishi T. Moser C.C. Page C.C. Dutton P.L. Yano T. Structure Fold Des. 2000; 8: R23-32Abstract Full Text Full Text PDF Scopus (71) Google Scholar). X-ray crystallographic structures are available for a number of members of the family. The available mitochondrial structures and representative bacterial examples are listed in Table 1.TABLE 1Tabulation of some x-ray structures available for members of the SQR/FRD familyEnzymeSourceReferenceProtein Data Bank codeResolutionRelevanceSQRChicken1YQ32.2Crystallized with OAA1YQ42.4Crystallized with 3-NP2FBW2.1Crystal soaked with carboxinPig24Sun F. Huo X. Zhai Y. Wang A. Xu J. Su D. Bartlam M. Rao Z. Cell. 2005; 121: 1043-1057Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar1ZOY2.4Chain A Arg298 out of active site1ZP03.5Crystallized with 3-NP and TTFAE. coli23Yankovskaya V. Horsefield R. Tornroth S. Luna-Chavez C. Miyoshi H. Leger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (684) Google Scholar1NEK2.6Chain A Arg286 out of active siteFCcShewanella38Taylor P. Pealing S.L. Reid G.A. Chapman S.K. Walkinshaw M.D. Nat. Struct. Biol. 1999; 6: 1108-1112Crossref PubMed Scopus (134) Google Scholar1QJD1.8Dicarboxylate site like SQRFRDWolinella49Lancaster C.R. Kroger A. Auer M. Michel H. Nature. 1999; 402: 377-385Crossref PubMed Scopus (310) Google Scholar1QLA/B2.2Open CAP domain46Lancaster C.R. Gross R. Simon J. Eur. J. Biochem. 2001; 268: 1820-1827Crossref PubMed Scopus (61) Google Scholar1QO83.0Chain A Arg301 disorderedFRDE. coli22Iverson T.M. Luna-Chavez C. Croal L.R. Cecchini G. Rees D.C. J. Biol. Chem. 2002; 277: 16124-16130Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar1KF62.7CAP domain slightly open Open table in a new tab The toxin 3-NP, structurally similar to and isoelectronic with the substrate succinate, is believed to be a suicide inactivator of Complex II. Alston et al. (19Alston T.A. Mela L. Bright H.J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3767-3771Crossref PubMed Scopus (336) Google Scholar) proposed, based on previous observations and on their own experience with another flavoprotein, that the normal reaction pathway involves a temporary adduct with the N-5 nitrogen of flavin, which in the case of 3-NP collapses to a stable adduct resulting in permanent inactivation. Irreversible covalent modification of the flavin was ruled out by later work (20Coles C.J. Edmondson D.E. Singer T.P. J. Biol. Chem. 1979; 254: 5161-5167Abstract Full Text PDF PubMed Google Scholar) examining the spectral change induced and showing that unmodified flavin peptide could be isolated from the inactivated complex by mild proteolysis. It was proposed that 3-NP is oxidized to 3-nitroacrylate, an unstable molecule that then reacts with some residue in the active site. A cysteine that was believed to be in the active site and essential for activity and for tight binding of another inhibitor, oxaloacetate (OAA), was suggested to be the residue involved. Later studies showed that this cysteine is not essential for activity or OAA binding. Recent elucidation of the structures of the E. coli FRD (21Iverson T.M. Luna-Chavez C. Cecchini G. Rees D.C. Science. 1999; 284: 1961-1966Crossref PubMed Scopus (366) Google Scholar, 22Iverson T.M. Luna-Chavez C. Croal L.R. Cecchini G. Rees D.C. J. Biol. Chem. 2002; 277: 16124-16130Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and SQR (23Yankovskaya V. Horsefield R. Tornroth S. Luna-Chavez C. Miyoshi H. Leger C. Byrne B. Cecchini G. Iwata S. Science. 2003; 299: 700-704Crossref PubMed Scopus (684) Google Scholar) proteins showed that in fact the cysteine in question (residue 247 in FRD and residue 257 in SQR) is some 7–8 Å from the active site. A recent report of the structure of porcine complex II reveals for the first time the overall architecture of the mitochondrial enzyme (24Sun F. Huo X. Zhai Y. Wang A. Xu J. Su D. Bartlam M. Rao Z. Cell. 2005; 121: 1043-1057Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar) at 2.4 Å resolution. The location of difference density in the substrate-binding site after 3-NP treatment was also reported; however, considering the lower resolution of that structure (3.5 Å), the specific model proposed for bound 3-NP has to be regarded as tentative. In any case the noncovalent binding described provides no explanation for the completely irreversible inactivation that is found with 3-NP. We recently developed a method for reproducible crystallization of mitochondrial Complex II from chicken (25Huang L.S. Borders T.M. Shen J.T. Wang C.J. Berry E.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 380-387Crossref PubMed Scopus (17) Google Scholar). We report here three structures of avian complex II: one treated with OAA, one treated with 3-NP, and one with no dicarboxylate site inhibitors but with the quinone site inhibitor carboxin. In the structure with added OAA, or in that with no added dicarboxylate ligand, the carboxylate site contains a malate-like ligand. The ligand and its surroundings are well ordered, allowing assignment of the residues involved in substrate binding and putative catalytic roles at this site. In particular, the structure confirms that Arg297 is positioned for the role of general acid-base catalyst abstracting a proton during conversion of succinate to fumarate, which has not been clearly seen in any of the membrane-bound SQR or FRD structures to date. In the structure of 3-NP-treated protein, the density for the ligand is quite different and can be modeled as a cyclic adduct of 3-NP with the catalytic Arg297. Although the chemistry involved has not yet been elucidated, we suppose that 3-nitroacrylate or some intermediate derived from it reacts with Arg297 in the active site to form a cyclic adduct such as obtained by treating arginine with 1,2- or 1,3-dicarbonyls (26Westwood M.E. Thornalley P.J. J. Protein Chem. 1995; 14: 359-372Crossref PubMed Scopus (197) Google Scholar, 27Toi K. Bynum E. Norris E. Itano H.A. J. Biol. Chem. 1965; 240: 3455-3457Abstract Full Text PDF Google Scholar, 28Toi K. Bynum E. Norris E. Itano H.A. J. Biol. Chem. 1967; 242: 1036-1043Abstract Full Text PDF PubMed Google Scholar, 29Gilbert III, H.F. O'Leary M.H. Biochemistry. 1975; 14: 5194-5199Crossref Scopus (55) Google Scholar, 30Oya T. Hattori N. Mizuno Y. Miyata S. Maeda S. Osawa T. Uchida K. J. Biol. Chem. 1999; 274: 18492-18502Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Purification, crystallization, and phasing of the avian complex II protein were described in a preliminary report (25Huang L.S. Borders T.M. Shen J.T. Wang C.J. Berry E.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 380-387Crossref PubMed Scopus (17) Google Scholar). As described, either one of two different crystal forms were obtained depending on conditions we have not yet determined. Type 1 crystals are orthorhombic with a monomer in the asymmetric unit (the same crystal form was reported (24Sun F. Huo X. Zhai Y. Wang A. Xu J. Su D. Bartlam M. Rao Z. Cell. 2005; 121: 1043-1057Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar) for the porcine enzyme), whereas type 2 crystals are monoclinic and contain two monomers in the asymetric unit. OAA and 3-NP were added to separate batches of the final purified protein in 2-fold molar excess before adding precipitant and additives, yielding type 1 crystals from both. Carboxin was soaked into a type 2 crystal by adding 0.5 μl of a 25 mm solution to a drop (initially set up with 15 μl each of protein solution and precipitant and supplemented with 1 μl of 15 mm MnCl2, 47.5% polyethylene glycol as described (25Huang L.S. Borders T.M. Shen J.T. Wang C.J. Berry E.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 380-387Crossref PubMed Scopus (17) Google Scholar)) after crystal growth was complete. The data were collected at the Advanced Light Source (Berkeley, CA) and the Stanford Synchrotron Radiation Laboratory (Stanford, CA). The data were processed using denzo and scalepack (31Otwinowski Z. Minor W. Carter J. Sweet R.M. Macromolecular Crystallography, Part A, Vol. 276. Academic Press, New York1997: 307-326Google Scholar). Other crystallographic calculations utilized the CCP4 suite (32COLLABORATIVE COMPUTATIONAL PROJECT N. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19762) Google Scholar) including molecular replacement by amore (33Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar). The initial model was built in a type 1 crystal by the ARP/wARP program (34Lamzin V.S. Perrakis A. Wilson K.S. Rossmann M.G. Arnold E. International Tables for Crystallography, Vol. F. Kluwer Academic Publishers, Dordrecht, The Netherlands2001: 720-722Google Scholar) using phases from the molecular replacement model (after considerable manual rebuilding) and was refined for each crystal by many cycles of automated refinement with CNS 1.1 (35Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16965) Google Scholar) alternating with manual inspection and rebuilding using the molecular graphics program O (36Jones T.A. Zhou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13010) Google Scholar). The monoclinic type 2 crystals were solved by molecular replacement using the structure from the type 1 crystals. The two molecules in the asymmetric unit are related by a 2-fold axis perpendicular to the crystallographic screw axis, resulting in pseudo-orthorhombic symmetry broken only by a slight screw component (0–6 Å) along the noncrystallographic 2-fold. The Protein Data Bank entry codes and final refinement statistics for the three structures presented here are shown in Table 2, with more detailed statistics in the on-line supplemental materials (part S1). Figs. 1, 2, 4, and 6 were made with molscript and raster3d. The electron density maps in Figs. 2, 4, and 6 were made using CCP4 programs, calculating structure factors from the model with sfall, scaling Fobs to Fcalc with rstats, and calculating the 2Fo - Fc maps with fft.TABLE 2Key refinement statistics for three Complex II structures used in this work Additional statistics from the data processing and refinement work are available in the supplemental materials.1YQ31YQ42FBWAdded ligandOAA3-NPCarboxinSpace groupP212121P212121P21Cell parameters70.0 × 84.4 × 289.569.6 × 83.5 × 288.6118.7 × 200.8 × 67.6, 90.0 90.0 90.0Resolution range38.66–2.2056.38–2.3364.09–2.10Last shell2.24–2.192.33–2.382.10–2.15Completeness89.2% (47.1%)93.9% (80.2%)88.2% (54.8%)No. of Reflections78719 (2623)68501 (3672)162208 (6675)Crystallographic R value0.176 (0.267)0.205 (0.32)0.187 (0.28)Free R value0.225 (0.313)0.259 (0.38)0.228 (0.31)B valuesFrom Wilson Plot34.4 Å230.9 Å211.9 Å2Mean atomic B Value47.8 Å247.1 Å231.3 Å2Root mean square deviations from idealityBond lengths0.022 Å0.019 Å0.032 ÅBond angles1.8°1.9°2.0°Dihedral angles22.4°22.3°22.8°Improper angles1.02°1.08°1.15° Open table in a new tab FIGURE 2The dicarboxylate site and its ligand in the untreated enzyme. The occupant is nonplanar, suggesting that if it is oxaloacetate, it is highly strained. Note the H-bond between C-2 of the ligand and catalytic base Arg297, and the close approach of C-3 to the flavin N-5 atom (blue dotted line). Essentially the same arrangement was seen when crystallization was carried out in the presence of excess OAA. The density is a 2Fo - Fc map contoured at 1.7 σ.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4The dicarboxylate site in the 3-NP-treated enzyme. The density attributed to the ligand has shrunk and moved away from the flavin toward Arg297 and His253. It can be modeled by assuming two atoms from the backbone of 3-NP (C-3 and N) fuse with the guanidino group to form a five-membered ring, with loss of the two nitro oxygens. The carboxylate at the other end fits into the clearly forked density branching off the ring. 2Fo - Fc map contoured at 1.7 σ.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Binding mode of carboxin and a H-bonded network involving heme propionate and residues from three subunits. The carbonyl oxygen of the inhibitor is H-bonded to Tyr58 of chain D and Trp173 of chain B. Chain D Tyr58 is also H-bonded to chain C Arg43, which makes H-bonds with a heme propionate and with chain D Asp57. The latter H-bonds chain B His216. The density is a 2Fo - Fc map contoured at 1.8 σ.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For mass spectral analysis, a sample of purified complex II (in 20 mm Tris-HCl, pH 7.5, 0.5 mm EDTA, 0.1 g/liter dodecyl maltoside) was concentrated to 0.9 mm, treated with 3-NPA at a 3 mm final concentration (from a 0.1 m stock solution in ethanol), and incubated for 4 days at 4 °C. Another sample was analyzed without treatment. The samples were electrophoresed on parallel lanes of a Tricine-SDS-PAGE gel as described previously (37Sun G. Kinter M.T. Anderson V.E. J. Mass Spectrom. 2003; 38: 531-539Crossref PubMed Scopus (17) Google Scholar). Following electrophoresis and Coomassie staining, the bands containing control and 3-NP-treated complex II subunits were sliced from the gel, and tryptic in-gel digestions were performed after reducing and alkylating cysteine (37Sun G. Kinter M.T. Anderson V.E. J. Mass Spectrom. 2003; 38: 531-539Crossref PubMed Scopus (17) Google Scholar). The tryptic peptides were extracted with 50% acetonitrile, 5% formic acid and analyzed by liquid chromatography/tandem MS using a Finnigan LTQ linear ion trap MS system, coupled with a nano-flow capillary high pressure liquid chromatography column (100× 0.18 mm; Biobasic-18, Thermoelectron) and a 10-μm-inner diameter PicoTip nanospray emitter (New Objective, Woburn, MA). After dilution with 0.1% formic acid, the tryptic peptides were chromatographed with a gradient of 0–80% CH3CN-0.1% formic acid at 500 nl/min with the ion source operated at 1.9 kV. The digest was analyzed by data-dependent acquisition of full scan mass spectra and tandem MS scans for the most abundant ion. The data obtained were processed by searching for modified residues in the tandem mass spectra against the chicken complex II sequence using TurboSequest. Further interpretation of the tandem MS spectrum of the modified peptide DLASR*DVVSR was performed manually with the aid of the web-based program MS-Product (www.prospector.ucsf.edu/ucsfhtml4.0/msprod.htm). The overall structure of the mitochondrial protein has been described for the porcine complex (24Sun F. Huo X. Zhai Y. Wang A. Xu J. Su D. Bartlam M. Rao Z. Cell. 2005; 121: 1043-1057Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar) and is generally confirmed by the present higher resolution structure of the avian complex (Fig. 1). Particularly noticeable is the packing of the N-terminal helix of chain C with the IP, which is not seen in the bacterial structures. The overall folds of the porcine and chicken enzymes are essentially identical, as expected from the phylogenetic proximity of these organisms. Comparing the porcine structure 1ZOY with a chicken structure in the same crystal form, the 1089 residues that were modeled in both structures can be superimposed with a root mean square deviation of 0.71 Å. Major differences are at the N and C termini; a loop around A259, the distal part (around A568) of a free floating loop of the flavoprotein between the helical domain and the C-terminal domain, and the transverse helix of chain C (comprising residues 66–79 in the chicken sequence). Excluding 17 residues with greatest differences gave a root mean square deviation of 0.57 Å, decreasing to 0.45, 0.33, 0.57, and 0.42 Å when the individual subunits were superimposed separately. Comparing the same 1072 residues in the porcine structure with the 3-NP-treated structure 1YQ4 and the two monomers of the carboxin-loaded structure 2FBW gave root mean square deviations of 0.60, 0.90, and 0.83 Å. In contrast to the backbone, a large number of side chains are clearly different than modeled in the porcine structure. This includes key residues of the dicarboxylate-binding site. Fig. 2 is a stereo view of this site in a crystal (Protein Data Bank entry 2FBW) to which no inhibitor of this site had been added. The same result was obtained when stoichiometrically excess OAA was present during crystallization (Protein Data Bank entry 1YQ3). The four-carbon backbone of the ligand, and the oxygens of the C-4 carboxylate and C-2 keto group, lie in a plane which is nearly parallel to the flavin isoalloxazine ring and in van der Waals’ contact with it. The C-1 carboxylate moiety (adjacent to the keto group), which extends beyond the edge of the ring, is rotated about 60° out of the plane of the rest of the molecule. In this conformation the resonance stabilization due to conjugation between the vicinal keto and carboxylate groups of OAA would be interrupted. The significance of this conformation and the specific identity of the ligand in the dicarboxylate site will be considered below. In any case superimposing our structure with fumarate-treated flavocytochrome c (FCc) from Shewanella (Protein Data Bank entry 1QJD) shows the ligand to be identical in conformation and orientation to the “malate-like intermediate” found in that structure. Furthermore all of the side chains in the active site superimpose well with those of 1QJD, and even though Met236 and Met375 are not conserved, the side chains of Phe130 and Leu263 that replace them occupy as nearly as possible the same space. These residues were proposed to provide steric constraints that together with the H-bonding pattern result in twisting the carboxylate of the substrate out of plane (38Taylor P. Pealing S.L. Reid G.A. Chapman S.K. Walkinshaw M.D. Nat. Struct. Biol. 1999; 6: 1108-1112Crossref PubMed Scopus (134) Google Scholar). That appears to be the case in Complex II as well. Each of the four carboxylate oxygens of the ligand makes two H-bonds with the protein, as illustrated schematically in Fig. 3. The ligand is correspondingly well ordered, with average B-factors of 38, 23, and 19 Å2 in 1YQ3 and the two monomers of 2FBW, well below the average for all atoms. This strong similarity with the relatively distantly related Shewanella FCc was unexpected, because previous SQR and FRD structures from pig, E. coli, and Wollinella are rather more different. The residue equivalent to Arg297 is believed from structural studies and site-directed mutagenesis to serve as a catalytic acid in the soluble FCc FRD (38Taylor P. Pealing S.L. Reid G.A. Chapman S.K. Walkinshaw M.D. Nat. Struct. Biol. 1999; 6: 1108-1112Crossref PubMed Scopus (134) Google Scholar, 39Doherty M.K. Pealing S.L. Miles C.S. Moysey R. Taylor P. Walkinshaw M.D. Reid G.A. Chapman S.K. Biochemistry. 2000; 39: 10695-10701Crossref PubMed Scopus (62) Google Scholar), donating a proton to one end of the double bond while a hydride is transferred from flavin to the other end. Assuming that succinate oxidation in SQR occurs by the reverse of this mechanism, Arg297 should act as a general base catalyst to abstract a proton from one of the central carbons, whereas a hydride is transferred from the other to the flavin. In fact Arg297 is well positioned to abstract a proton, if it is assumed that succinate and fumarate bind as does the ligand in the 1YQ3 structure, in which a terminal nitrogen atom of Arg297 is ∼3.0 Å from C-3 of OAA. Furthermore the other two nitrogens of the guanidino group H-bond to carboxylates, which would tend to make Arg297 a stronger base; NH1 binds to the substrate carboxylate oxygen O-1, whereas N∈ binds the carboxylate of conserved Glu266. A third H-bond to Gln251, together with that to Glu266, serves to position the guanidino group. At the same time, C-2 of OAA is about 3.1 Å from N-5 of the flavin moiety (Fig. 2, blue dotted lines). This presumably represents the path of the hydride transfer. If in fact the same intermediate is obtained starting with fumarate (as in structure 1QJD) or with OAA (as in the present studies), it suggests that SQR can carry out the Kreb’s cycle reactions normally catalyzed by fumarase and malate dehydrogenase, although perhaps at very low rate. It is known that malate can be oxidized to OAA by FRD or SQR (40Dervartanian D.V. Veeger C. Biochim. Biophys. Acta. 1965; 105: 424-436Crossref PubMed Google Scholar, 41Belikova Y.O. Kotlyar A.B. Vinogradov A.D. Biochim. Biophys. Acta. 1988; 936: 1-9Crossref PubMed Scopus (18) Google Scholar), and there is evidence that OAA is bound as the enol tautomer (42Panchenko M.V. Vinogradov A.D. FEBS Lett. 1991; 286: 76-78Crossref PubMed Scopus (11) Google Scholar), with the double bond between C-2 and C-3, rather than the keto form, which is more stable in aqueous solution. The geometry about C-2 (the oxygen-bearing central carbon) is nearly planar rather than tetrahedral, which suggests sp2 hybridization. This would be consistent with either tautomer of OAA but would imply that the C-1 carboxylate, which is about 60° out of plane of the C-2 center, is in a strained conformation; resonance stabilization would favor an in-plane conformation. The unsaturated “malate-like intermediate” proposed for the ligand in 1QJD (Fig. 3b of Ref. 38Taylor P. Pealing S.L. Reid G.A. Chapman S.K. Walkinshaw M.D. Nat. Struct. Biol. 1999; 6: 1108-1112Crossref PubMed Scopus (134) Google Scholar) would allow the out-of-plane C-1 carboxylate, because the double bond between C-3 and C-4 is not conjugated with the carbonyl of C-1. This would however result in sp3 hybridization for C-2. C-2 in the ligand of 1QJD in fact has (R) chirality as reported (38Taylor P. Pealing S.L. Reid G.A. Chapman S.K. Walkinshaw M.D. Nat. Struct. Biol. 1999; 6: 1108-1112Crossref PubMed Scopus (134) Google Scholar); however, the improper angle is only 11°, which is more nearly planar than tetrahedral. Regardless of the nature of the dicarboxylate in the active site of the OAA-inhibited enzyme, acid denaturation (43Schroder I. Gunsalus R.P. Ackrell B.A. Cochran B. Cecchini G. J. Biol. Chem. 1991; 266: 13572-13579Abstract Full Text PDF PubMed Google Scholar) or reductive activation (44Ackrell B.A. Kearn