Structure of the Yeast Cytochrome bc 1 Complex with a Hydroxyquinone Anion Qo Site Inhibitor Bound

Bifurcated electron transfer during ubiquinol oxidation is the key reaction of cytochrome bc 1 complex catalysis. Binding of the competitive inhibitor 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole to the Qo site of the cytochrome bc 1 complex from Saccharomyces cerevisiae was analyzed by x-ray crystallography. This alkylhydroxydioxobenzothiazole is bound in its ionized form as evident from the crystal structure and confirmed by spectroscopic analysis, consistent with a measured pK a = 6.1 of the hydroxy group in detergent micelles. Stabilizing forces for the hydroxyquinone anion inhibitor include a polarized hydrogen bond to the iron-sulfur cluster ligand His181 and on-edge interactions via weak hydrogen bonds with cytochrome b residue Tyr279. The hydroxy group of the latter contributes to stabilization of the Rieske protein in the b-position by donating a hydrogen bond. The reported pH dependence of inhibition with lower efficacy at alkaline pH is attributed to the protonation state of His181 with a pK a of 7.5. Glu272, a proposed primary ligand and proton acceptor of ubiquinol, is not bound to the carbonyl group of the hydroxydioxobenzothiazole ring but is rotated out of the binding pocket toward the heme b L propionate A, to which it is hydrogen-bonded via a single water molecule. The observed hydrogen bonding pattern provides experimental evidence for the previously proposed proton exit pathway involving the heme propionate and a chain of water molecules. Binding of the alkyl-6-hydroxy-4,7-dioxobenzothiazole is discussed as resembling an intermediate step of ubiquinol oxidation, supporting a single occupancy model at the Qo site. Bifurcated electron transfer during ubiquinol oxidation is the key reaction of cytochrome bc 1 complex catalysis. Binding of the competitive inhibitor 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole to the Qo site of the cytochrome bc 1 complex from Saccharomyces cerevisiae was analyzed by x-ray crystallography. This alkylhydroxydioxobenzothiazole is bound in its ionized form as evident from the crystal structure and confirmed by spectroscopic analysis, consistent with a measured pK a = 6.1 of the hydroxy group in detergent micelles. Stabilizing forces for the hydroxyquinone anion inhibitor include a polarized hydrogen bond to the iron-sulfur cluster ligand His181 and on-edge interactions via weak hydrogen bonds with cytochrome b residue Tyr279. The hydroxy group of the latter contributes to stabilization of the Rieske protein in the b-position by donating a hydrogen bond. The reported pH dependence of inhibition with lower efficacy at alkaline pH is attributed to the protonation state of His181 with a pK a of 7.5. Glu272, a proposed primary ligand and proton acceptor of ubiquinol, is not bound to the carbonyl group of the hydroxydioxobenzothiazole ring but is rotated out of the binding pocket toward the heme b L propionate A, to which it is hydrogen-bonded via a single water molecule. The observed hydrogen bonding pattern provides experimental evidence for the previously proposed proton exit pathway involving the heme propionate and a chain of water molecules. Binding of the alkyl-6-hydroxy-4,7-dioxobenzothiazole is discussed as resembling an intermediate step of ubiquinol oxidation, supporting a single occupancy model at the Qo site. Ubiquinol:cytochrome c oxidoreductase (cytochrome bc 1 complex, EC 1.10.2.2 (bc 1 complex)) is a multisubunit membrane protein complex, which is one of the fundamental components of respiratory and photosynthetic electron transfer chains. The enzyme catalyzes electron transfer from ubiquinol to cytochrome c and couples this process to electrogenic translocation of protons across the membrane (1Brandt U. Trumpower B. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 165-197Crossref PubMed Scopus (295) Google Scholar, 2Berry E.A. Guergova K. Huang L.S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (399) Google Scholar). Each functional unit of the homodimeric complex consists of three catalytic subunits: cytochrome b with two b type hemes, cytochrome c 1 with one c type heme, and the Rieske protein containing a [2Fe-2S] cluster. Mitochondrial bc 1 complexes contain up to eight additional subunits. Structures of vertebrate and yeast bc 1 complexes were determined, providing a breakthrough in understanding the enzyme mechanism and structure-function relationships within the enzyme (3Xia D. Yu C.A. Kim H. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Science. 1997; 277: 60-66Crossref PubMed Scopus (876) Google Scholar, 4Zhang Z. Huang L. Shulmeister V.M. Chi Y.I. Kim K.K. Hung L.W. Crofts A.R. Berry E.A. Kim S.H. Nature. 1998; 392: 677-684Crossref PubMed Scopus (943) Google Scholar, 5Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Crossref PubMed Scopus (1071) Google Scholar, 6Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Struct. Fold Des. 2000; 8: 669-684Abstract Full Text Full Text PDF Scopus (516) Google Scholar, 7Lange C. Nett J.H. Trumpower B.L. Hunte C. EMBO J. 2001; 20: 6591-6600Crossref PubMed Scopus (366) Google Scholar). The 2.3-Å resolution structure from the yeast Saccharomyces cerevisiae has the highest resolution available so far. It allowed a detailed description of substrate and inhibitor binding sites, elucidating parts of the enzyme mechanism and suggesting pathways for proton transfer. The mechanism of the enzyme known as the protonmotive Q cycle (8Mitchell P. J. Theor. Biol. 1976; 62: 327-367Crossref PubMed Scopus (927) Google Scholar) involves separate catalytic sites for ubiquinol oxidation (Qo site) and ubiquinone reduction (Qi site). Protons are taken up from the matrix side when ubiquinone is reduced and released to the intermembrane side when ubiquinol is oxidized. The key reaction, ubiquinol oxidation, involves a bifurcated electron transfer. One electron is passed via the [2Fe-2S] cluster to heme c 1, subsequently reducing the substrate cytochrome c. The electron transfer to cytochrome c 1 involves a large scale domain movement of the extrinsic part of the Rieske protein (4Zhang Z. Huang L. Shulmeister V.M. Chi Y.I. Kim K.K. Hung L.W. Crofts A.R. Berry E.A. Kim S.H. Nature. 1998; 392: 677-684Crossref PubMed Scopus (943) Google Scholar). The second electron from ubiquinol is transferred via the low and the high potential b type hemes to ubiquinone. The resulting stable semiquinone is fully reduced after a second ubiquinol molecule is oxidized at the Qo site. Whereas the main features of catalysis are understood, the molecular mechanism of ubiquinol oxidation is not clear. Also, pathways for proton uptake and release are hypothetical (7Lange C. Nett J.H. Trumpower B.L. Hunte C. EMBO J. 2001; 20: 6591-6600Crossref PubMed Scopus (366) Google Scholar, 9Berry E.A. Zhang Z. Huang L.S. Kim S.H. Biochem. Soc. Trans. 1999; 27: 565-572Crossref PubMed Scopus (17) Google Scholar, 10Crofts A.R. Hong S. Ugulava N. Barquera B. Gennis R. Guergova K. Berry E.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10021-10026Crossref PubMed Scopus (151) Google Scholar). Several hypotheses have been proposed to explain the divergent transfer of electrons into thermodynamically different pathways (see Ref. 2Berry E.A. Guergova K. Huang L.S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (399) Google Scholar). The double occupancy model suggests synergistic interaction between two quinone molecules that occupy the Qo site simultaneously (11Ding H. Robertson D.E. Daldal F. Dutton P.L. Biochemistry. 1992; 31: 3144-3158Crossref PubMed Scopus (175) Google Scholar, 12Ding H. Moser C.C. Robertson D.E. Tokito M.K. Daldal F. Dutton P.L. Biochemistry. 1995; 34: 15979-15996Crossref PubMed Scopus (158) Google Scholar, 13Bartoschek S. Johansson M. Geierstanger B.H. Okun J.G. Lancaster C.R. Humpfer E. Yu L. Yu C.A. Griesinger C. Brandt U. J. Biol. Chem. 2001; 276: 35231-35234Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The protongated charge transfer mechanism proposes that the activation barrier is a function of the deprotonation of ubiquinol (14Brandt U. FEBS Lett. 1996; 387: 1-6Crossref PubMed Scopus (106) Google Scholar), but this mechanism is not supported by other kinetic studies (15Hong S. Ugulava N. Guergova K. Crofts A.R. J. Biol. Chem. 1999; 274: 33931-33944Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Single occupancy models include simultaneous as well as sequential electron transfer to the acceptors. For the latter, a proton-gated affinity change mechanism claims the presence of a relatively stable intermediate in the transition state with the rate-limiting step at the second electron transfer (16Link T.A. FEBS Lett. 1997; 412: 257-264Crossref PubMed Scopus (140) Google Scholar). Since a semiquinone radical has not been detected at the Qo site, this has been explained by an EPR silent anti-ferromagnetically coupled semiquinone-[2Fe-2S]reduced pair (16Link T.A. FEBS Lett. 1997; 412: 257-264Crossref PubMed Scopus (140) Google Scholar, 17Junemann S. Heathcote P. Rich P.R. J. Biol. Chem. 1998; 273: 21603-21607Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Another explanation for the undetectable semiquinone is provided by Crofts et al. (10Crofts A.R. Hong S. Ugulava N. Barquera B. Gennis R. Guergova K. Berry E.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10021-10026Crossref PubMed Scopus (151) Google Scholar, 18Crofts A.R. Barquera B. Gennis R.B. Kuras R. Guergova K. Berry E.A. Biochemistry. 1999; 38: 15807-15826Crossref PubMed Scopus (153) Google Scholar), who suggest rapid dissociation of the product after the first electron transfer and movement of the semiquinone within the bilobal Qo binding pocket to allow rapid reduction of heme b L. Recent kinetic data show that the midpoint potentials of b type hemes control the rate of cytochrome c 1 reduction. This is consistent with the view that ubiquinol oxidation is a concerted reaction (19Snyder C.H. Gutierrez C. Trumpower B.L. J. Biol. Chem. 2000; 275: 13535-13541Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Inhibitors are important tools to analyze the molecular mechanism of Qo site catalysis. Three types of inhibitors can be distinguished: ligands binding at the proximal domain and therefore perturbing the spectroscopic properties of heme b L (Qo-I; e.g. myxothiazol and MOA-stilbene), those binding to the distal domain and affecting the Rieske [2Fe-2S] EPR line shape (Qo-II; e.g. UHDBT), 1The abbreviations used are: UHDBT, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole; HHDBT, 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole; THDBT, 5-n-tridecyl-6-hydroxy-4,7-dioxobenzothiazole; UM, n-undecyl-β-d-maltopyranoside; UQ6, coenzyme Q6; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid. or compounds exhibiting both effects (Qo-III; e.g. stigmatellin) (20Kim H. Xia D. Yu C.A. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8026-8033Crossref PubMed Scopus (259) Google Scholar). Kinetic data indicate that occupation of these inhibitors at the Qo site is mutually exclusive, suggesting overlapping binding sites. This was observed in crystal structures where these inhibitors are found to bind in different but overlapping domains of the bilobal Qo site, termed distal and proximal to heme b L (20Kim H. Xia D. Yu C.A. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8026-8033Crossref PubMed Scopus (259) Google Scholar). Analysis of anomalous scattering data indicated a high occupancy of the catalytic Rieske domain in the b-position in the presence of inhibitors that bind to the distal domain, such as stigmatellin and UHDBT (20Kim H. Xia D. Yu C.A. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8026-8033Crossref PubMed Scopus (259) Google Scholar). However, in previous crystallographic studies on UHDBT binding (18Crofts A.R. Barquera B. Gennis R.B. Kuras R. Guergova K. Berry E.A. Biochemistry. 1999; 38: 15807-15826Crossref PubMed Scopus (153) Google Scholar, 20Kim H. Xia D. Yu C.A. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8026-8033Crossref PubMed Scopus (259) Google Scholar), the inhibitor could not be refined, and high resolution structural information about UHDBT binding at the active site has not been available up to now. The substrate ubiquinol has not been detected in the Qo site by x-ray structural analysis. Therefore, the analysis of structural analogs of the substrate that function as competitive inhibitors is important. Here, we present the three-dimensional structure of a UHDBT analog, HHDBT-inhibited bc 1 complex from the yeast S. cerevisiae at 2.5-Å resolution. This hydroxyquinone binds in its ionized form, and its binding is discussed as resembling an intermediate step of ubiquinol oxidation. Conformational changes at the binding site confirm the previously postulated proton transfer pathway and reveal plasticity at the active site. Protein Purification and Crystallization—The bc 1 complex from the yeast S. cerevisiae was purified, and a co-complex with the antibody fragment Fv18E11 was formed and crystallized as previously described with the following minor modifications (6Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Struct. Fold Des. 2000; 8: 669-684Abstract Full Text Full Text PDF Scopus (516) Google Scholar, 21Palsdottir H. Hunte C. Hunte C. von Jagow G. Schagger H. Membrane Protein Purification and Crystallization: A Practical Guide. 2nd Ed. Academic Press, Inc., New York2003: 191-203Crossref Google Scholar). The buffer volume for detergent exchange in the second DEAE anion exchange chromatographic step was reduced by 5-fold. HHDBT was added at a final concentration of 100 μm to the purified bc 1 complex-Fv co-complex after size exclusion chromatography (TSK4000; TosoHaas) prior to crystallization. The final purification step was performed at pH 7.5. The crystals were obtained using a microseeding and vapor diffusion technique against polyethylene glycol 4000 at 4 °C. The protein solution (50 mg/ml) was mixed with precipitation agent (5% polyethylene glycol 4000, Tris-HCl pH 7.5 (adjusted at room temperature), 0.05% n-undecyl-β-d-maltopyranoside, 10 μm HHDBT), resulting in a pH of 8.0 at 4 °C; i.e. 0.5 pH unit lower than the structure of the stigmatellin-inhibited enzyme (6Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Struct. Fold Des. 2000; 8: 669-684Abstract Full Text Full Text PDF Scopus (516) Google Scholar). Crystals grew within a few days to a size suitable for x-ray analysis (∼0.5 × 0.5 × 1.0 mm). Total protein determination was performed with a modified Lowry procedure, using the BC Assay protein quantitation kit (Uptima) (22Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18708) Google Scholar). The bc 1 complex content was estimated as half the amount from spectroscopic quantification of the two b-type hemes using an extinction coefficient of 28.5 mm–1 cm–1 for the dithionite-reduced minus ferricyanide-oxidized difference spectra (262–275 nm). Enzyme activity was determined by monitoring cytochrome c reduction in a spectrophotometric assay at 550 nm using an extinction coefficient of 18.5 mm–1 cm–1 for cytochrome c. Turnover numbers refer to mol of cytochrome c reduced mol–1 of bc 1 complex s–1 under conditions of continuous turnover, where the catalytic reaction is zero order with respect to decyl ubiquinol and cytochrome c. A detailed description is reported elsewhere (21Palsdottir H. Hunte C. Hunte C. von Jagow G. Schagger H. Membrane Protein Purification and Crystallization: A Practical Guide. 2nd Ed. Academic Press, Inc., New York2003: 191-203Crossref Google Scholar). pK a Determination of Hydroxydioxobenzothiazoles—For determination of the pK a in detergent micelles, the longer tridecyl side chain analog was used to retain partitioning of the inhibitor into the micelle. THDBT was dissolved at 5 μm in a buffered mixture containing 20 mm MES, 20 mm MOPS, 20 mm TAPS, 100 mm NaCl, and 400 μm n-dodecyl-β-d-maltopyranoside, pH 3.5. The pH was adjusted by increments of 0.2–0.5 pH units from pH 3.5 to 8.5 by adding 5 m KOH. Optical spectra were recorded from 250–350 nm with a slit width of 1.5 nm on a computerized DW2a dual wavelength spectrophotometer controlled by OLIS software (On-Line Instruments Systems, Bogart, GA). Determination of the Apparent K m of Yeast bc 1 Complex for Ubiquinol in the Absence and Presence of UHDBT—The reaction mixture consisted of 25 μm cytochrome c in 50 mm potassium phosphate, pH 7, 250 mm sucrose, 1 mm sodium azide, 200 μm EDTA, and 0.01% Tween 20. Prior to the reaction, the complex was diluted to 15 μm in the same buffer and incubated for ∼45 min on ice. The concentration of decyl ubiquinol was varied, and the activity was measured in the absence or presence of UHDBT. Determination of the Ionization State of Bound THDBT—The bc 1 complex was diluted to a concentration of 2.84 μm in a buffer at pH 6.0 (50 mm MES, 50 mm MOPS, 50 mm TAPS, 250 mm sucrose, 200 μm EDTA, 2 mm NaN3, 0.1% Tween 20). The complex had an absorbance at 280 nm = 2.7 and a 280:414 absorbance ratio of 3. Spectra were recorded from 250 to 350 nm on the DW2a dual wavelength spectrophotometer. In order to obtain maximum illumination, the slit was set at 6 nm, and the UV filter, quartz diffuser and beam scrambler were removed from the spectrophotometer. Data Collection and Refinement—X-ray diffraction data were collected at 4 °C at the synchroton beamline ID14EH3 at the European Synchroton Radiation Facility (Grenoble, France), using a charge-coupled device detector (marCCD; Mar USA, Evanston, IL). Data were processed with the program DENZO and merged using SCALEPACK from the HKL package (HKL Research, Charlottesville, NC) (23Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38609) Google Scholar). The crystals belong to the space group C2, with unit cell parameters a = 215.0 Å, b = 165.1 Å, c = 147.5 Å, and β = 117.3°. The structure was refined using the coordinates of the stigmatellin-inhibited enzyme as a model (Protein Data Bank code 1KB9) after excluding all nonprotein molecules (7Lange C. Nett J.H. Trumpower B.L. Hunte C. EMBO J. 2001; 20: 6591-6600Crossref PubMed Scopus (366) Google Scholar). Energy minimization and B-factor refinement were performed using the CNS program package (version 1.0) (24Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse K. 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. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). The maximum likelihood function was used as the target for refinement. The model was improved based on F o – F c and 3F o – 2F c electron density maps, using the program O (version 8.0.4) (25Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Amino acid displacements were manually adjusted, followed by stepwise inclusion of the energy-minimized structure of the inhibitor HHDBT, UQ6, phospholipids, and UM and finally manually repositioning a displaced loop. Each step of model building was followed by a refinement cycle. Topology and parameter files were generated using the program Xplo2d and torsion data blocks prepared with the program Moleman2 (X-UTIL package; available on the World Wide Web at x-ray.bmc.uu.se/usf/) (26Kleywegt G.J. Jones T.A. Methods Enzymol. 1997; 277: 208-230Crossref PubMed Scopus (309) Google Scholar). The difference electron density map (F o – F c) indicated the presence of several additional phospholipids, manifested as elongated hairpin-like features. One phospholipid bound close to the Qo site was identified and refined in addition to the previously assigned phospholipids (7Lange C. Nett J.H. Trumpower B.L. Hunte C. EMBO J. 2001; 20: 6591-6600Crossref PubMed Scopus (366) Google Scholar). Finally, water molecules were included according to peaks observed in the F o – F c electron density map contoured at 3σ. Their positions were refined yielding 326 molecules of which 203 are the same as in the original model (1KB9), and their numbering was kept. New water molecules were numbered as starting from Wat500. Refinement resulted in final R factor and free R factor of 22.8 and 25.2%, respectively (Table I). Coordinates of the HHDBT-inhibited enzyme have been deposited in the Protein Data Bank data base (entry 1P84).Table IData collection and refinement statisticsParameterValueSpace groupC2a,b,c [Å]215.0 165.1 147.5β (degrees)117.3No of nonhydrogen atoms in the modelAtoms18,069Amino acid residues2169Nonprotein molecules13Solvent molecules326Data collectionResolution range (outer shell) [Å]25.0-2.50 (2.56-2.50)Measured reflections372,220 (19,194)Unique reflections149,103 (9128)Completeness (%)92.4 (84.6)R merge (%)aR merge = ΣhΣi|Ii (h) - 〈I(h)〉|/Σh Σi I i(h), where Ii (h) is intensity of ith measurement, 〈I(h)〉 is average intensity of a reflection.6.6 (37.3)I/σ(I) < 113.4 (1.2)RefinementResolution range (outer shell) (Å)25.0-2.50 (2.52-2.50)R factor (%)bR factor = Σh∥F(h)obs| - |F(h)calc ∥/Σh|F(h)|.22.8 (41.0)R free (%)cR free calculated for 2.5% of reflections.25.2 (41.1)B wilson [Å2]dB wilson was calculated using TRUNCATE, CCP4 package (53). PDB entry: 1P84.50.3Average B-factor72.0Root mean square deviations from ideal valuesBond lengths [Å]0.007Bond angles (degrees)1.298Ramachandran plot (non-Gly, non-Pro)Most favored regions (%)86.8Additional allowed (%)12.5Generously allowed (%)0.5Disallowed (%)0.2a R merge = ΣhΣi|Ii (h) - 〈I(h)〉|/Σh Σi I i(h), where Ii (h) is intensity of ith measurement, 〈I(h)〉 is average intensity of a reflection.b R factor = Σh∥F(h)obs| - |F(h)calc ∥/Σh|F(h)|.c R free calculated for 2.5% of reflections.d B wilson was calculated using TRUNCATE, CCP4 package (53Collaborative Computational ProjectActa Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). PDB entry: 1P84. Open table in a new tab For comparison, stigmatellin-inhibited bc 1 complex was crystallized at the same pH as the HHDBT-containing enzyme. The control data set was collected with 2.8-Å resolution, 93% completeness, and R sym of 5.8%. Refinement resulted in final R factor of 20.8% and R free of 24.5%. Lowering the pH by 0.5 units does not affect the structure of the catalytic subunits of the stigmatellin-inhibited enzyme, as judged by positional root mean square deviation (rmsd; Å) of superimposed atoms with LSQMAN yielding rmsdall/rmsdCα of 0.142/0.098, 0.175/0.118, and 0.204/0.133 for cytochrome b, cytochrome c 1, and the Rieske protein, respectively (Dejavuu package; available on the World Wide Web at x-ray.bmc.uu.se/usf/). Comparison between the HHDBT- and the stigmatellin-inhibited enzyme was therefore based on the recently published 2.3-Å resolution structure (1KB9) (6Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Struct. Fold Des. 2000; 8: 669-684Abstract Full Text Full Text PDF Scopus (516) Google Scholar). The structures were superimposed using the explicit least squares option in LSQMAN and inspected in the program O. By-residue analysis of root mean square deviation in Cα trace position and orientation was performed using the McLachlan algorithm as implemented in the program ProFit version 2.2 (available on the World Wide Web at bioinf.org.uk/software/profit). Analysis of neighboring atoms and hydrogen bond interactions was performed using the program HBPlus (27McDonald I.K. Thornton J.M. J. Mol. Biol. 1994; 238: 777-793Crossref PubMed Scopus (1885) Google Scholar) and contact analysis from CNS. Accessibility was estimated, and buried surface calculations were performed using the program NACCESS (28Hubbard S.J. Thornton J.M. Naccess, Version 2.1.1. Department of Biochemistry, University College, Londoncular Biology1993Google Scholar). PROCHECK (version 3.2) analysis verified the stereochemical quality of the coordinates (Table I) (29Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Hydrogen bonds were assigned according to appropriate distance and geometry. For analysis of weak hydrogen bonds, an estimation of hydrogen atom position was made by generating a structural model with hydrogens added using CNS (version 1.0). Criteria for identifying weak hydrogen bonds were extracted from Ref. 30Desiraju G.R. Steiner T. The Weak Hydrogen Bond. Oxford University Press, NY1999Google Scholar. Hydrogen bond angle is denoted as θ (X-H···A), and the bending angle at acceptor atom is shown as ϕ (H···A-C). Figures were prepared using the programs O (25Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar), LIGPLOT version 4.0 (31Wallace A.C. Laskowski R.A. Thornton J.M. Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4428) Google Scholar), MolScript version 1.4 (32Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), BobScript (33Esnouf R.M. Acta Crystallogr. Sect. D. 1999; 55: 938-940Crossref PubMed Scopus (850) Google Scholar), and Raster3D (34Merritt E.A. Murphy M.E.P. Acta Crystallogr. D. 1994; 50: 869-873Crossref PubMed Scopus (2859) Google Scholar). Crystallization of HHDBT-inhibited bc 1 Complex—The optimized purification of yeast bc 1 complex resulted in a pure and more active membrane protein complex with a higher turnover number of 82 s–1 compared with the previously reported activity of 64 s–1 (21Palsdottir H. Hunte C. Hunte C. von Jagow G. Schagger H. Membrane Protein Purification and Crystallization: A Practical Guide. 2nd Ed. Academic Press, Inc., New York2003: 191-203Crossref Google Scholar). The increase is most likely due to higher phospholipid content of the modified protein preparation (Fig. 1) (35Yu C.A. Yu L. Biochemistry. 1980; 19: 5715-5720Crossref PubMed Scopus (57) Google Scholar, 36Schagger H. Hagen T. Roth B. Brandt U. Link T.A. von J. Eur. J. Biochem. 1990; 190: 123-130Crossref PubMed Scopus (80) Google Scholar). Effective inhibition of the complex has been shown for 5-n-alkyl-6-hydroxy-4,7-dioxobenzothiazoles containing 7–15 carbon alkyl side chains (37Bowyer J.R. Edwards C.A. Ohnishi T. Trumpower B.L. J. Biol. Chem. 1982; 257: 8321-8330Abstract Full Text PDF PubMed Google Scholar). Here, the shorter heptyl side chain analog of UHDBT was used for crystallization in order to avoid nonspecific binding that might occur with the longer alkyl side chains at the high concentrations used. The inhibitory efficacy of UHDBT was shown to depend on the oxidation-reduction poise of the catalytic subunits, demonstrated by enhanced binding when the Rieske protein is reduced (37Bowyer J.R. Edwards C.A. Ohnishi T. Trumpower B.L. J. Biol. Chem. 1982; 257: 8321-8330Abstract Full Text PDF PubMed Google Scholar). The purified bc 1 complex used in this study has a partially reduced Rieske and is fully inhibited by the applied amount of HHDBT (results not shown). A pK a of 6.5 has been determined for the weakly acidic hydroxy group of UHDBT, which was measured in phosphate buffer containing 1% ethanol, and deprotonation of the hydroxy group is manifested by a color change from yellow to rose-violet (38Trumpower B.L. Haggerty J.G. J. Bioenerg. Biomembr. 1980; 12: 151-164Crossref PubMed Scopus (53) Google Scholar). Here, the complex was crystallized at pH 8.0 as a protein-detergent complex; therefore, the acidity of the hydroxy group was measured in detergent micelles by monitoring the blue shift in the optical spectrum upon ionization. The pK a determined by spectrophotometric titration in detergent micelles was 6.1 (results not shown). This suggests that 98% of the inhibitor is ionized in the crystallization mixture, demonstrated by the violet color of the solution and finally a purple tint of the crystals. A difference spectrum of bc 1 complex with bound inhibitor versus the complex alone shows that the inhibitor is ionized when bound to the enzyme, when the pH of the buffer is close to the pK a of the unbound inhibitor (Fig. 2). The difference spectrum of the complex at pH 6.0 with inhibitor bound at a substoichiometric amount is similar to that of the inhibitor alone at pH 8.7. As inhibitor is added in molar excess, the mixture consists of bound and unbound inhibitor, and the absorbance maximum shifts to longer wavelengths. Analysis of HHDBT Binding at the Q o Site—The difference electron density map (F o – F c) calculated prior to inclusion of HHDBT clearly showed the localization of the bound inhibitor at the Qo site (Figs. 1 and 3). The clear cut and asymmetric form of the difference density for the head moiety allowed unambiguous orientation of the hydroxydioxobenzothiazole ring. Furthermore, the position of the alkyl side chain was defined in the full length. The inhibitor binds in the distal domain of the Qo site, located between the two electron acceptors of ubiquinol oxidation, namely heme b L and the [2Fe-2S] cluster of the Rieske protein. The catalytic domain of the latter is docked onto cytochrome b (i.e. in the b-position). The hydroxydioxobenzothiazole head group is stabilized by a network of weak and strong hydrogen bonds and numerous van der Waals interactions with neighboring residues (Table II). Importantly, the oxygen atom of the ionized 6-hydroxy group (O6) is in close contact with the