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• W2091220416 abstract "In cellular respiration, cytochrome c transfers electrons from cytochrome bc1 complex (complex III) to cytochrome c oxidase by transiently binding to the membrane proteins. Here, we report the structure of isoform-1 cytochrome c bound to cytochrome bc1 complex at 1.9Å resolution in reduced state. The dimer structure is asymmetric. Monovalent cytochrome c binding is correlated with conformational changes of the Rieske head domain and subunit QCR6p and with a higher number of interfacial water molecules bound to cytochrome c1. Pronounced hydration and a “mobility mismatch” at the interface with disordered charged residues on the cytochrome c side are favorable for transient binding. Within the hydrophobic interface, a minimal core was identified by comparison with the novel structure of the complex with bound isoform-2 cytochrome c. Four core interactions encircle the heme cofactors surrounded by variable interactions. The core interface may be a feature to gain specificity for formation of the reactive complex. In cellular respiration, cytochrome c transfers electrons from cytochrome bc1 complex (complex III) to cytochrome c oxidase by transiently binding to the membrane proteins. Here, we report the structure of isoform-1 cytochrome c bound to cytochrome bc1 complex at 1.9Å resolution in reduced state. The dimer structure is asymmetric. Monovalent cytochrome c binding is correlated with conformational changes of the Rieske head domain and subunit QCR6p and with a higher number of interfacial water molecules bound to cytochrome c1. Pronounced hydration and a “mobility mismatch” at the interface with disordered charged residues on the cytochrome c side are favorable for transient binding. Within the hydrophobic interface, a minimal core was identified by comparison with the novel structure of the complex with bound isoform-2 cytochrome c. Four core interactions encircle the heme cofactors surrounded by variable interactions. The core interface may be a feature to gain specificity for formation of the reactive complex. Electron transfer processes are essential for all living organisms. Most energy equivalents in eukaryotic cells are generated by the mitochondrial respiratory chain. In cellular respiration, the soluble protein cytochrome c (cyt c) 3The abbreviations used are: cyt c, cytochrome c; iso-1, isoform 1; M3L, trimethyllysine. transports electrons from the cytochrome bc1 complex (cyt bc1) to cytochrome c oxidase (1Saraste M. Science. 1999; 283: 1488-1493Crossref PubMed Scopus (1030) Google Scholar). The interaction of cyt bc1 and cyt c is transient and amazingly efficient, enabling turnover rates higher than 100/s (2Hunte C. Solmaz S. Lange C. Biochim. Biophys. Acta-Bioenerg. 2002; 1555: 21-28Crossref PubMed Scopus (47) Google Scholar, 3Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (312) Google Scholar). The mitochondrial cyt bc1 is a homodimeric multisubunit integral membrane protein complex with a molecular mass close to 500 kDa. The enzyme catalyzes the electron transfer from ubiquinol to cyt c coupled to the net translocation of protons over the membrane (4Berry E.A. Guergova-Kuras M. Huang L.S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (397) Google Scholar). A key feature of the mechanism is the large scale domain movement of the Rieske protein by 20 Å, which facilitates electron transfer from oxidation of ubiquinol at center P to subunit cyt c1 (5Zhang Z.L. Huang L.S. 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 (939) Google Scholar). Cyt c docks onto the latter subunit and takes up the electron. An x-ray structure of yeast cyt bc1 with cyt c and an antibody fragment bound has been previously determined at 2.97 Å resolution (3Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (312) Google Scholar). A single cyt c molecule is bound to the homodimeric complex. Direct and specific interactions of the electron transfer complex visualized in the x-ray structure are mediated by non-polar forces and a cation-π interaction with charged residues positioned peripherally to the interface. These interactions appear to be the dominant features of transient electron transfer complexes and are also observed for the interface of the yeast cyt c peroxidase·cyt c (6Pelletier H. Kraut J. Science. 1992; 258: 1748-1755Crossref PubMed Scopus (712) Google Scholar) and the bacterial reaction center·cyt c2 complexes (7Axelrod H.L. Abresch E.C. Okamura M.Y. Yeh A.P. Rees D.C. Feher G. J. Mol. Biol. 2002; 319: 501-515Crossref PubMed Scopus (130) Google Scholar). In general, a two-step model for formation of transient electron transfer complexes is today strongly supported. A short-lived, dynamic encounter complex steered by long-range electrostatic interactions precedes a dominant well defined bound state based mainly on non-polar interactions (8Ubbink M. Ejdebäck M. Karlsson B.G. Bendall D.S. Structure. 1998; 6: 323-335Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 9Volkov A.N. Worrall J.A.R. Holtzmann E. Ubbink M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 18945-18950Crossref PubMed Scopus (225) Google Scholar). However, electron transport proteins such as cyt c do have several structurally unrelated reaction partners, and it is not understood how the high degree of specificity for the reaction partners is achieved in combination with the necessary weak binding and whether redox state-dependent alterations are required to facilitate association and dissociation processes. No large structural changes are known to explain these requirements. These features most likely have their molecular causes in subtle structural differences, including specific contributions of water molecules. Here, the structure of yeast cyt bc1 with its substrate cyt c and an Fν fragment bound was determined at 1.9 Å resolution in reduced state. It is the highest resolution for a cyt bc1 structure so far, and it allows the accurate description of the complex interface, focusing especially on the role of electrostatic and water-mediated interactions as well as on differences related to the monovalent binding mode of cyt c. A second structure of cyt bc1 with isoform-2 cyt c was determined, and comparison of the structures resulted in the identification of common binding interactions, the core interface, important for specificity of binding. Preparation of cyt bc1·cyt c Crystals—The ternary complex of cyt bc1, Fν fragment, and isoform-1 (iso-1) cyt c was prepared as described in Ref. 3Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (312) Google Scholar with the following modifications. To reduce nuclei formation, sucrose was added (1 m) to the buffer of the cyt bc1·Fν fragment complex prior to concentrating the protein to 30 mg/ml. The derivatized yeast cyt c was added in a molar ratio of 2.6/cyt bc1 dimer. An ultracentrifugation step (40 min at 106,000 × g) was introduced. 2 μl of the ternary complex were mixed with 1 μl of precipitant in a microbatch setup under paraffin oil. The crystallization buffer was 1 m sucrose, 10% DMSO, 20 mm Tris, pH 7.5, 66 mm NaCl, 1.67% polyethylene glycol 4000, 0.05% n-undecyl-β-d-maltopyranoside, and 1 μm stigmatellin (final concentrations). Cyt bc1 is highly active in this buffer if the inhibitor stigmatellin is omitted. Crystal grew up to 0.4 mm within 6 weeks to 6 months at 4 °C. Crystals were soaked for 5 min in crystallization buffer with 1 mm ascorbate, 80 mm NaCl, and 5% polyethylene glycol 4000 prior to flash-freezing in liquid nitrogen. The reduced state of the c-type hemes was spectroscopically confirmed (supplemental Fig. S5A). Isoform-2 (iso-2) cyt c was prepared as described (2Hunte C. Solmaz S. Lange C. Biochim. Biophys. Acta-Bioenerg. 2002; 1555: 21-28Crossref PubMed Scopus (47) Google Scholar); the crystals were prepared as for iso-1 cyt c, but ascorbate was omitted. Structure Determination and Refinement—Diffraction data were collected from single crystals at 100 K at the European Synchrotron Facility (Grenoble, France) at beam line ID23EH1 for the high resolution structure (MarmosaicCCD Q225 detector) and at beam line ID14EH1 for the structure with iso-2 cyt c bound (ADSC CCD Q4 detector). Data were processed and scaled with XDS (10Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3233) Google Scholar). The structures were solved by molecular replacement as described in Ref. 3Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (312) Google Scholar. For iso-2 cyt c, Protein Data Bank accession code 1YEA (11Murphy M.E.P. Nall B.T. Brayer G.D. J. Mol. Biol. 1992; 227: 160-176Crossref PubMed Scopus (47) Google Scholar) was used as a starting model. The models were refined in CNS (12Brünger 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 (16967) Google Scholar) and by manual remodeling in program O (13Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar). The two monomers were refined independently without non-crystallographic symmetry restraints. The final statistics are listed in supplemental Table S1. Hydrogen bonds and non-bonded atom contacts were analyzed with the programs LIGPLOT (14Wallace A.C. Laskowski R.A. Thornton J.M. Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4368) Google Scholar) and CNS. All binding pairs were checked for well defined electron density. Least-squares superimpositions were performed in LSQMAN, ESCET (25Schneider T. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2269-2275Crossref PubMed Scopus (33) Google Scholar), or CNS as indicated. Root mean square deviations were calculated with CNS. For the crystal contact analysis, a model of cyt c bound at the empty binding site was created by superimposition of the two monomers via cyt b with the program O. Figures were created with MolScript (15Kraulis P. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), Raster3D (16Merritt E.A. Murphy M.E.P. Acta Cryst. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2857) Google Scholar), and PyMOL (42DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA2002Google Scholar). Structure of cyt bc1 with Bound cyt c in Reduced State at 1.9 Å Resolution—The electron transfer complex of cyt c bound to cyt bc1 from the yeast Saccharomyces cerevisiae was crystallized with the antibody fragment Fν18E11, and the structure was determined at 1.9 Å resolution (Rfree = 26.2%, Rcryst = 24.5%, see supplemental Table S1). Optimized crystallization conditions and cryo-cooling for data collection provided the basis for the improved quality as compared with the previously published structure of this complex at 2.97 Å resolution, for which data collection was carried out at 277 K (3Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (312) Google Scholar). In agreement with the initial structure, only one molecule cyt c is bound to the homodimeric cyt bc1. As a result of the higher resolution, 1648 water, 13 lipid, and 2 detergent molecules were included in the model and refined (Fig. 1). A mixed redox state has to be assumed for the initial structure, as the complex was used as isolated with cyt c1 reduction below 25% and oxidized cyt c (3Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (312) Google Scholar). For the high resolution structure, a defined redox state was achieved by soaking the crystals in ascorbate solution prior to flash cooling and data collection at 100 K. Ascorbate reduces cyt c and the high potential chain subunits cyt c1 and the Rieske protein (17Vanneste W.H. Biochim. Biophys. Acta. 1966; 113: 175-178Crossref PubMed Google Scholar, 18Yu C.A. Yu L. King T.E. J. Biol. Chem. 1974; 249: 4905-4910Abstract Full Text PDF PubMed Google Scholar). The reduced state of the crystals was spectroscopically confirmed (supplemental Fig. S5, A and B). The high resolution allows an accurate description of the complex interface present between cyt c and subunit cyt c1 (Fig. 2A, Table 1). At the center of the interface, heme c1 and heme c are in close contact, with 4.1 Å distance only between two carbon atoms in the respective thioether-bonded substituents of the tetrapyrrol rings. Nine pairs of interacting atoms surround the heme clefts separated by 3-4 Å, permitting van der Waals contacts and hydrophobic interactions (Table 1, Fig. 3, A and B). Four contacts including the cation-π interaction Phe-230/Arg-19 of cyt c1/cyt c are the same as in the 2.97 Å structure, for which only a total of five interacting pairs was observed (Table 1). An energy-based analysis with the program CaPTURE (19Gallivan J.P. Dougherty D.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9459-9464Crossref PubMed Scopus (1706) Google Scholar) indicates that the Phe-230/Arg-19 pair contributes a strong cation-π interaction with a calculated interaction energy of E = -7.07 kcal/mol. Residues Ala-103/Ala-87, Ala-103/M3L78 and Met-233/Gly-89 of cyt c1/cyt c mediate hydrophobic interactions with the respective carbon atoms in van der Waals distance (M3L: trimethyllysine). The contribution of the modified amino acid residue trimethyllysine (M3L78) of cyt c is noteworthy. In addition, Phe-230/Thr-18, Ala-168/Val-34, Gln-170/Gln-22, and Gln-170/Lys-33 of cyt c1/cyt c form van der Waals contacts.TABLE 1Interactions in the cyt bc1 · cyt c interface and heme-to-heme distances for three structures of the electron transfer complex with either iso-1 cyt c or iso-2 cyt ccyt c1cyt bc1·iso-1 cyt cac-type heme groups of the complex reduced (see “Experimental Procedures”).cyt bc1·iso-1 cyt cbRedox state of the complex as isolated.cyt bc1·iso-2 cyt cbRedox state of the complex as isolated.iso-1 cyt cd(Å)iso-1 cyt cd(Å)iso-2 cyt cd(Å)1.9 Å resolution3 Å resolutioncProtein Data Bank accession code 1KYO (3). Residues are numbered according to the yeast protein data base. Homologous residues of iso-1 cyt c are given in parentheses for iso-2 cyt c.2.5 Å resolutionCore interface (d ≤ 4 Å)Ala-103 CBAla-87 CB3.6Ala-87 CB3.4Ala-91 (87) CB3.8Ala-168 OVal-34 CG13.4Val-34 CG14.0Val-38 (34) CG13.5Ala-168 OVal-34 CG23.5Val-38 (34) CG23.8Phe-230 CZThr-18 O3.6Thr-18 O3.3Thr-22 (18) O3.7Phe-230 CE1Arg-19 NE3.3Arg-19 NE3.8Arg-23 (19) NH13.2Variable interactions (d ≤ 4 Å)Ala-103 CBM3L-78 CH34.0M3L-82 (78) CH33.9Met-233 CEGly-89 CA3.7Ala-93 (89) CB3.7Gln-170 NE2Gln-22 O3.8Gln-170 OE1Lys-33 CD3.7Met-233 CEArg-19 NH23.8Arg-23 (19) NH23.4Glu-99 OAla-93 (89) CB3.7Asp-232 OLys-96 (92) CB3.6Met-233 OLys-96 (92) NZ3.5Glu-201 OE1Lys-21 (17) NZ3.6Glu-201 OE2Lys-21 (17) NZ3.0Long-range electrostatic interactions (4-9.6 Å)Asp-200 OD1Lys-17 NZ#7.8Lys-21 (17) NZ8.2Glu-201 OE2Lys-17 NZ#3.3§Asp-231 OD1Arg-19 NH19.2Arg-19 NH18.8Asp-232 OD1Arg-19 NH19.3Glu-99 OE1Lys-92 NZ*6.8Lys-92 NZ*6.9Lys-96 (92) NZ5.3Asp-232 OD2Lys-92 NZ*9.1Glu-235 OE1*Lys-92 NZ*7.6Lys-92 NZ*3.0Lys-96 (92) NZ7.1Asp-231 OD1Lys-93 NZ*7.8Lys-97 (93) NZ*5.5Asp-232 OD1Lys-93 NZ*4.7Lys-93 NZ*7.7Lys-97 (93) NZ*4.1Glu-235 OE1*Lys-93 NZ*9.5Asp-232 OD1Lys-95 NZ*9.2Lys-99 (95) NZ*7.9Heme-to-heme distancesHeme c1 CBCHeme c CBC4.1Heme c CBC4.5Heme c CBC4.2Heme c1 FEHeme c FE17.4Heme c FE17.4Heme c FE17.4Edge-to-edge9.19.39.0a c-type heme groups of the complex reduced (see “Experimental Procedures”).b Redox state of the complex as isolated.c Protein Data Bank accession code 1KYO (3Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (312) Google Scholar). Residues are numbered according to the yeast protein data base. Homologous residues of iso-1 cyt c are given in parentheses for iso-2 cyt c. Open table in a new tab FIGURE 3The building blocks of the interface. The cyt c1 (A)-cyt c (B) interface is shown in open book view. The heme groups (black) are encircled by the core interface (orange area, interacting atoms as orange spheres). The variable interface (yellow) surrounds the latter. Residues mediating long-range electrostatic interactions (pink and blue for negative and positive charges, respectively) form a semicircle around the central hydrophobic contact site. C, the semitransparent surface representation of cyt c1 (gray) and cyt c (green) shows the position of these long-range electrostatic interactions in the high resolution structure (Table 1) as highlighted in blue-green and pink (positive and negative charges, respectively). Heme groups are shown in black. A high number of water molecules (cyan spheres) are bound at the interface of cyt c1 (D) but not of cyt c (E). Only two of these water molecules form hydrogen bonds to both cyt c1 and cyt c (green spheres). F, interface water molecules bound to cyt c1 colored in cyan and green (as in D) are superimposed with all surface water molecules that are present at the other cyt c1 with the non-occupied cyt c binding site (dark blue spheres). Surface water molecules up to a distance of 3.5 Å are included.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The interface is not built from modules of closely interacting residues (20Reichmann D. Rahat O. Albeck S. Meged R. Dym O. Schreiber G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 57-62Crossref PubMed Scopus (215) Google Scholar), but the individual contacts are dispersed over the small interface and are separated from each other by distances longer than 4 Å. Interacting residues close enough for hydrogen bonds or salt bridges were not observed in the interface. Water Molecules at the Interface—For the first time, water molecules at the cyt bc1·cyt c interface were analyzed. The ring of non-polar interactions around the heme clefts does not seal off the interface from the aqueous environment. The hydration of the interface was analyzed for residue pairs from cyt c1/cyt c with an interatom distance of less than 7 Å. 30 water molecules bind within this distance constraint to either surface by hydrogen bonds. The interface is well hydrated, and water molecules are either in hydrogen bond distance to cyt c or to cyt c1 (Fig. 2, A and B, and Fig. 3, D and E). Eight water molecules mediate a hydrogen bond either to cyt c1 or cyt c and are in van der Waals contact distance to atoms of the respective interaction partner. Two water molecules mediate an interaction by hydrogen bonds between the two interaction partners, although the interaction is weak due to the extended bond length: water molecule X1708 is bound to Thr-18 of cyt c (distance 2.9 Å) and to the backbone carbonyl oxygen atom of Arg-227 of cyt c1 (distance 3.2 Å); water molecule X1845 is bound to the main chain nitrogen atom of Ala-226 of cyt c1 (distance 3 Å) and the Gln-22 OE1 atom of cyt c (distance 3.3 Å). Interestingly, most of the water molecules are stabilized by interactions with cyt c1 and not with cyt c (Fig. 3, D and E). Extending the analysis of the interface to a cutoff distance of 10 Å between cyt c1 and cyt c showed that there are no interactions mediated by two water molecules. Considerable rearrangement of water molecules is observed upon binding of cyt c. The simultaneous presence of an occupied and a free cyt c binding site in the cyt bc1 dimer permitted the comparison of water molecule positions between the two sites in the same crystal structure (Fig. 3F). 9 water positions are the same in both sites. 5 and 10 water molecules are only present on the free and occupied surface, respectively. The latter include the two water molecules with hydrogen bonds to both cyt c1 and cyt c. Overall, more crystallographically defined water molecules are located on the surface of cyt c1 with bound cyt c. The Electrostatic Interactions Form a Semicircle around the Non-polar Interface—The non-polar interface is surrounded by oppositely charged residue pairs, which are not in close contact. For the analysis of long-range electrostatic interactions, a cutoff distance of 4-9.6 Å was chosen. The latter is known as Debye length between single-charged ions at an ionic strength of 100 mm (21Wedler G. Lehrbuch der Physikalischen Chemie. 5th Ed. Wiley-VCH, Weinheim, Germany2004Google Scholar). Above this distance, the charge of an ion is shielded by counter ions of the solvent. Eight oppositely charged residue pairs were identified in the structure; they are separated by distances of 4.7-9.2 Å (Table 1). Three of five of the identified cyt c residues have been shown by selective labeling to be important for binding of horse heart cyt c (homologous yeast residues are in parentheses): Lys13 (Arg-19), Lys-86 (Lys-92), and Lys-87 (Lys-93) (22Speck S.H. Ferguson-Miller S. Osheroff N. Margoliash E. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 155-159Crossref PubMed Scopus (123) Google Scholar). Whereas the selected residues from cyt c1 are overall well defined by the electron density map, most of the positively charged side chains of cyt c appear to be flexible, indicating that the long-range electrostatic interactions are weak (Table 1). Analysis of the hydration shell clearly showed that there are no electrostatic interactions mediated by one or two bridging water molecules. The charged residue pairs are clustered at one side of the interface and form a semicircle around it (Fig. 3, A-C). Structure of cyt bc1 with Isoform-2 cyt c Bound—Yeast cyt c exists in two isoforms that share 83% sequence identity (supplemental Fig. S6). Depending on growth conditions, yeast cells contain typically 95% iso-1 and 5% iso-2 (23Laz T.M. Pietras D.F. Sherman F. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4475-4479Crossref PubMed Scopus (39) Google Scholar). Both the high resolution and the previous structure (3Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (312) Google Scholar) contain iso-1 cyt c. To challenge the specificity of the binding interaction and the monovalent binding mode, we crystallized the complex of yeast cyt bc1 and iso-2 cyt c with the antibody fragment Fν18E11. The crystals were directly used for x-ray diffraction data collection at cryo conditions without any redox treatment, and the structure was determined at 2.5 Å resolution (Rfree = 25.6%, Rcryst = 22.5%, see supplemental Table S1). As for the complexes with iso-1 cyt c, only one molecule of iso-2 cyt c is bound. A slight shift within the error limits was observed for the position of iso-2 cyt c. The heme c1 to heme c distances of the electron transfer complex are nearly identical to the complexes with iso-1 cyt c bound. Six residue pairs of the non-polar interface are the same for iso-2 and iso-1 cyt c: Ala-103/Ala-91(87), Ala-103/M3L82(78), Ala168/Val38 (34), Phe-230/Thr-22(18), Phe-230/Arg-23(19), and Met-233/Ala-93(Gly-89) of cyt c1/iso-2 cyt c (iso-1 cyt c) (Table 1). This includes the cation-π interaction and the trimethyllysine of cyt c. Only one of these residues differs in iso-2; Ala-93 is substituted with glycine in iso-1 cyt c (Gly-89). Seven additional contacts, including two close (<4Å) electrostatic interactions between oppositely charged residue pairs (Glu-201/Lys-21) were identified; all involve residues that are conserved between the two isoforms. The coinciding monovalent binding mode and the highly similar interfaces could be enforced by protein-protein interactions between neighboring molecules in the crystal lattice. Therefore, crystal contact analysis was carried out using models in which cyt c binding to the second monomer was generated by superimposition. In the occupied binding site of the high resolution structure, atoms of His-45 from iso-1 cyt c are in contact with subunit QCR2p of the neighboring molecule. The strongest interaction is a polar hydrogen bond between His-45 NE2 of cyt c and Leu-368 OT2 of QCR2p with a distance of 2.9 Å. At the second binding site, the nearest atom of the neighboring molecule would be 7 Å apart from cyt c. If both sites were occupied, the two cyt c molecules would be separated by a minimal distance of 18 Å. In conclusion, no steric hindrance impedes the binding at the second binding site. Furthermore, His-49 of iso-2 cyt c (homologous to His-45 of iso-1 cyt c) does not mediate any crystal contacts. In the respective structure, the only contacts present are van der Waals contacts between Asn-66 from cyt c and the neighboring cyt bc1 at the occupied binding site. There is no indication for steric hindrance at the modeled (empty) binding site, but Asn-66 of cyt c could potentially form a polar hydrogen bond with Asp-366 of cyt bc1 that could stabilize cyt c at the empty binding site. Taken together, the contacts of cyt c to neighboring molecules in the crystal lattice are very weak and are mediated by different residues in the two structures. This strongly indicates that the interface, which has a larger contact area, dominates and specifies the binding interaction. The Core Interface Is Defined by Interactions Occurring in Three cyt bc1·cyt c Structures—Three structures of the electron transfer complex have now been determined, the structures with iso-1 cyt c at 1.9 Å and 2.97 Å resolution and with iso-2 cyt c. The structures differ in crystallization conditions, redox state, data collection temperature, and cyt c amino acid sequence. However, when comparing the respective interfaces (see Table 1), four interacting residue pairs of cyt c1/cyt c were identified in all three structures: Ala-103/Ala-87, Ala-168/Val-34, Phe-230/Arg-19, and Phe-230/Thr-18. These pairs are apparently important for the stabilization. They define an area around the heme cleft, which we term the core interface (Fig. 2B). Besides the core interface, the high resolution structure and the structure with iso-2 cyt c bound share two more interacting pairs (Ala-103/M3L78 and Met-233/Gly-89 of cyt c1/cyt c). The 2.97 Å resolution structure and the structure with iso-2 cyt c share one additional interaction (Met-233/Arg-19 of cyt c1/cyt c). Two of the variable interactions appear only in the high resolution structure, and five pairs appear only in the structure with iso-2 cyt c (Table 1). The core interface interactions (orange in Fig. 3, A and B) are all located in close vicinity to the heme clefts whereas the variable interactions are farther away from the hemes and surround the core interface (pale yellow in Fig. 3, A and B). The heme-to-heme distances are consistent between the three structures. The edge-to-edge distances of the porphyrin rings that govern the electron transfer rates range from 9.0 to 9.3 Å (Table 1). The resulting calculated electron transfer rates (3Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (312) Google Scholar, 24Page C.C. Moser C.C. Chen X.X. Dutton P.L. Nature. 1999; 402: 47-52Crossref PubMed Scopus (1516) Google Scholar) are very fast and in the range of 1.0*106 to 2.6*107 s-1, affected by subtle differences in edge-edge distance and packing density. A comparison of the long-range electrostatic interactions for the three complex structures shows that, with few exceptions, the same residues are involved (Table 1). Asp-231, Asp-232, Glu-99, and Glu-235 of cyt c1 and Lys-92 and Lys-93 of cyt c mediate long-range electrostatic interactions in all three structures. The other residue pairs mediate long-range electrostatic interactions in two out of three structures. Comparison of cyt bc1 Monomers with Free and Occupied cyt c Binding Sites Reveals Rigid Body Movement of the Rieske Protein Head Domain—The resolution of 1.9 Å and the independent refinement of the two monomers allowed a detailed comparison of monomer A and B with empty and occupied binding site, respectively. As previously observed (3Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (312) Google Scholar), the occupancy of the Qi site with ubiquinone was higher for the monomer with bound cyt c. However, the overall occupancy was too low at both binding sites to refine ubiquinone. No significant structural differences were observed for the structures of the two ubiquinone binding sites at center N. Also, the two binding sites for cyt c do not show significant structural differences, indicating that the cyt c binding site does not rearrange upon substrate binding. The B-factors, and thus also the coordinate error, showed very large differences for the different regions of the dimer, imposing limitations for assigning the significance of conformational changes. Thus, the monomers were compared with the program ESCET (25Schneider T. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2269-2275Crossref PubMed Scopus (33) Google Scholar), which uses the diffraction precision index DPI (26Cruickshank D. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 583-601Crossref PubMed Scopus (501) Google Scholar) that includes B-factors in the error model. For the analysis, a sigma cutoff of two was used. Three rigid bodies were identified (Fig. 4A). The major part of the monomer structure is equivalent between the two monomers and forms the blue rigid body. The second rigid body contains residues of subunit QCR6p (74-104 and 141-174, green in Fig. 4A) and is likely to have a different conformation. QCR6p shows overall increased per-residue root mean" @default.
• W2091220416 created "2016-06-24" @default.
• W2091220416 creator A5059146662 @default.
• W2091220416 creator A5069744756 @default.
• W2091220416 date "2008-06-01" @default.
• W2091220416 modified "2023-10-03" @default.
• W2091220416 title "Structure of Complex III with Bound Cytochrome c in Reduced State and Definition of a Minimal Core Interface for Electron Transfer" @default.
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