Functional Implications from an Unexpected Position of the 49-kDa Subunit of NADH:Ubiquinone Oxidoreductase
2003; Elsevier BV; Volume: 278; Issue: 31 Linguagem: Inglês
10.1074/jbc.m302713200
ISSN1083-351X
AutoresVolker Zickermann, Mihnea Bostina, Carola Hunte, Teresa Ruíz, Michael Radermacher, Ulrich Brandt,
Tópico(s)ATP Synthase and ATPases Research
ResumoMembrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the "catalytic core" conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer. Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the "catalytic core" conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer. Complex I (NADH:ubiquinone oxidoreductase) 1The abbreviations used are: complex I, NADH:ubiquinone oxidoreductase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TXPBS, Triton X-100 PBS.1The abbreviations used are: complex I, NADH:ubiquinone oxidoreductase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TXPBS, Triton X-100 PBS. is the major entry point for electrons into the respiratory chain. By linking redox chemistry to vectorial proton translocation, complex I converts up to 40% of the energy used in mitochondria to make ATP. The molecular mechanism of catalysis and its structural basis is not at all understood. Complex I from bovine heart mitochondria is composed of more than 40 different subunits adding to a molecular mass of almost 1000 kDa (1Walker J.E. Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (681) Google Scholar). The enzyme from the strictly aerobic yeast Yarrowia lipolytica is of similar size and has been established as a model system to study eucaryotic complex I employing yeast genetics (2Djafarzadeh R. Kerscher S. Zwicker K. Radermacher M. Lindahl M. Schägger H. Brandt U. Biochim. Biophys. Acta. 2000; 1459: 230-238Crossref PubMed Scopus (82) Google Scholar, 3Kerscher S. Dröse S. Zwicker K. Zickermann V. Brandt U. Biochim. Biophys. Acta-Bioenerg. 2002; 1555: 83-91Crossref PubMed Scopus (87) Google Scholar). A major obstacle to progress in understanding this extremely large and complicated enzyme complex has been the lack of detailed structural information. Resolutions in the 20–30 Å range have been obtained by electron microscopy of single particles from different organisms that show an L-shaped structure with a hydrophobic arm residing in the membrane and a peripheral arm protruding into the mitochondrial matrix space (2Djafarzadeh R. Kerscher S. Zwicker K. Radermacher M. Lindahl M. Schägger H. Brandt U. Biochim. Biophys. Acta. 2000; 1459: 230-238Crossref PubMed Scopus (82) Google Scholar, 4Hofhaus G. Weiss H. Leonard K. J. Mol. Biol. 1991; 221: 1027-1043Crossref PubMed Scopus (166) Google Scholar, 5Guenebaut V. Schlitt A. Weiss H. Leonard K. Friedrich T. J. Mol. Biol. 1998; 276: 105-112Crossref PubMed Scopus (204) Google Scholar, 6Grigorieff N. J. Mol. Biol. 1998; 277: 1033-1046Crossref PubMed Scopus (299) Google Scholar). Recently, projection maps from two-dimensional crystals of the bovine enzyme have been obtained at 13 Å resolution (7Sazanov L.A. Walker J.E. J. Mol. Biol. 2000; 392: 455-464Crossref Scopus (63) Google Scholar). Böttcher et al. (8Böttcher B. Scheide D. Hesterberg M. Nagel-Steger L. Friedrich T. J. Biol. Chem. 2002; 277: 17970-17977Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) have reported that the enzyme from Escherichia coli may change from an L shape to a horseshoe-like shape at zero ionic strength. The minimal form of the enzyme consists of 14 subunits that are found in both bacterial and mitochondrial complex I (9Friedrich T. Biochim. Biophys. Acta. 1998; 1364: 134-146Crossref PubMed Scopus (179) Google Scholar). In higher eucaryotes seven highly hydrophobic polypeptides are encoded by the mitochondrial genome. All redox prosthetic groups that have been identified so far (eight iron-sulfur clusters and one FMN) are associated with a total of seven hydrophilic and nuclear-coded polypeptides. This poses the problem of how the redox chemistry taking place in the peripheral arm can drive proton translocation across the membrane arm. Evidence from different laboratories (10Schuler F. Yano T. Di Bernardo S. Yagi T. Yankovskaya V. Singer T.P. Casida J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4149-4153Crossref PubMed Scopus (163) Google Scholar, 11Darrouzet E. Issartel J.P. Lunardi J. Dupuis A. FEBS Lett. 1998; 431: 34-38Crossref PubMed Scopus (109) Google Scholar) and the identification of several pathogenic mutations (12Smeitink J. Van den Heuvel L. DiMauro S. Nat. Rev. Genet. 2001; 2: 342-352Crossref PubMed Scopus (557) Google Scholar) suggested a key mechanistic role for the 49-kDa, PSST, and TYKY subunits. Based on these indications and a well established homology (13Böhm R. Sauter M. Böck A. Mol. Microbiol. 1990; 4: 231-243Crossref PubMed Scopus (245) Google Scholar) between the 49-kDa and PSST subunits of complex I and the large and small subunits of [NiFe] hydrogenases, we proposed that at least part of the ubiquinone binding pocket and possibly the proton translocation machinery of complex I have evolved from the domains surrounding the [NiFe] site of the hydrogenase (14Kashani-Poor N. Zwicker K. Kerscher S. Brandt U. J. Biol. Chem. 2001; 276: 24082-24087Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). This catalytic core hypothesis was substantiated experimentally by a series of site-directed mutations in the 49-kDa subunit of complex I from Y. lipolytica (3Kerscher S. Dröse S. Zwicker K. Zickermann V. Brandt U. Biochim. Biophys. Acta-Bioenerg. 2002; 1555: 83-91Crossref PubMed Scopus (87) Google Scholar). The same studies also provided further support for the previously demonstrated ligation of iron-sulfur cluster N2 by subunit PSST (15Rasmussen T. Scheide D. Brors B. Kintscher L. Weiss H. Friedrich T. Biochemistry. 2001; 40: 6124-6131Crossref PubMed Scopus (67) Google Scholar, 16Duarte M. Populo H. Videira A. Friedrich T. Schulte U. Biochem. J. 2002; 364: 833-839Crossref PubMed Scopus (41) Google Scholar), placing this redox center at the interface between the 49-kDa and PSST subunits close to the former [NiFe] binding domain. For further progress in understanding the function of complex I it is essential to localize these key subunits within the structure of this very large membrane protein complex. Monoclonal Antibodies—A female BALB/c mouse (Harlan/Winkelmann, Borchen, Germany) was immunized by intraperitoneal injection of complex I from Y. lipolytica using a long-term protocol. The initial immunization with 100 μl of protein suspension (100 μg of purified protein in 20% (v/v) Gerbu Adjuvant MM, Gerbu Biotechnik GmbH, Gaiberg, Germany) was followed by five injections of 50 μl of protein suspension (50 μg of protein in 20% of the same adjuvant) in 4-week intervals. The final injection was repeated the following day, and the mouse was sacrificed after 2 days for removal of the spleen. The subsequent cell fusion and cell culture work was done according to standard protocols (17Padan E. Venturi M. Michel H. Hunte C. FEBS Lett. 1998; 441: 53-58Crossref PubMed Scopus (41) Google Scholar). A specific immune response was detected in the serum up to a 105-fold dilution, and 4800 clones were obtained from 50% of the cells after cell fusion. The clones were tested by a standard ELISA procedure. For the approach presented here, it was critical to identify antibodies that recognized the native enzyme. Therefore, a His-tag-based ELISA (17Padan E. Venturi M. Michel H. Hunte C. FEBS Lett. 1998; 441: 53-58Crossref PubMed Scopus (41) Google Scholar) was established to avoid denaturing complex I upon binding to the plates under alkaline conditions and in the absence of detergent. In this native ELISA protocol, the antigen was bound to a nickel-nitrilotriacetic acid-coated support via its hexahistidine sequence (18Kashani-Poor N. Kerscher S. Zickermann V. Brandt U. Biochim. Biophys. Acta. 2001; 1504: 363-370Crossref PubMed Scopus (74) Google Scholar). All incubation and washing steps were done under mild conditions in the presence of detergent: His-tagged complex I was diluted to 15 μg/ml in coating buffer (50 mm potassium phosphate, pH 7.5, 300 mm NaCl, 0.1% laurylmaltoside) applied to Ni2+-NTA-coated ELISA plates (Qiagen) and incubated for 1 h at room temperature under gentle shaking. After washing (4 cycles, 20 s each) with 20 mm Tris/Cl, pH 7.5, 300 mm NaCl, 0.03% laurylmaltoside 0.2% bovine serum albumin, the cell culture supernatant was adjusted to 50 mm Tris/Cl, pH 8.0, 0.03% laurylmaltoside and incubated for 1 h as above. After washing, a secondary antibody (anti-mouse IgG conjugated to alkaline phosphatase; Sigma) was used for detection of bound antibodies as described in Ref. 17Padan E. Venturi M. Michel H. Hunte C. FEBS Lett. 1998; 441: 53-58Crossref PubMed Scopus (41) Google Scholar. About half of the cell lines that gave strong signals after single cell cloning turned out to be of the IgM subtype; others were mainly IgG1. The majority of cell culture supernatants was Western blot-positive, and no cross reactivity with bovine complex I could be observed. Antibodies were purified from cell culture supernatants by hydrophobic charge induction and ion exchange chromatography; the supernatant was applied to an MEP Hypercell column (Kronlab) equilibrated with 50 mm Tris/Cl, pH 8.0. After washing with pure water and 50 mm Tris/Cl, pH 8.0, 25 mm sodium-caprylate, the antibody was eluted with 50 mm sodium-acetate, pH 4.5. Antibody containing fractions were applied to an S-Hyper D column (Biosepra) equilibrated with 50 mm sodium-acetate, pH 4.5, and eluted by changing conditions in a linear gradient to 150 mm NaCl, 50 mm Tris/Cl, pH 7.5. Epitope Mapping of the 49-kDa Subunit—Synthesis of 218 overlapping decapeptides frameshifted by 2 amino acids was carried out on a cellulose membrane (Abimed) with an ASP222 robot (Abimed) as described in Ref. 19Venturi M. Rimon A. Gerchman Y. Hunte C. Padan E. Michel H. J. Biol. Chem. 2002; 275: 4734-4742Abstract Full Text Full Text PDF Scopus (50) Google Scholar. After complete deprotection the membrane was incubated for 30 min in PBS (100 mm sodium phosphate, pH 7.5, 100 mm NaCl) containing 0.5% Triton X-100 washed with PBS containing 0.1% Triton X-100 (TXPBS) and incubated for several hours to overnight with 5 μg/ml purified antibody in TXPBS. After washing with TXPBS an anti-mouse antibody conjugated to peroxidase (Sigma) was added at a dilution of 1:10000 in TXPBS and incubated for 1 h. Bound antibody was detected by chemoluminescence (ECL system, Amersham Biosciences). For reprobing the membrane with other antibodies, the membrane was washed with 8 m urea, 1% SDS in PBS at room temperature (two times, 30 min) and at 50 °C (1 h), followed by 10% acetic acid, 50% ethanol, 40% water (two times, 10 min). After washing with methanol (two times, 10 min), the membrane was either dried and stored at –20 °C or reprobed starting with the blocking step. Purification of Complex I from Y. lipolytica and Decoration with Antibodies—Mitochondrial membranes were isolated essentially as described previously (18Kashani-Poor N. Kerscher S. Zickermann V. Brandt U. Biochim. Biophys. Acta. 2001; 1504: 363-370Crossref PubMed Scopus (74) Google Scholar). However, cells were broken by a glass bead mill operating under continuous flow of material and efficient cooling (Bernd Euler Biotechnology, Frankfurt, Germany). Up to 400 g of cells were processed for 2 h in one liter of 600 mm sucrose, 20 mm Na/Mops, pH 7.0, 1 mm EDTA, 2 mm phenylmethylsulfonyl fluoride. Complex I was purified via His-tag affinity chromatography as described earlier (18Kashani-Poor N. Kerscher S. Zickermann V. Brandt U. Biochim. Biophys. Acta. 2001; 1504: 363-370Crossref PubMed Scopus (74) Google Scholar) from a Y. lipolytica strain containing a chromosomal copy of the NUGM gene carrying a carboxyl-terminal extension coding for a six-alanine and six-histidine tag (3Kerscher S. Dröse S. Zwicker K. Zickermann V. Brandt U. Biochim. Biophys. Acta-Bioenerg. 2002; 1555: 83-91Crossref PubMed Scopus (87) Google Scholar). Complex I was reactivated at a concentration of 1 mg/ml by incubation with an equal volume of 1 mg/ml polar lipids (Avanti) solubilized with 1.6% octyl glucoside. NADH:decylubioquinone activity was measured as described in Ref. 20Dröse S. Zwicker K. Brandt U. Biochim. Biophys. Acta. 2002; 1556: 65-72Crossref PubMed Scopus (74) Google Scholar. Complex I antibody complexes were prepared by addition of a 2-fold molar excess of antibody to 220 μl of purified complex I at 2 mg/ml. In control samples, the same volume of buffer without antibody was added. The mixture was kept on ice for 30 min. To check for the formation of antigen-antibody complexes, the samples were applied to a TSK 4000 FPLC gel filtration column (Toso-Haas), and the retention time was compared with a chromatogram of complex I without antibody. Electron Microscopy—For preparing grids, complex I with or without bound antibody was diluted to 0.06 mg/ml, and 6 μl were applied to 400-mesh copper grid coated with a thin carbon film. The specimen was stained with 2% ammonium molybdate using a deep stain technique (21Stoops J.K. Kolodziej S.J. Schroeter J.P. Bretaudiere J.P. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6585-6589Crossref PubMed Scopus (32) Google Scholar, 22Radermacher M. Ruiz T. Wieczorek H. Gruber G. J. Struct. Biol. 2001; 135: 26-37Crossref PubMed Scopus (94) Google Scholar). Micrographs were recorded under low dose condition on a Philips CM120 electron microscope (FEI) equipped with a LaB6 cathode at an accelerating voltage of 100 kV and a calibrated magnification of ×58300. The micrographs were recorded at a defocus of ∼1.0 μm. Selected micrographs were scanned on a Zeiss SCAI flat bed scanner (Zeiss) with 7-μm raster size. Images were converted to spider format and reduced three times by binning to a final pixel size of 3.6 Å on the scale of the sample. Particles were interactively selected and windowed into 128 × 128 pixel images. All particles, except obvious small fragments, were selected from micrographs of complex I without antibody. In the case of complex I decorated with antibodies only clear L-shaped particles with an antibody visibly attached were selected. Image processing was carried out using SPIDER (version 5.0; modified) and WEB (23Frank J. Radermacher M. Penzcek P. Zhu J. Li Y. Ladjadj M. Leith A. J. Mol. Biol. 1996; 161: 134-137Crossref Scopus (149) Google Scholar). All alignments were performed using a simultaneous translational/rotational alignment algorithm, based on correlation of Radon transforms (22Radermacher M. Ruiz T. Wieczorek H. Gruber G. J. Struct. Biol. 2001; 135: 26-37Crossref PubMed Scopus (94) Google Scholar). The average image of unlabelled complex I was calculated from a data set of 6000 images. A first alignment was carried out using 900 images and one L-shaped particle as reference. The first alignment result was analyzed using the neural network technique (24Marabini R. Masegosa I.M. San Martin M.C. Marco S. Fernandez J.J. de la Fraga L.G. Vaquerizo C. Carazo J.M. J. Struct. Biol. 1996; 116: 237-240Crossref PubMed Scopus (171) Google Scholar), and two nodes were selected representing the "flip" (left-handed) and "flop" (right-handed) L-shaped views. The two node images were used for multireference alignment, again followed by neural network analysis. Four node images were selected from this second analysis and used as starting references for multireference alignment of the full data set (6000 images). The multireference alignment was iterated, recalculating the reference images after each step by averaging the corresponding aligned particle images, which resulted in four class averages. Correspondence analysis (26Frank J. van Heel M.G. J. Mol. Biol. 1982; 161: 134-137Crossref PubMed Scopus (156) Google Scholar, 27van Heel M. Frank J. Ultramicroscopy. 1981; 6: 187-194PubMed Google Scholar), combined with Diday's classification by moving centers (28Diday E. Rev. Stat. Appl. 1971; : 19-34Google Scholar) was applied separately to the four classes. Incomplete particles were excluded. After this process the two major classes (one for the flip orientation, the other for the flop orientation) contained ∼1300 particles each. The contrast transfer function was determined for each micrograph, and every single image was corrected by phase flipping. Each data set was split into two halves, and the corresponding averages were compared by Fourier ring correlation (25Saxton W.O. Baumeister W. J. Microsc. 1982; 127: 127-138Crossref PubMed Scopus (694) Google Scholar). A cutoff value of five times the noise correlation was used as resolution criterion. The images of complex I labeled with antibodies were aligned in a multireference alignment procedure using the two averages obtained for the native complex I as references. All aligned particles were visually inspected; only those that were well aligned and exhibited a high similarity with the reference were chosen for the final average. For assessment of the significance of the differences, Student's t test was applied. His-tag-purified complex I was analyzed as single particles in deep negative stain (ammonium molybdate) by electron microscopy. The micrographs (Fig. 1A) showed a homogenous distribution of particles and very little aggregation. The angle between the membrane and peripheral arm was found to be variable to a certain extent, ranging from 90° to 150°. For analysis of the heterogeneity of the data set we used a combination of multivariate statistical analysis tools. Self-organizing maps from a neural network algorithm (24Marabini R. Masegosa I.M. San Martin M.C. Marco S. Fernandez J.J. de la Fraga L.G. Vaquerizo C. Carazo J.M. J. Struct. Biol. 1996; 116: 237-240Crossref PubMed Scopus (171) Google Scholar) were chosen for a first classification of the data set into four classes differentiated by the orientation relative to the specimen plane and by the angle between the two arms. After multireference alignment correspondence analysis and classification, two major classes containing complete particles were retained totaling 2600 particles and representing ∼63% of the image set. The angle between the two arms was 90°, and the particles were almost equally distributed into two classes. They were interpreted as flip view (Fig. 1B, left-handed L) and the mirrored flop view (Fig. 1C, right-handed L). Both views are from the side of the supporting carbon on the specimen grid. About the same number of particles were used to calculate two-dimensional averages for the flip and flop views. The resolution was estimated using the Fourier ring correlation with a cutoff value of five times noise correlation. For both averages the resolution was determined to be 25 Å (Fig. 2). Despite the careful classification, the resolution of the two-dimensional averages was limited, most probably because of a residual angular variation and deviations from perfect horizontality of the particles on the grid. The two-dimensional averages were in good agreement with those obtained earlier with complex I from Y. lipolytica purified by conventional chromatographic steps (2Djafarzadeh R. Kerscher S. Zwicker K. Radermacher M. Lindahl M. Schägger H. Brandt U. Biochim. Biophys. Acta. 2000; 1459: 230-238Crossref PubMed Scopus (82) Google Scholar) and resembled those reported for complex I from bovine heart, Neurospora crassa, and E. coli (4Hofhaus G. Weiss H. Leonard K. J. Mol. Biol. 1991; 221: 1027-1043Crossref PubMed Scopus (166) Google Scholar, 5Guenebaut V. Schlitt A. Weiss H. Leonard K. Friedrich T. J. Mol. Biol. 1998; 276: 105-112Crossref PubMed Scopus (204) Google Scholar, 7Sazanov L.A. Walker J.E. J. Mol. Biol. 2000; 392: 455-464Crossref Scopus (63) Google Scholar). An unambiguous assignment of the two perpendicular arms of complex I to a membrane and a peripheral arm, respectively, had been made for N. crassa complex I by comparison of single particles of whole complex I and purified membrane arm (4Hofhaus G. Weiss H. Leonard K. J. Mol. Biol. 1991; 221: 1027-1043Crossref PubMed Scopus (166) Google Scholar). The very high structural similarity of the two fungal complexes allowed application of this assignment to the Y. lipolytica two-dimensional averages: the peripheral arm of complex I particles from both organisms can be identified most easily by a characteristic protrusion on its inner side (Ref. 4Hofhaus G. Weiss H. Leonard K. J. Mol. Biol. 1991; 221: 1027-1043Crossref PubMed Scopus (166) Google Scholar, Fig. 1C). Based on this assignment, the membrane arm was oriented horizontally in Fig. 1 of this study and all following figures showing two-dimensional averages of complex I.Fig. 2Resolution of unlabelled complex I averages. Fourier ring correlation curves of averages shown in Fig. 1, flip (a) and flop (b) positions. The resolution was 25 Å using the five times noise correlation criterion, curve (c) and 21 Å using the three times noise correlation criterion, curve (d).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the flop view (Fig. 1C) we noted a novel feature, namely a thin bridge-like structure reaching from a protrusion of the peripheral arm to the distal end of the membrane arm. Further studies are under way to investigate the significance of this observation. From a combined conventional and His-tag-based ELISA screen (17Padan E. Venturi M. Michel H. Hunte C. FEBS Lett. 1998; 441: 53-58Crossref PubMed Scopus (41) Google Scholar) of 4800 clones from mice immunized with native complex I, 38 clones were found to produce antibodies binding to Y. lipolytica complex I. Four of these clones (35C5, 37F3, 42A10, and 34C10) produced IgG-type antibodies against the 49-kDa subunit. In a Western blot all antibodies recognized the 49-kDa subunit and no cross-reactivity with other subunits was observed (not shown). The epitopes of the antibodies were identified by testing their binding to overlapping decapeptides of the 49-kDa subunit sequence. Peptide arrays made by spot synthesis were probed with the individual antibodies. Fig. 3A shows the labeling of the two epitopes identified in the 49-kDa subunit: antibodies from three clones (35C5, 37F3, 42A10) recognized the same epitope (designated 49.1) close to the amino terminus, whereas the antibodies from clone 34C10 bound to an epitope (designated 49.2) 55 amino acids downstream of epitope 49.1. Antibody 49.1 from clone 42A10 and antibody 49.2 from clone 34C10 (both subclass IgG1) were used for further analysis. None of the antibodies affected NADH: decylubiquinone oxidoreductase activity of purified complex I that had been reactivated by the addition of phospholipids (20Dröse S. Zwicker K. Brandt U. Biochim. Biophys. Acta. 2002; 1556: 65-72Crossref PubMed Scopus (74) Google Scholar) (data not shown). The sequence homology between the 49-kDa subunit of complex I and the large subunit from Desulfovibrio fructosovorans [NiFe] hydrogenase is low but significant. Several characteristic sequence patterns, e.g. the essentially invariant RGXE motif (see Fig. 3B), are found in virtually all known sequences from both enzyme families (see also alignments in supplemental data). The membrane-bound hydrogenase from Methanosarcina barkeri can be considered an evolutionary link to complex I because it still contains a [NiFe] site but shows a much higher degree of homology to complex I, allowing an unambiguous alignment of the subunits from two rather distant families of enzymes. Sequence alignment of the amino-terminal part of the 49-kDa sequence from different organisms and the large subunit of two different [NiFe] hydrogenases revealed that the portion of the protein around epitope 49.1 is missing in the bacterial homologues of the 49-kDa subunit and that it has no similarity to the corresponding sequence of the large hydrogenase subunit. The sequence around epitope 49.2 of Y. lipolytica complex I exhibited a high degree of homology between 49-kDa subunits of different origin and could be aligned to the corresponding part of the hydrogenase subunit (Fig. 3B). Notably, the decapeptide identified as epitope 49.2 matched the middle strand of a three-stranded β-sheet on the surface of the protein in the known structure of D. fructosovorans [NiFe] hydrogenase (Fig. 3C). The cascaded multiple classifiers algorithm for secondary structure prediction (29Ouali M. King R.D. Protein Sci. 2000; 9: 1162-1176Crossref PubMed Scopus (299) Google Scholar) predicted three matching β-strands in this part of mitochondrial and bacterial 49-kDa subunits (Fig. 3B), strongly suggesting that, as shown previously for the fold around the [NiFe] site (3Kerscher S. Dröse S. Zwicker K. Zickermann V. Brandt U. Biochim. Biophys. Acta-Bioenerg. 2002; 1555: 83-91Crossref PubMed Scopus (87) Google Scholar), the β-sheet and the overall structure around epitope 49.2 has been preserved in complex I. In E. coli there is a direct fusion of the carboxyl terminus of the 30-kDa subunit of complex I to the amino terminus of the 49-kDa subunit forming the NuoCD protein (9Friedrich T. Biochim. Biophys. Acta. 1998; 1364: 134-146Crossref PubMed Scopus (179) Google Scholar). The homology of the NuoCD subunit to individual subunits from other organisms is high, and there are no insertions. It follows for the structure of complex I from Y. lipolytica that the carboxyl-terminal end of the 30-kDa subunit is expected to reside in the vicinity of the amino-terminal epitope 49.2. To test this prediction, we included a commercially available anti-His-tag antibody in this study to locate the His-tag sequence that had been attached to the carboxyl terminus of the 30-kDa subunit to purify complex I from Y. lipolytica (18Kashani-Poor N. Kerscher S. Zickermann V. Brandt U. Biochim. Biophys. Acta. 2001; 1504: 363-370Crossref PubMed Scopus (74) Google Scholar). The antibody-complex I complexes used for electron microscopy were prepared by incubating a 2-fold excess of antibody with purified complex I. Formation of stable complexes with the native enzyme was confirmed for all antibodies used in this study as clear shifts of retention time in analytical gel filtration in comparison to complex I alone (data not shown). Gel filtration was also used in some experiments to remove excess antibody before preparing the grids for electron microscopy, without any detectable effect on the results (not shown). About 10–25% of complex I particles were found by visual inspection of the micrographs (Fig. 4) to be labeled with an antibody. Only those particles were aligned and averaged that exhibited a high similarity with the 90° flip or flop reference (Fig 5, A–D). For antibody 49.2 a 40:60 distribution of flip and flop views of decorated complex I was observed. The vast majority of particles was in flip view orientation when labeled with antibody 49.1 and in flop view orientation when labeled with the His-tag antibody. All three antibodies against hydrophilic subunits of complex I were found to bind to the part of the particles previously identified as peripheral arm, confirming the assignment of these two major parts of the complex I structure.Fig. 5Two-dimensional averages of single particles decorated with antibodies. A, average of 60 complex I particles decorated with antibody 49.1 in flip view. B and C, average of 60 complex I particles decorated with antibody 49.2 in flip (B) and flop (C) view. D, average of 60 complex I particles decorated with anti-His-tag antibody in flop view. Because of a clear preference for either the flip or flop orientation, mirrored averages were not calculated for antibody 49.1 and the anti-His-tag antibody. E–H, Student's t test of the averages shown in panels A–D. Light areas indicate a statistical difference between labeled and unlabeled particles (confidence level >95%). The contour lines of the unlabelled averages (Fig. 1) were superimposed over the Student's t test images.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To better define the contact site between the antibodies and complex I, a Student's t test was performed to compare the native with the labeled complex. Images were obtained by averaging previously aligned particles, and the corresponding variance images were calculated. The coincidence of the mean values of each pixel in both images was tested. The pixels for which the confidence level was greater than 95% were set to zero, and only those with a statistical significant diff
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