Exploring the Ubiquinone Binding Cavity of Respiratory Complex I
2007; Elsevier BV; Volume: 282; Issue: 40 Linguagem: Inglês
10.1074/jbc.m704519200
ISSN1083-351X
AutoresMaja A. Tocilescu, Uta Fendel, Klaus Zwicker, Stefan Kerscher, Ulrich Brandt,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoProton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by site-directed mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues. Proton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by site-directed mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues. Respiratory chain NADH:ubiquinone oxidoreductase (complex I) is a large membrane protein complex that catalyzes electron transfer from NADH to ubiquinone and thereby pumps protons across the inner mitochondrial or bacterial plasma membrane (1Brandt U. Annu. Rev. Biochem. 2006; 75: 69-92Crossref PubMed Scopus (642) Google Scholar). Electron microscopy revealed that complex I is L-shaped (2Leonard K. Haiker H. Weiss H. J. Mol. Biol. 1987; 194: 277-286Crossref PubMed Scopus (85) Google Scholar, 3Guenebaut V. Schlitt A. Weiss H. Leonard K. Friedrich T. J. Mol. Biol. 1998; 276: 105-112Crossref PubMed Scopus (203) Google Scholar, 4Grigorieff N. J. Mol. Biol. 1998; 277: 1033-1046Crossref PubMed Scopus (298) Google Scholar, 5Djafarzadeh 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, 6Böttcher 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, 7Peng G. Fritzsch G. Zickermann V. Schägger H. Mentele R. Lottspeich F. Bostina M. Radermacher M. Huber R. Stetter K.O. Michel H. Biochemistry. 2003; 42: 3032-3039Crossref PubMed Scopus (73) Google Scholar, 8Sazanov L.A. Carroll J. Holt P. Toime L. Fearnley I.M. J. Biol. Chem. 2003; 278: 19483-19491Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 9Radermacher M. Ruiz T. Clason T. Benjamin S. Brandt U. Zickermann V. J. Struct. Biol. 2006; 154: 269-279Crossref PubMed Scopus (103) Google Scholar) and is composed of a hydrophobic arm embedded in the membrane and a peripheral arm protruding into the mitochondrial matrix or the bacterial cytosol. The peripheral arm contains all known redox centers, one FMN and eight or nine iron-sulfur clusters. Based on sequence comparisons (10Böhm R. Sauter M. Böck A. Mol. Microbiol. 1990; 4: 231-243Crossref PubMed Scopus (241) Google Scholar, 11Albracht S.P.J. Biochim. Biophys. Acta. 1993; 1144: 221-224Crossref PubMed Scopus (90) Google Scholar), mutational analysis (12Darrouzet E. Issartel J.P. Lunardi J. Dupuis A. FEBS Lett. 1998; 431: 34-38Crossref PubMed Scopus (107) Google Scholar, 13Prieur I. Lunardi J. Dupuis A. Biochim. Biophys. Acta. 2001; 1504: 173-178Crossref PubMed Scopus (58) Google Scholar, 14Kashani-Poor N. Zwicker K. Kerscher S. Brandt U. J. Biol. Chem. 2001; 276: 24082-24087Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 15Loeffen J. Elpeleg O. Smeitink J. Smeets R. Stöckler-Ipsiroglu S. Mandel H. Sengers R. Trijbels F. Van den Heuvel L. Ann. Neurol. 2001; 49: 195-201Crossref PubMed Scopus (161) Google Scholar), and photoaffinity labeling studies (16Schuler 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 (162) Google Scholar), we have proposed previously (14Kashani-Poor N. Zwicker K. Kerscher S. Brandt U. J. Biol. Chem. 2001; 276: 24082-24087Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 17Kerscher S. Kashani-Poor N. Zwicker K. Zickermann V. Brandt U. J. Bioenerg. Biomembr. 2001; 33: 187-196Crossref PubMed Scopus (45) Google Scholar) that the PSST and the 49-kDa subunit that are homologous to the small and large subunit of [NiFe] hydrogenases form part of the quinone reducing catalytic core of complex I (note that the bovine nomenclature will be used for the central subunits of complex I throughout). Recently the crystal structure of the peripheral domain of complex I from Thermus thermophilus has been solved at 3.3 Å resolution (18Sazanov L.A. Hinchliffe P. Science. 2006; 311: 1430-1436Crossref PubMed Scopus (657) Google Scholar). This structure (Fig. 1) shows a wire of iron-sulfur clusters connecting the NADH-binding site near FMN with a broad cavity formed by the PSST and the 49-kDa subunit that should comprise the active site for ubiquinone reduction and the binding region for the large number of inhibitors that have been found for complex I (19Degli Esposti M. Biochim. Biophys. Acta. 1998; 1364: 222-235Crossref PubMed Scopus (446) Google Scholar).Because reduction of ubiquinone is likely to be a key event in the energy-coupling mechanism of complex I (1Brandt U. Annu. Rev. Biochem. 2006; 75: 69-92Crossref PubMed Scopus (642) Google Scholar, 20Zwicker K. Galkin A. Dröse S. Grgic L. Kerscher S. Brandt U. J. Biol. Chem. 2006; 281: 23013-23017Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), the quinone-binding site in the PSST and the 49-kDa subunit next to iron sulfur-cluster N2 is of particular interest. To identify the domains essential for catalytic activity and inhibitor binding, we introduced a set of point mutations in the PSST and the 49-kDa subunits of complex I from our model organism, the strictly aerobic yeast Yarrowia lipolytica. Positions for point mutations were chosen by analyzing the T. thermophilus structure so that they would probe all parts of the proposed quinone binding cavity and some surrounding residues.EXPERIMENTAL PROCEDURESStrains and Site-directed Mutagenesis—The Y. lipolytica nucmΔ and nukmΔ deletion strains described earlier (21Grgic L. Zwicker K. Kashani-Poor N. Kerscher S. Brandt U. J. Biol. Chem. 2004; 279: 21193-21199Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Garofano A. Zwicker K. Kerscher S. Okun P. Brandt U. J. Biol. Chem. 2003; 278: 42435-42440Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) were transformed with the replicative plasmids pUB26 containing a genomic fragment of the NUCM gene or pUB4 containing a genomic fragment of the NUKM gene. All point mutations were generated in Escherichia coli by PCR mutagenesis. After transformation (23Chen D.-C. Beckerich J.-M. Gaillardin C. Appl. Biochem. Biotechnol. 1997; 48: 232-235Google Scholar) into Y. lipolytica strain nucmΔ or nukmΔ, plasmids were recovered, and the entire open reading frames were sequenced to verify the introduced point mutations and exclude other sequence changes.Small Scale Preparation of Mitochondrial Membranes—Mitochondrial membranes were isolated essentially according to published protocols (24Kerscher S. Okun J.G. Brandt U. J. Cell Sci. 1999; 112: 2347-2354Crossref PubMed Google Scholar). After phenylmethylsulfonyl fluoride was added to a final concentration of 2 mm, mitochondrial membranes were homogenized, shock-frozen, and stored in liquid nitrogen. Aliquots of preparations were used for activity measurements and gel electrophoresis. Protein concentration was determined colorimetrically using the DC protein assay (Bio-Rad).Measurement of Catalytic Activity—NADH:HAR 2The abbreviations used are:HARhexaammineruthenium(III)-chlorideDBQn-decylubiquinonedNADHdeamino-NADH (reduced form)DQA2-decyl-4-quinazolinylamineMops3-(N-morpholino)propanesulfonic acid. oxidoreductase activity was measured as NADH oxidation (ϵ340–400 nm = 6.22 mm-1 cm-1) in the presence of the artificial electron acceptor HAR using a Molecular Devices SPECTRAmax PLUS384 plate reader spectrometer. The activity was measured at 30 °C in 20 mm Na+/Hepes, pH 8.0, with 250 mm sucrose, 2 mm NaN3, 0.2 mm EDTA, 0.2 mm NADH, and 2 mm HAR. The reaction was initiated by the addition of mitochondrial membranes (final concentration 25 μg of protein per ml). Specific NADH:HAR oxidoreductase activity was used to estimate complex I content because it is not affected by changes in the ubiquinone binding pocket.dNADH:DBQ oxidoreductase activity of mitochondrial membranes was determined as the fraction of dNADH oxidation activity (ϵ340–400 nm = 6.22 mm-1 cm-1) sensitive to the complex I inhibitor DQA in the presence of DBQ as electron acceptor. Measurements were carried out on a SPECTRAmax PLUS384 plate reader spectrometer (Molecular Devices) at 30 °C in 20 mm Na+/Mops, pH 7.4, with 50 mm NaCl, 2 mm KCN, 0.1 mm dNADH, and 0.07 mm DBQ. The final concentration of mitochondrial membranes was 50 μg/ml. The reaction was started by adding DBQ. The inhibitor-sensitive fraction of the ubiquinone reductase activity was calculated by subtracting the residual rate in the presence of 27 μm DQA that was usually 5–10% of the dNADH:DBQ oxidoreductase activity of the parental strain. To allow comparison between different membrane preparations, all activities were normalized for complex I content. The results are given as mean ± S.E. (n = 5–15).I50 values and apparent Km values were determined under essentially the same conditions as dNADH:DBQ oxidoreductase activity by varying the inhibitor and DBQ concentrations, respectively. The I50 value is defined as the concentration of inhibitor that decreased the inhibitor-sensitive complex I activity by 50%. For determination of the apparent Km, data were fitted using the program "Enzfitter" (version 2.0.17.0, Biosoft 1999, Cambridge, UK) and a modified Michaelis-Menten equation (25Eschemann A. Galkin A. Oettmeier W. Brandt U. Kerscher S. J. Biol. Chem. 2005; 280: 3138-3142Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar).Gel Electrophoresis—Blue-native PAGE was carried out according to Schägger (26Schägger H. Hunte C. von Jagow G. Schägger H. Membrane Protein Purification and Crystallization: A Practical Guide. Academic Press, San Diego2003: 105-130Crossref Google Scholar). Complex I in gel activity was preformed as described previously (27Wittig I. Karas M. Schagger H. Mol. Cell. Proteomics. 2007; 6: 1215-1225Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar).EPR Spectroscopy—X-band EPR spectra were obtained with a Bruker ESP 300E spectrometer equipped with an HP 53159A frequency counter (Hewlett Packard), ER 035 M NMR gaussmeter (Bruker, BioSpin), and a liquid helium continuous flow cryostat (Oxford Instruments). Spectra were recorded using the following parameters: microwave frequency 9.47 GHz, microwave power 5 milliwatts, modulation amplitude 0.64 millitesla, and modulation frequency 100 kHz.Mitochondrial membranes (10–25 mg/ml, depending on the specific mutant) were reduced with 2 mm NADH or 2 mm NADH, 5 mm sodium dithionite. Samples were frozen in cold isopentane/methylcyclohexane (5:1, ∼120 K) and stored in liquid nitrogen. Usually, spectra were recorded at temperatures of 40 K to analyze binuclear clusters only or at 12 K to analyze binuclear and tetranuclear clusters.Structure Images—Images of the structural model and interpretation of the results from mutagenesis were based on the x-ray structure of the peripheral arm of complex I from T. thermophilus at 3.3 Å resolution (18Sazanov L.A. Hinchliffe P. Science. 2006; 311: 1430-1436Crossref PubMed Scopus (657) Google Scholar). The software package PyMol (version 0.99) was used for visualizing the coordinates (Protein Data Bank 2FUG) and preparing the figures in which the amino acids were labeled using Y. lipolytica numbering that was deduced from aligning the sequences from the two organisms (supplemental Fig. S1). In cases where a residue was not conserved between T. thermophilus and Y. lipolytica, the side chain was exchanged to match the Y. lipolytica sequence using the mutagenesis wizard of the PyMol package.RESULTSMutations in the Ubiquinone Binding Cavity Did Not Interfere with Complex I Assembly—To gain more insight into the function of amino acid residues that line the putative ubiquinone binding pocket (18Sazanov L.A. Hinchliffe P. Science. 2006; 311: 1430-1436Crossref PubMed Scopus (657) Google Scholar), we generated a set of 39 point mutations covering 20 different residues of the 49-kDa and the PSST subunits of complex I from Y. lipolytica (Tables 1 and 2). In many positions the residues were exchanged by several different amino acids and at least one conservative, and one more drastic exchange was introduced if possible. Tables 1 and 2 also list data on point mutations that we had generated and analyzed before (14Kashani-Poor N. Zwicker K. Kerscher S. Brandt U. J. Biol. Chem. 2001; 276: 24082-24087Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 21Grgic L. Zwicker K. Kashani-Poor N. Kerscher S. Brandt U. J. Biol. Chem. 2004; 279: 21193-21199Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Garofano A. Zwicker K. Kerscher S. Okun P. Brandt U. J. Biol. Chem. 2003; 278: 42435-42440Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 28Ahlers P. Zwicker K. Kerscher S. Brandt U. J. Biol. Chem. 2000; 275: 23577-23582Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), providing information on a total of 52 mutations at 26 positions. Mitochondrial membranes were isolated from all newly generated mutant Y. lipolytica strains, and complex I content was estimated as NADH:HAR oxidoreductase activity (Tables 1 and 2) and by blue-native PAGE with subsequent complex I in gel activity stain (data not shown). Membranes from most mutants contained fully assembled complex I in amounts comparable with the parental strain indicating that complex I was not destabilized in these mutants. Only very few mutations led to moderately decreased complex I contents, but even in these mutants complex I appeared to be fully assembled.TABLE 1Effects of point mutations introduced into the 49-kDa subunit ND indicates not determined.StrainComplex I contenta100% of complex I content corresponds to 1.25 μmol·min-1·mg-1 NADH:HAR oxidoreductase activityComplex I activitybComplex I activity of the parental strain was 0.58 μmol·min-1·mg-1Apparent Km (DBQ)I50DQARotenone%%μmnmParental100 ± 3100 ± 51516530A94I85 ± 314 ± 2NDNDNDH95AcData are from Ref. 21130<5NDNDNDH95McData are from Ref. 21120<5NDNDNDH95RcData are from Ref. 21100<5NDNDNDV97W78 ± 210 ± 2NDNDNDL98F101 ± 322 ± 2NDNDNDL98K71 ± 215 ± 2NDNDNDR99D58 ± 214 ± 2NDNDNDR99T71 ± 115 ± 2NDNDNDR141KcData are from Ref. 211404513551500R141A130dData are from Ref. 1417dData are from Ref. 1410eData are from N. Kashani-Poor, unpublished data21dData are from Ref. 14570dData are from Ref. 14Y144W129 ± 28 ± 4NDNDNDY144HdData are from Ref. 1470<5NDNDNDV145F127 ± 17 ± 3NDNDNDV145T122 ± 492 ± 81217550S146CfData are from L. Grgic, unpublished data10010012801500M188Y104 ± 212 ± 2NDNDNDS192I110 ± 316 ± 2NDNDNDS192R102 ± 58 ± 2NDNDNDS192Y111 ± 224 ± 2NDNDNDF207W99 ± 258 ± 21221850R210I100 ± 263 ± 21416500E211Q124 ± 483 ± 61621530E218Q97 ± 297 ± 41221550R224D97 ± 2107 ± 41312550R224I93 ± 3102 ± 51412650R224K86 ± 2103 ± 31011550R224N124 ± 299 ± 71415700L225A102 ± 3103 ± 61313550L225F95 ± 3110 ± 61311460L225H84 ± 3107 ± 51214700L225V95 ± 291 ± 31212700K407H80 ± 278 ± 21313500K407R93 ± 3105 ± 41275550K407W66 ± 112 ± 2NDNDNDG455I71 ± 316 ± 2NDNDNDG455S98 ± 172 ± 41122700D458A93dData are from Ref. 1428dData are from Ref. 1412eData are from N. Kashani-Poor, unpublished data520dData are from Ref. 145200dData are from Ref. 14L459I92 ± 792 ± 101613620L459K50 ± 224 ± 2NDNDNDV460AdData are from Ref. 1490 75%, blue), and in others, the mutant activities were markedly reduced (25–75%, green). Yellow was used if at least one mutant with an activity below 25% was found. If several exchanges all resulted in complex I activities below <25% of the parental strain, this was indicated by red color. A color-coded representation of our results in the structure around cluster N2 (Fig. 2) illustrates that the effects of the mutations nicely correlated with their positions within the ubiquinone binding cavity. The most severe reductions of activity were observed along a path leading from the first strand of the N-terminal β-sheet of the 49-kDa subunit toward a region adjacent to iron-sulfur cluster N2.FIGURE 2Effects of point mutations on complex I activity. The putative ubiquinone binding cavity of complex I is shown as a schematic with exchanged amino acids highlighted in stick representation. Amino acids mutated within the putative quinone binding cavity are color-coded to illustrate the effect on dNADH: ubiquinone oxidoreductase activity observed for an amino acid exchange in a given position in complex I from Y. lipolytica. Red, several exchanges all resulted in very low activity (<25% of parental); yellow, at least one exchange resulted in very low activity ( 75% of parental) for all exchanges. Residues of subunit PSST are marked with an asterisk. Iron-sulfur cluster N2 is shown as gray spheres. A, surface representation. B, schematic representation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of Point Mutations on Apparent Km Value for DBQ—To test whether the mutations had altered the ubiquinone-binding site of complex I, we determined their apparent Km value for the ubiquinone derivative DBQ (Tables 1 and 2). However, this was only possible for those mutants that had a residual activity of well above 20% of the parental strain. Many mutants exhibited a slightly lower apparent Km value than the parental strain, but in most cases this seemed to go parallel with reduced ubiquinone reductase activity, a trend that we had observed previously with complex I mutations. Overall none of the mutants analyzed here that had retained appreciable ubiquinone reductase activities exhibited marked changes in the apparent Km value for DBQ.Effects of Point Mutations on I50 of Specific Complex I Inhibitors—Many different complex I inhibitors are known that act on the quinone-binding site. Because of their kinetic properties these inhibitors are divided into three classes, A–C (19Degli Esposti M. Biochim. Biophys. Acta. 1998; 1364: 222-235Crossref PubMed Scopus (446) Google Scholar), with different but partially overlapping binding sites (29Okun J.G. Lümmen P. Brandt U. J. Biol. Chem. 1999; 274: 2625-2630Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Because DQA and rotenone are very potent representatives of class A and B, respectively, we measured the I50 values for these inhibitors in our complex I mutants. Again, reliable determination of this parameter was only possible if the residual activity of a given mutant was well above 20%. Mutation K407R in the 49-kDa subunit caused a 4.7-fold increase of the I50 value for DQA, but no change in the I50 value for rotenone. In contrast, for mutant F207W we observed a significant increase in the I50 value only for rotenone. Mutation V88M in the PSST subunit exhibited a slight hypersensitivity for rotenone and some resistance to DQA. The latter was also observed to a somewhat lesser extent in PSST mutant V88L. All other mutants studied here exhibited I50 values for both inhibitors that were not significantly different from the parental strain (Table 1 and 2). Table 1 also contains several previously generated and published mutations, which change I50 for DQA and rotenone. All resistance data are illustrated in Fig. 3. Amino acid positions where mutants exhibited resistance or hypersensitivity to DQA or rotenone are shown in orange in Fig. 3. For orientation, the residues that were most critical for catalytic activity are shown again in red in Fig. 3.FIGURE 3Effects of point mutations on I50 value of complex I inhibitors. The same view into the ubiquinone binding cavity of complex I as in Fig. 2 is shown. However, amino acids that, when exchanged, had an effect on I50 of DQA or rotenone are now shown in orange. For better orientation, other residues that were most critical for catalytic activity are shown again in red. The other mutated residues highlighted in Fig. 2 are now shown in the same color as the secondary structure schematic of the subunit. (A and B as in Fig. 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONWe have probed the proposed ubiquinone binding cavity within the peripheral arm of complex I by site-directed mutagenesis. Of the 39 mutations that were analyzed here, a significant number resulted in a marked reduction of inhibitor-sensitive ubiquinone reductase activity. Several mutations changed inhibitor sensitivity. By localizing the corresponding residues in the partial structure of complex I from T. thermophilus (18Sazanov L.A. Hinchliffe P. Science. 2006; 311: 1430-1436Crossref PubMed Scopus (657) Google Scholar) and by combining these results with information from earlier studies (14Kashani-Poor N. Zwicker K. Kerscher S. Brandt U. J. Biol. Chem. 2001; 276: 24082-24087Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 21Grgic L. Zwicker K. Kashani-Poor N. Kerscher S. Brandt U. J. Biol. Chem. 2004; 279: 21193-21199Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 28Ahlers P. Zwicker K. Kerscher S. Brandt U. J. Biol. Chem. 2000; 275: 23577-23582Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), we could identify functionally important regions within this central domain of complex I (Figs. 2 and 3). The region identified as being most critical for activity included a group of residues that (except for Tyr-144, Ser-192, and Val-460) were not located immediately in the spacious cavity around cluster N2 but rather seemed to form a path of entry for ubiquinone (Fig. 2A). This path starts with Ala-94 at a distance of about 24 Å from the ubiquinone-reducing iron-sulfur cluster N2 within the first strand of the N-terminal three-stranded β-sheet of the 49-kDa subunit. The amphipathic loop connecting the first and second strand of this β-sheet reaches into the proposed ubiquin
Referência(s)