FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2068754574> ?p ?o ?g. }

• W2068754574 endingPage "25737" @default.
• W2068754574 startingPage "25731" @default.
• W2068754574 abstract "An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 °C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184–193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane. An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 °C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184–193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane. The NADH:ubiquinone oxidoreductase (also known as respiratory complex I) is the first segment of the energy-conserving electron transfer chains of mitochondria and many respiratory and photosynthetic bacteria. This complex catalyzes electron transfer from NADH to ubiquinone and concomitantly translocates protons across the membrane to generate a membrane potential and proton gradient for ATP synthesis (1Anraku Y. Gennis R.B. Trends Biochem. Sci. 1987; 12: 262-266Abstract Full Text PDF Scopus (238) Google Scholar, 2Walker J.E. Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (681) Google Scholar). Whereas the mitochondrial enzyme contains up to 46 different subunits (3Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Mol. Cell. Proteomics. 2003; 2: 117-126Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar), the bacterial enzyme is made up of 13–14 subunits (4Friedrich T. Biochim. Biophys. Acta. 1998; 1364: 134-146Crossref PubMed Scopus (180) Google Scholar) and thus can be considered as the minimal core complex I. The Escherichia coli NADH-Q oxidoreductase has been purified to homogeneity by liquid chromatography in the presence of detergent alkyl polyglucosides (5Leif H. Sled V.D. Ohnishi T. Weiss H. Friedrich T. Eur. J. Biochem. 1995; 230: 538-548Crossref PubMed Scopus (254) Google Scholar) or dodecyl maltoside (6Spehr V. Schlitt A. Scheide D. Guénebaut V. Friedrich T. Biochemistry. 1999; 38: 16216-16267Crossref Scopus (42) Google Scholar). The purified complex has a molecular mass of approximately 550 kDa and contains 13 subunits encoded by the nuo genes (7Weidner U. Geier S. Ptock A. Friedrich T. Leif H. Weiss H. J. Mol. Biol. 1993; 233: 109-122Crossref PubMed Scopus (288) Google Scholar). This bacterial complex can adopt either a conserved L-shaped or horseshoe-shaped quaternary structure made of a peripheral arm and a membrane arm (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). The horseshoe-shaped structure is believed to be the active form. Six subunits, NuoB, NuoCD, NuoE, NuoF, NuoG, and NuoI, constitute the peripheral arm (5Leif H. Sled V.D. Ohnishi T. Weiss H. Friedrich T. Eur. J. Biochem. 1995; 230: 538-548Crossref PubMed Scopus (254) Google Scholar). The remaining seven subunits, NuoA, NuoH, NuoJ, NuoL, NuoM, and NuoN, are hydrophobic proteins, which comprise the membrane arm. The peripheral arm can be subdivided into an NADH dehydrogenase domain composed of subunits NuoE, NuoF, and NuoG, which catalyzes the oxidation of NADH, and a connecting domain composed of subunits NuoB, NuoCD, and NuoI. The dehydrogenase domain contains the FMN-binding site and the EPR-detectable FeS clusters, N1a, N1b, N3, and N4 (5Leif H. Sled V.D. Ohnishi T. Weiss H. Friedrich T. Eur. J. Biochem. 1995; 230: 538-548Crossref PubMed Scopus (254) Google Scholar), whereas the connecting domain contains the EPR-detectable FeS cluster N2 (5Leif H. Sled V.D. Ohnishi T. Weiss H. Friedrich T. Eur. J. Biochem. 1995; 230: 538-548Crossref PubMed Scopus (254) Google Scholar) and the UV-visible-detectable clusters N6a and N6b (9Rasmussen T. Scheide D. Brors B. Kintscher L. Weiss H. Friedrich T. Biochemistry. 2001; 40: 6124-6131Crossref PubMed Scopus (67) Google Scholar). The subunits of the membrane arm contain no known cofactors, such as flavin or iron-sulfur clusters, and are thought to be involved in quinone binding and proton translocation (10Friedrich T. Weiss H. Baltscheffsky H. Origin and Evolution of Biological Energy Conversion. VCH Publishers, New York1996: 205-220Google Scholar, 11Friedrich T. Weiss H. J. Theor. Biol. 1997; 187: 529-540Crossref PubMed Scopus (183) Google Scholar). The mechanism of electron transfer and its coupling to proton translocation in NADH-Q oxidoreductase is poorly understood. Ubiquinone is the final electron acceptor in NADH-Q oxidoreductase and may take part in electron recycling and/or proton transport processes. Knowledge of ubiquinone binding is essential for mechanistic studies of this complex. However, the number of binding sites for ubiquinone in NADH-Q oxidoreductase is an unsolved question subject to intense controversy (12Vinogradov A. J. Bioenerg. Biomembr. 1993; 25: 367-375Crossref PubMed Scopus (74) Google Scholar, 13Degli Esposti M. Ghelli A. Biochim. Biophys. Acta. 1994; 1187: 116-120Crossref PubMed Scopus (74) Google Scholar, 14Brandt U. Biochim. Biophys. Acta. 1997; 1318: 79-91Crossref PubMed Scopus (198) Google Scholar). Up to three sites have been proposed (14Brandt U. Biochim. Biophys. Acta. 1997; 1318: 79-91Crossref PubMed Scopus (198) Google Scholar). Most suggestions derived from studies involving labeled inhibitor analogues. The Q-binding site has long been thought to be in the membrane domain of the enzyme due to its lipophilic nature. The ND1 in mitochondria (NuoH in E. coli) was identified as a quinone- and rotenone-binding protein by photoaffinity labeling using rotenone analogs (15Earley F.G.P. Ragan C.I. Biochem. J. 1984; 224: 525-534Crossref PubMed Scopus (62) Google Scholar, 16Earley F.G.P. Patel S.D. Ragan C.I. Attardi G. FEBS Lett. 1987; 219: 108-112Crossref PubMed Scopus (181) Google Scholar) and by mutational studies of human mitochondrial DNA (17Degli Esposti M. Carelli V. Ghelli A. Ratta M. Crimi M. Sangiorgi S. Montagna P. Lenaz G. Luagresi E. Cortelli P. FEBS Lett. 1994; 352: 375-379Crossref PubMed Scopus (114) Google Scholar, 18Majander A. Finel M. Savontaus M.L. Nikoskelaninen E. Wikström M. Eur. J. Biochem. 1996; 239: 201-207Crossref PubMed Scopus (65) Google Scholar, 19Lunardi J. Darrrouzet E. Dupuis A. Issartel J.P. Biochim. Biophys. Acta. 1998; 1407: 114-124Crossref PubMed Scopus (24) Google Scholar). The results of rotenone binding were used to indicate that ubiquinone binds to the same site. Recently, the inhibitor/quinone-binding site was postulated to be located at the interface between the peripheral and membrane domains of complex I and to involve subunits NuoH and NuoD (20Dupuis A. Prieur I. Lunardi J. J. Bioenerg. Biomembr. 2001; 33: 159-168Crossref PubMed Scopus (41) Google Scholar, 21Darrouzet E. Issartel J.P. Lunardi J. Dupuis A. FEBS Lett. 1998; 431: 34-38Crossref PubMed Scopus (110) Google Scholar). This suggestion stemmed from identification of a missense mutation in the hydrophilic subunit NuoD of Rhodobacter capsulatus that conferred resistance of complex I activity to inhibition by piericidin and rotenone and from the assumption that complex I contains only one ubiquinone-binding site. Pyridaben is another potent inhibitor of complex I. Using a pyridaben photoaffinity ligand, the hydrophilic subunit PSST (NuoB in E. coli) was specifically labeled (22Schuler F. Yano T. DiBernardo S. Yagi T. Yankovskaya V. Singer T. Casida J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4149-4153Crossref PubMed Scopus (162) Google Scholar). It should be noted that rotenone, piericidin A, and pyridaben inhibition of electron transfer from cluster N2 to Q is noncompetitive with Q. Therefore, the binding sites for these inhibitors may not be the same as that for ubiquinone. One way to unambiguously identify the Q-binding site(s) in complex I is to use azido-Q 1The abbreviations used are: azido-Q, 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone; Q1, 2,3-dimethoxy-5-methyl-6-isoprenoyl-1,4-benzoquinone; Q0C10Br, 2,3-dimethoxy-5-methyl-6-(10-bromodecyl)-1,4-benzoquinone; HPLC, high performance liquid chromatography; [3H]azido-Q, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone; MES, 2-morpholinoethanesulfonic acid. derivatives that possess electron acceptor activity for complex I or competitively inhibit complex I activity with Q. The availability in our laboratory of a photoactivatable azido-Q derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone, which has partial electron acceptor activity for complex I, encouraged us to study the Q/protein interaction and to identify the Q-binding site in E. coli complex I. Herein, we report conditions for photoaffinity labeling of complex I with azido-Q derivatives and a detailed isolation procedure for the Q-binding peptide. Materials—Sodium cholate was obtained from Sigma and re-crystallized from methanol. n-Dodecyl-β-d-maltoside was from Anatrace. Insta-Gel liquid scintillation mixture was from ICN. Other chemicals were of the highest purity commercially available. The ubiquinone derivatives, 2,3-dimethoxy-5-methyl-6-isoprenoyl-1,4-benzoquinone (Q1), 2,3-dimethoxy-5-methyl-6-(10-bromodecyl)-1,4-benzoquinone (Q0C10Br), azido-Q, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), and 5-azido-2,3-dimethoxy-6-decyl-1,4-benzoquinone, were synthesized by methods reported previously (23Yu L. Yang F.D. Yu C.A. J. Biol. Chem. 1985; 260: 963-973Abstract Full Text PDF PubMed Google Scholar). Enzyme Preparations and Assays—E. coli complex I was prepared and assayed essentially as reported previously (6Spehr V. Schlitt A. Scheide D. Guénebaut V. Friedrich T. Biochemistry. 1999; 38: 16216-16267Crossref Scopus (42) Google Scholar). Complex I, azido-Q-treated or untreated, was mixed with asolectin at a ratio of 1:20 (by weight) and incubated at 4 °C for 15 min before assaying for activity. The reaction mixture (1 ml) contained 50 mm Tris-Cl buffer, pH 7.5, 5 mm NaN3, 0.15% dodecyl maltoside, 100 μm NADH, and 60 μm Q1. The reaction was started by addition of an appropriate amount of azido-Q-treated or untreated complex I. The oxidation of NADH was followed by measuring the absorption decrease at 340 nm, using a millimolar extinction coefficient of ϵ340 nm = 6.22 mm–1 cm–1. Identification of Endogenous Quinone in E. coli Complex I—Quinones were extracted from purified E. coli complex I with hexane as reported previously (24Redfearn E.R. Methods Enzymol. 1967; 10: 381-384Crossref Scopus (78) Google Scholar). The concentration of quinone was determined by the method of Redfearn (24Redfearn E.R. Methods Enzymol. 1967; 10: 381-384Crossref Scopus (78) Google Scholar). A millimolar extinction coefficient of 12.25 mm–1cm–1 was used as the difference in absorption of the oxidized and reduced forms of Q at 275 nm. Quinone identity was determined by matching the retention time of quinone obtained from complex I with those of reference quinones, Q2, Q6, Q8, and Q10, in a HPLC system using a Nova-Pak® reverse phase column (C18; 3.9 × 150 mm) from Waters, eluting with a linear gradient of methanol from 90% to 100% (v/v) in 20 ml at a flow rate of 0.8 ml/min. Photoaffinity Labeling of E. coli Complex I with [3H]Azido-Q—The dodecyl maltoside present in purified complex I was replaced with sodium cholate by repeated dilution-concentration using centriprep-30 as described previously (25Bader M.W. Xie T. Yu C.A. Bardwell J.C. J. Biol. Chem. 2000; 275: 26082-26088Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Complex I, as prepared (specific activity, 0.301 μmol of NADH oxidized/min/mg of protein), was in 50 mm NaCl, 0.15% dodecyl maltoside, 50 mm MES/NaOH, pH 6.0. This complex was diluted with 1% sodium cholate to a protein concentration of 1 mg/ml in 50 mm K+/Na+ phosphate buffer, pH 7.5, and concentrated to 10 mg/ml by centrifugation for 30 min at 3,000 rpm, using a JS-42 rotor in a Beckman centrifuge J6-HC. The concentrated complex I was diluted again with the same buffer containing 1% sodium cholate and concentrated again to 10 mg/ml. This process was repeated eight times. The complex (specific activity, 0.267 μmol of NADH oxidized/min/mg of protein) was then adjusted to a protein concentration of 4 mg/ml in the same buffer containing 1% sodium cholate. 300 μl of this solution was mixed with 5 μl of [3H]azido-Q (9.0 mm in 95% ethanol) and incubated at 0 °C for 30 min in the dark. The specific radioactivity of [3H]azido-Q used was 9.7 × 103 cpm/nmol in 95% ethanol and 3.6 × 103 cpm/nmol in the 50 mm K+/Na+ phosphate buffer, pH 7.5, containing 1.0% sodium cholate in the presence of E. coli complex I. This mixture was transferred to a 2-mm light path quartz cuvette that was sealed with paraffin film and mounted on an illuminating apparatus. This assembly was immersed in ice water in a container with a quartz window and illuminated with long-wavelength UV light (Spectroline EN-14, 365-nm wavelength, 23 watts) for 10 min at a distance of 4 cm from the light source. NADH-Q oxidoreductase activity was assayed after reconstitution with asolectin, before and after the illumination. To determine the amount of [3H]azido-Q incorporated into complex I, illuminated samples were spotted on Whatman filter paper and developed with a mixture of chloroform and methanol (2:1, v/v) to remove non-protein-bound [3H]azido-Q. After the paper was air-dried, the origin spot was cut into small pieces and subjected to liquid scintillation counting. Determination of the Distribution of 3H Radioactivity among the Subunits of E. coli Complex I—The illuminated, [3H]azido-Q-treated sample was digested with 1% SDS and 1% β-mercaptoethanol at 37 °C for 2 h before being subjected to SDS-PAGE. The SDS-polyacrylamide gel was prepared according to Schägger and von Jagow (26Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar), except that bisacrylamide was substituted for the cleavable cross-linker, N,N′-diallytartardiamide. Electrophoresis was run at 30 V for 2 h and then at 80 V for another 6 h. After electrophoresis, the gel was stained and de-stained (26Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar). Gels were sliced according to the stained protein bands. The portion containing no protein was also sliced to the same size as that of the protein bands. Gel slices were completely dissolved by incubation in 0.3 ml of 3% periodic acid at room temperature for 1 h. 5 ml of Insta-Gel counting fluid was added, and radioactivity was determined. Isolation of [3H]Azido-Q-labeled NuoM—Part of the digest (see above) was subjected to preparative SDS-PAGE. A small portion (10%) of the SDS-digested protein solution was treated with fluorescamine (100-fold molar excess) for 10 min at 37 °C and placed in the reference wells, located on both edges and the middle of the gel. The SDS-PAGE gel and electrophoresis conditions were as described in the preceding section. The protein bands were visualized by the UV fluorescence in the reference lanes. The SDS-PAGE pattern of the fluorescamine-treated sample was identical to that of the untreated sample, as established by Coomassie Blue staining. The NuoM protein band was excised from the SDS-PAGE gel. The protein was eluted from the combined gel slices with an electro-eluter from Bio-Rad. Protease K Digestion of [3H]Azido-Q-labeled NuoM—Purified [3H]azido-Q labeled NuoM obtained by electro-elution was subjected to a repeated dilution and concentration process using centriprep-30 with a dilution buffer of 30 mm Tris-Cl, pH 7.5, to remove SDS. The final protein concentration was about 1 mg/ml, with the SDS concentration around 0.5%. Protein was then digested with protease K at 37 °Cfor6h using a protease K:NuoM ratio of 1:50 (w/w). Isolation of Ubiquinone-binding Peptides—Aliquots (100 μl) of the protease K-digested NuoM were separated by HPLC on a Supelcosil LC-308 column (C8; 5-μm particles; 300 Å pores; inner diameter, 4.6 mm; length, 25 cm) using a gradient formed from 0.1% trifluoroacetic acid and 90% acetonitrile containing 0.1% trifluoroacetic acid with a flow rate of 0.8 ml/min. 0.8-ml fractions were collected. For each fraction, the absorbance (from 200 to 400 nm) was recorded with a Waters 996 Diode Array Detector, and radioactivity was measured. Peaks with high specific radioactivity were collected, dried, and subjected to peptide sequence analysis. Amino Acid Sequence Determination—Amino acid sequence analyses were done at the Molecular Biology Resource Facility, Saint Francis Hospital of Tulsa Medical Research Institute, University of Oklahoma Health Sciences Center, under the supervision of Dr. Ken Jackson. Effect of Cofactors on the Labeling—Aliquots of 0.94 nmol of NADH-Q oxidoreductase from E. coli in 50 mm K+/Na+ phosphate buffer, pH 7.5, containing 1.0% sodium cholate were incubated with [3H]azido-Q (18.7 nmol) for 30 min at 0 °C in the dark, and then NADH, NAD, or ATP (400 mm, final concentration) plus 1 mm sodium pyruvate were added for activation, and the incubation was continued for 30 min. Residual NADH was oxidized by l-lactate dehydrogenase (type II, from rabbit muscles) to NAD shortly before photoirradiation because it would quench the UV light responsible for photoactivation of [3H]azido-Q. Then, the mixtures were illuminated under UV light for 10 min at 0 °C as described above. Preparation of NADH-Q Oxidoreductase—NADH-Q oxidoreductase, prepared according to the procedure described previously (6Spehr V. Schlitt A. Scheide D. Guénebaut V. Friedrich T. Biochemistry. 1999; 38: 16216-16267Crossref Scopus (42) Google Scholar), contains 0.5 mol of bound coenzyme Q-8 (ubiquinone-40)/mol of protein. When this preparation is titrated with exogenous Q0C10Br, no Q binding is observed, suggesting that the vacant Q-binding site(s) are masked by the detergent dodecyl maltoside or phospholipids and that the binding affinity of Q0C10Br is weaker than that of endogenous Q, phospholipid, and detergent used. Because the binding affinity of azido-Q derivatives to the Q-binding sites of several Q-binding proteins was reported to be weaker than that of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone or Q0C10Br (23Yu L. Yang F.D. Yu C.A. J. Biol. Chem. 1985; 260: 963-973Abstract Full Text PDF PubMed Google Scholar, 27Lee G.Y. He D.Y. Yu L. Yu C.A. J. Biol. Chem. 1995; 270: 6193-6198Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Yang X. Yu L. He D.Y. Yu C.A. J. Biol. Chem. 1998; 273: 31916-31923Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 29Xie T. Yu L. Bader M.W. Bardwell J.C. Yu C.A. J. Biol. Chem. 2002; 277: 1649-1652Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), study of the Q/protein interaction in NADH-Q oxidoreductase, using azido-Q derivatives, requires prior removal of endogenous Q from the complex and an unmasking of the Q-binding sites. When NADH-Q oxidoreductase is subjected to a repeated dilution-centrifugation/concentration process as described under “Experimental Procedures,” endogenous Q8 is partially removed (from 0.5 mol/mol of protein down to 0.2 mol/mol of protein), and the Q-binding site is unmasked. It is evident that unmasking occurs during detergent exchange from the binding of 0.7 mol of exogenous Q0C10Br to 1 mol of NADH-Q oxidoreductase. The binding of exogenous Q0C10Br was determined by titration according to the reported method (25Bader M.W. Xie T. Yu C.A. Bardwell J.C. J. Biol. Chem. 2000; 275: 26082-26088Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The SDS-PAGE pattern of the cholate containing NADH-Q oxidoreductase is similar to that of dodecyl maltoside containing enzyme, except that a protein band with apparent molecular mass of 75 kDa becomes more apparent (see Fig. 1). This protein band was identified by partial NH2-terminal amino acid sequence analysis as a proteolytic digestion product of subunit NuoG. Because sodium cholate-replaced NADH-Q oxidoreductase has its Q-binding site unmasked, it is therefore suitable for use in photoaffinity labeling studies. When the purified and cholate-containing NADH-Q oxidoreductase is incubated with a 20-fold molar excess of azido-Q or 5-azido-2,3-dimethoxy-6-decyl-1,4-benzoquinone for 30 min at 0 °C in the dark and then illuminated with long-wavelength UV light for 10 min, only the azido-Q-treated sample shows inactivation, indicating that azido-Q is suitable for studying the Q/protein interaction in this complex. This azido-Q derivative was used previously to identify the Q-binding sites in succinate-Q oxidoreductases (27Lee G.Y. He D.Y. Yu L. Yu C.A. J. Biol. Chem. 1995; 270: 6193-6198Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Yang X. Yu L. He D.Y. Yu C.A. J. Biol. Chem. 1998; 273: 31916-31923Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 29Xie T. Yu L. Bader M.W. Bardwell J.C. Yu C.A. J. Biol. Chem. 2002; 277: 1649-1652Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 30Shenoy S.K. Yu L. Yu C.A. J. Biol. Chem. 1997; 272: 17867-17872Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and cytochrome bc 1 complexes (23Yu L. Yang F.D. Yu C.A. J. Biol. Chem. 1985; 260: 963-973Abstract Full Text PDF PubMed Google Scholar) from several sources. Azido-Q Concentration-dependent Inactivation of NADH-Q Oxidoreductase— Fig. 2 shows that when NADH-Q oxidoreductase is incubated with various concentrations of azido-Q and illuminated, activity decreases as the concentration of azido-Q is increased. Maximum inactivation of 40% is obtained when 20 mol of azido-Q per mol of NADH-Q oxidoreductase is used. Inactivation is not due to the inhibition of NADH-Q oxidoreductase by photolyzed products of azido-Q because when azido-Q is photolyzed in the absence of NADH-Q oxidoreductase and then mixed with the enzyme, no inhibition is observed. Inactivation is also not due to protein damage by UV radiation because when the enzyme alone is illuminated, no activity loss is observed. Because the activity of the azido-Q-treated NADH-Q oxidoreductase, after illumination, is assayed in the presence of excess Q1 (60 μm), the extent of inactivation should be proportional to the fraction of the Q-binding sites covalently linked to azido-Q. Correlation between Azido-Q Incorporation and Inactivation of NADH-Q Oxidoreductase—To further confirm that the inactivation observed results from covalent linkage of azido-Q to protein in the complex, azido-Q uptake and the extent of inactivation were determined for different periods of illumination. As shown in Fig. 3, when the complex is treated with a 20-fold molar excess of azido-Q and illuminated for different time periods, activity decreases as illumination time increases; maximum inactivation (40%) is reached at 10 min. Moreover, the amount of azido-Q incorporated into protein parallels the extent of inactivation, until the maximum is reached, suggesting that inactivation results from binding of azido-Q to the Q-binding site. Although illumination for longer than 10 min causes no further decrease in activity, azido-Q uptake continues, but at a slower rate, indicating that this incorporation is due to nonspecific binding of azido-Q to protein. It should be mentioned that a control sample containing the same amount of ethanol, illuminated under identical conditions, shows little (<5%) activity loss over the time periods studied. Identification of Q-binding Subunit in NADH-Q Oxidoreductase by Photoaffinity Labeling with [3H]Azido-Q Derivatives— Because the uptake of azido-Q derivative by NADH-Q oxidoreductase upon illumination is correlated to the enzymatic inactivation, it is reasonable to assume that the azido-Q derivative is bound specifically to the Q-binding site(s). Thus, the distribution of the covalently bound azido-Q among subunits of NADH-Q oxidoreductase after SDS-PAGE indicates the specific Q-binding protein in this enzyme complex. Fig. 4 shows the 3H radioactivity distribution among subunits of NADH-Q oxidoreductase. The advantage of using the acrylamide/N,N′-diallytartardiamide gel system, rather than the commonly used acrylamide/bisacrylamide system, is that the gel slices can be completely dissolved in 3% periodic acid, and this solution can be used directly for radioactivity determination. The acrylamide/N,N′-diallytartardiamide gel system has been used to identify Q-binding proteins in a bacterial reaction center (31Marinetti T.D. Okamura M.Y. Feher G. Biochemistry. 1979; 18: 3126-3133Crossref PubMed Scopus (33) Google Scholar) and in mitochondrial ubiquinol-cytochrome c reductase (23Yu L. Yang F.D. Yu C.A. J. Biol. Chem. 1985; 260: 963-973Abstract Full Text PDF PubMed Google Scholar). The electrophoretic pattern of illuminated, azido-Q-treated NADH-Q oxidoreductase obtained with the acrylamide/N,N′-diallytartardiamide gel system is similar to that obtained from the acrylamide/bisacrylamide gel system; 11 major protein bands are observed in each (by Coomassie Blue staining). Radioactivity is found in protein band 5, suggesting that this subunit provides the Q-binding site. No radioactivity is found in slices from a gel loaded with illuminated buffer containing [3H]azido-Q and 1% sodium cholate. Because the amount of radioactivity in protein band 5 was directly proportional to the extent of inactivation of the oxidoreductase, participation of this protein in Q binding is established. This radioactive protein band in SDS-PAGE of the illuminated [3H]azido-Q-treated NADH-Q oxidoreductase is identified as NuoM, based on the identification of two NuoM peptides in the protease K digest of the labeled protein. Peptide peaks with retention times of 29.9 and 45.8 min obtained from HPLC separation of protease K-digested labeled protein have the partial NH2-terminal amino acid sequence of NH2-SAAGLFI- and NH2-LPDAH- corresponding to residues 351–357 and 244–248 of NuoM subunit, respectively. The identification of NuoM as the ubiquinone-binding subunit of NADH-Q oxidoreductase is consistent with the report (32Hofhaus G. Attardi G. EMBO J. 1993; 12: 3043-3048Crossref PubMed Scopus (87) Google Scholar) that human complex I lacking the mtDNA-encoded subunit ND4, due to a frameshift mutation in the gene, has no NADH:Q1 oxidoreductase activity but has normal NADH: Fe(CN)6 oxidoreductase activity. ND4 of human complex I is the counterpart of NuoM of E. coli enzyme (33Yagi T. Matsuno-Yagi A. Biochemistry. 2003; 42: 2266-2274Crossref PubMed Scopus (265) Google Scholar). Isolation and Characterization of Ubiquinone-binding Peptides of NuoM—In order to identify the Q-binding domain in NuoM through isolation and sequencing of an azido-Q-linked peptide, it is absolutely necessary that the isolated azido-Q-labeled NuoM be free from contamination with unbound azido-Q and completely susceptible to proteolytic enzyme digestion. [3H]Azido-Q-labeled NuoM was isolated from illuminated, [3H]azido-Q-treated NADH-Q oxidoreductase by a procedure involving preparative SDS-PAGE, electrophoretic elution, and repeated dilution/concentration with centriprep-30. The SDS-PAGE step removes non-protein-bound azido-Q adducts. The SDS conce" @default.
• W2068754574 created "2016-06-24" @default.
• W2068754574 creator A5013929528 @default.
• W2068754574 creator A5015246033 @default.
• W2068754574 creator A5025917396 @default.
• W2068754574 creator A5031977237 @default.
• W2068754574 creator A5060633211 @default.
• W2068754574 creator A5062250993 @default.
• W2068754574 creator A5074681938 @default.
• W2068754574 date "2003-07-01" @default.
• W2068754574 modified "2023-10-06" @default.
• W2068754574 title "The Ubiquinone-binding Site in NADH:Ubiquinone Oxidoreductase from Escherichia coli" @default.
• W2068754574 cites W142797207 @default.
• W2068754574 cites W1487169814 @default.
• W2068754574 cites W1565276006 @default.
• W2068754574 cites W1570597278 @default.
• W2068754574 cites W1967694260 @default.
• W2068754574 cites W1975085160 @default.
• W2068754574 cites W1981628967 @default.
• W2068754574 cites W1983487087 @default.
• W2068754574 cites W1998730765 @default.
• W2068754574 cites W1999660119 @default.
• W2068754574 cites W2000389514 @default.
• W2068754574 cites W2001063971 @default.
• W2068754574 cites W2001083394 @default.
• W2068754574 cites W2005969857 @default.
• W2068754574 cites W2008750261 @default.
• W2068754574 cites W2012003686 @default.
• W2068754574 cites W2014960917 @default.
• W2068754574 cites W2018287750 @default.
• W2068754574 cites W2026333217 @default.
• W2068754574 cites W2033555996 @default.
• W2068754574 cites W2036607640 @default.
• W2068754574 cites W2041867957 @default.
• W2068754574 cites W2041950514 @default.
• W2068754574 cites W2044417730 @default.
• W2068754574 cites W2054252561 @default.
• W2068754574 cites W2058182576 @default.
• W2068754574 cites W2061745332 @default.
• W2068754574 cites W2065619661 @default.
• W2068754574 cites W2067434074 @default.
• W2068754574 cites W2076472217 @default.
• W2068754574 cites W2087524379 @default.
• W2068754574 cites W2090813109 @default.
• W2068754574 cites W2092999001 @default.
• W2068754574 cites W2108436623 @default.
• W2068754574 cites W2120684043 @default.
• W2068754574 cites W2121147116 @default.
• W2068754574 cites W2122437957 @default.
• W2068754574 cites W2146396614 @default.
• W2068754574 cites W315113715 @default.
• W2068754574 cites W4245994416 @default.
• W2068754574 cites W85724712 @default.
• W2068754574 cites W984281319 @default.
• W2068754574 doi "https://doi.org/10.1074/jbc.m302361200" @default.
• W2068754574 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12730198" @default.
• W2068754574 hasPublicationYear "2003" @default.
• W2068754574 type Work @default.
• W2068754574 sameAs 2068754574 @default.
• W2068754574 citedByCount "62" @default.
• W2068754574 countsByYear W20687545742012 @default.
• W2068754574 countsByYear W20687545742013 @default.
• W2068754574 countsByYear W20687545742014 @default.
• W2068754574 countsByYear W20687545742017 @default.
• W2068754574 countsByYear W20687545742023 @default.
• W2068754574 crossrefType "journal-article" @default.
• W2068754574 hasAuthorship W2068754574A5013929528 @default.
• W2068754574 hasAuthorship W2068754574A5015246033 @default.
• W2068754574 hasAuthorship W2068754574A5025917396 @default.
• W2068754574 hasAuthorship W2068754574A5031977237 @default.
• W2068754574 hasAuthorship W2068754574A5060633211 @default.
• W2068754574 hasAuthorship W2068754574A5062250993 @default.
• W2068754574 hasAuthorship W2068754574A5074681938 @default.
• W2068754574 hasBestOaLocation W20687545741 @default.
• W2068754574 hasConcept C104317684 @default.
• W2068754574 hasConcept C107824862 @default.
• W2068754574 hasConcept C181199279 @default.
• W2068754574 hasConcept C185592680 @default.
• W2068754574 hasConcept C2779268744 @default.
• W2068754574 hasConcept C547475151 @default.
• W2068754574 hasConcept C55493867 @default.
• W2068754574 hasConcept C86803240 @default.
• W2068754574 hasConceptScore W2068754574C104317684 @default.
• W2068754574 hasConceptScore W2068754574C107824862 @default.
• W2068754574 hasConceptScore W2068754574C181199279 @default.
• W2068754574 hasConceptScore W2068754574C185592680 @default.
• W2068754574 hasConceptScore W2068754574C2779268744 @default.
• W2068754574 hasConceptScore W2068754574C547475151 @default.
• W2068754574 hasConceptScore W2068754574C55493867 @default.
• W2068754574 hasConceptScore W2068754574C86803240 @default.
• W2068754574 hasIssue "28" @default.
• W2068754574 hasLocation W20687545741 @default.
• W2068754574 hasOpenAccess W2068754574 @default.
• W2068754574 hasPrimaryLocation W20687545741 @default.
• W2068754574 hasRelatedWork W1797475586 @default.
• W2068754574 hasRelatedWork W1953757588 @default.
• W2068754574 hasRelatedWork W1967796626 @default.
• W2068754574 hasRelatedWork W1968348916 @default.

• next