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• W2049632955 abstract "Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross-linked domain was identified by reverse-phase high performance liquid chromatography of subunit I peptides produced by in-gel double digestion with lysyl endopeptidase and endoproteinase Asp-N. Electrospray ionization quadrupole time-of-flight mass spectrometry determined the amino acid sequence of the peptide (m/z 1047.5) to be Glu278–Lys283, where a photoproduct of azido-Q2 was linked to the carboxylic side chain of I-Glu280. This study demonstrated directly that the N-terminal region of periplasmic loop VI/VII (Q-loop) is a part of the quinol oxidation site and indicates that the 2- and 3-methoxy groups of the quinone ring are in the close vicinity of I-Glu280. Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross-linked domain was identified by reverse-phase high performance liquid chromatography of subunit I peptides produced by in-gel double digestion with lysyl endopeptidase and endoproteinase Asp-N. Electrospray ionization quadrupole time-of-flight mass spectrometry determined the amino acid sequence of the peptide (m/z 1047.5) to be Glu278–Lys283, where a photoproduct of azido-Q2 was linked to the carboxylic side chain of I-Glu280. This study demonstrated directly that the N-terminal region of periplasmic loop VI/VII (Q-loop) is a part of the quinol oxidation site and indicates that the 2- and 3-methoxy groups of the quinone ring are in the close vicinity of I-Glu280. Cytochrome bd (CydAB) is a heterodimeric ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and is predominantly expressed under microaerophilic growth conditions (see Refs. 1Ingledew W.J. Poole R.K. Microbiol. Rev. 1984; 48: 222-271Crossref PubMed Google Scholar, 2Jünemann S. Biochim. Biophys. Acta. 1997; 1321: 107-127Crossref PubMed Scopus (228) Google Scholar, 3Mogi T. Tsubaki M. Hori H. Miyoshi H. Nakamura H. Anraku Y. J. Biochem. Mol. Biol. Biophys. 1998; 2: 79-110Google Scholar for reviews). It catalyzes dioxygen reduction with two molecules of ubiquinol-8 (Q8H2), 2The abbreviations used are: Qn, 2,3-dimethoxy-5-methyl-6-prenyl-1,4-benzoquinone; QnH2, a reduced form of Qn; 2-azido-Q2, 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone; 3-azido-Q2, 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone; 3-azido-2-methyl-5-methoxy-BQ2, 2-methyl-3-azido-5-methoxy-6-geranyl-1,4-benzoquinone; 3-azido-2-methyl-5-methoxy-BQ2s, 2-methyl-3-azido-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone; 3-azido-2-methyl-5-methoxy-dBQ, 2-methyl-3-azido-5-methoxy-6-n-decyl-1,4-benzoquinone; ESI, electrospray ionization; HPLC, high performance liquid chromatography; HQNO, 2-heptyl-hydroxyquinoline-N-oxide; Lep, lysyl endopeptidase; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; TOF, time-of-flight. leading to the release of four protons from quinols to the periplasm. Through a putative proton channel, four protons used for dioxygen reduction are taken up from the cytoplasm and delivered to the dioxygen reduction site at the periplasmic side of the cytoplasmic membrane (4Zhang J. Barquera B. Gennis R.B. FEBS Lett. 2004; 561: 58-62Crossref PubMed Scopus (32) Google Scholar). During dioxygen reduction, cytochrome bd generates an electrochemical proton gradient (ΔpH and membrane potential) across the membrane through apparent transmembrane movement of four chemical protons (5Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 6Miller M.J. Gennis R.B. J. Biol. Chem. 1985; 260: 14003-14008Abstract Full Text PDF PubMed Google Scholar, 7Jasaitis A. Borisov V.B. Belevich N.P. Morgan J.E. Konstantinov A.A. Verkohsky M.I. Biochemistry. 2000; 39: 13800-13809Crossref PubMed Scopus (72) Google Scholar). In contrast to cytochrome bo, an alternative oxidase under highly aerated growth conditions, cytochrome bd has no proton pumping activity and does not belong to the heme-copper terminal oxidase superfamily. On the basis of spectroscopic and ligand binding studies, three distinct redox metal centers have been identified as heme b558, heme b595, and heme d (see Ref. 8Tsubaki M. Hori H. Mogi T. J. Inorg. Biochem. 2000; 82: 19-25Crossref PubMed Scopus (28) Google Scholar for a review). Unlike cytochrome bo, cytochrome bd does not contain a tightly bound Q8. Heme b558 is a low spin protoheme IX and is ligated by I-His186 (helix V) and I-Met393 (helix VII) of subunit I (CydA) (9Fang G.H. Lin R.J. Gennis R.B. J. Biol. Chem. 1989; 264: 8026-8032Abstract Full Text PDF PubMed Google Scholar). Heme b595 is a high spin protoheme IX bound to I-His19 (helix I) of subunit I (9Fang G.H. Lin R.J. Gennis R.B. J. Biol. Chem. 1989; 264: 8026-8032Abstract Full Text PDF PubMed Google Scholar) and mediates electron transfer from heme b558 to heme d, where dioxygen is reduced to water (10Poole R.K. Williams H.D. FEBS Lett. 1987; 217: 49-52Crossref PubMed Scopus (38) Google Scholar, 11Hill B.C. Hill J.J. Gennis R.B. Biochemistry. 1994; 33: 15110-15115Crossref PubMed Scopus (57) Google Scholar, 12Kobayashi K. Tagawa S. Mogi T. Biochemistry. 1999; 38: 5913-5917Crossref PubMed Scopus (27) Google Scholar, 13Zhang J. Hellwig P. Osborne J.P. Huang H. Moenne-Loccoz P. Konstantinov A.A. Gennis R.B. Biochemistry. 2001; 40: 8548-8556Crossref PubMed Scopus (33) Google Scholar). Heme d is a high spin chlorin bound to an unidentified nitrogenous ligand (14Hirota S. Mogi T. Ogura T. Anraku Y. Gennis R.B. Kitagawa T. Biospectroscopy. 1995; 1: 305-311Crossref Scopus (20) Google Scholar, 15Sun J. Kahlow M.A. Kaysser T.M. Osborne J. Hill J.J. Rohlfs R.J. Hille R. Gennis R.B. Loehr T.M. Biochemistry. 1996; 35: 2403-2412Crossref PubMed Scopus (47) Google Scholar, 16Hori H. Tsubaki M. Mogi T. Anraku Y. J. Biol. Chem. 1996; 271: 9254-9258Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and forms a di-heme binuclear center with heme b595 (16Hori H. Tsubaki M. Mogi T. Anraku Y. J. Biol. Chem. 1996; 271: 9254-9258Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 17Hill J.J. Alben J.O. Gennis R.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5863-5867Crossref PubMed Scopus (109) Google Scholar). Topological analysis suggests that all of the hemes are located at the periplasmic end of transmembrane helices (4Zhang J. Barquera B. Gennis R.B. FEBS Lett. 2004; 561: 58-62Crossref PubMed Scopus (32) Google Scholar). In loop VI/VII (Q-loop) of subunit I, binding of monoclonal antibodies to 252KLAAIEAEWET262 (18Kranz R.G. Gennis R.B. J. Biol. Chem. 1984; 259: 7998-8003Abstract Full Text PDF PubMed Google Scholar, 19Dueweke T.J. Gennis R.B. J. Biol. Chem. 1990; 265: 4273-4277Abstract Full Text PDF PubMed Google Scholar) and proteolytic cleavage with trypsin at I-Tyr290 or chymotrypsin at I-Arg298 (20Lorence R.M. Carter K. Gennis R.B. Matsushita K. Kaback H.R. J. Biol. Chem. 1988; 263: 5271-5276Abstract Full Text PDF PubMed Google Scholar, 21Dueweke T.J. Gennis R.B. Biochemistry. 1991; 30: 3401-3406Crossref PubMed Scopus (55) Google Scholar) suppressed ubiquinol oxidase activity. Photoaffinity labeling studies with 2-methyl-3-azido-5-methoxy-6-(3,7-dimethyl-[3H]octyl)-1,4-benzoquinone ([3H]3-azido-2-methyl-5-methoxy-BQ2s) indicate the presence of the quinol oxidation site in subunit I (22Yang F.D. Yu L. Yu C.A. Lorence R.M. Gennis R.B. J. Biol. Chem. 1986; 261: 14987-14990Abstract Full Text PDF PubMed Google Scholar). These results suggest that periplasmic loop VI/VII in subunit I is involved in the ubiquinol oxidation site. Inhibitor binding studies indicated the close proximity of the quinol oxidation site to heme b558 (23Jünemann S. Wrigglesworth J.M. FEBS Lett. 1994; 345: 198-202Crossref PubMed Scopus (28) Google Scholar, 24Jünemann S. Wrigglesworth J.M. Rich P.R. Biochemistry. 1997; 36: 9323-9331Crossref PubMed Scopus (40) Google Scholar). Recently, the x-ray structure of the E. coli cytochrome bo has been solved at 3.5-Å resolution, but the structure of the quinol oxidation site remains unknown (25Abramson J. Riistama S. Larsson G. Jasaitis A. Svensson-Ek M. Laakkonen L. Puustinen A. Iwata S. Wikström M. Nat. Struct. Biol. 2000; 7: 910-917Crossref PubMed Scopus (358) Google Scholar). A low affinity for substrates and the difficulty in anaerobically keeping substrates as reduced forms make it difficult to crystallize a quinol-bound form. Currently, no atomic structure for cytochrome bd is available; thus photoaffinity cross-linking with photolabile substrate analogs is an alternative approach to identify the substrate-binding site. We have developed an efficient method for synthesis of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone (2-azido-Q2) and 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone (3-azido-Q2) (26Sakamoto K. Nomura K. Miyoshi H. J. Pesticide Sci. 2002; 27: 147-149Crossref Scopus (10) Google Scholar), which are more closely related to native ubiquinone (2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone) than azidoquinones used for previous studies (3-azido-2-methyl-5-methoxy-BQ2s for cytochrome bd (22Yang F.D. Yu L. Yu C.A. Lorence R.M. Gennis R.B. J. Biol. Chem. 1986; 261: 14987-14990Abstract Full Text PDF PubMed Google Scholar), 2-methyl-3-azido-5-methoxy-6-geranyl-1,4-benzoquinone (3-azido-2-methyl-5-methoxy-BQ2) for bovine Complex III (27Yu L. Yang F.-D. Yu C-A. J. Biol. Chem. 1985; 260: 963-973Abstract Full Text PDF PubMed Google Scholar), and the E. coli cytochrome bo (28Welter R. Gu L.Q. Yu L. Yu C.A. Rumbley J. Gennis R.B. J. Biol. Chem. 1994; 269: 28834-28838Abstract Full Text PDF PubMed Google Scholar, 29Tsatsos P.H. Reynolds K. Nickels E.F. He D.-Y. Yu C.-A. Gennis R.B. Biochemistry. 1998; 37: 9884-9888Crossref PubMed Scopus (34) Google Scholar) and the E. coli Complex II (30Yang X. Yu L. He D. Yu C.-A. J. Biol. Chem. 1998; 273: 31916-31923Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and 2-methyl-3-azido-5-methoxy-6-n-decyl-1,4-benzoquinone (3-azido-2-methyl-5-methoxy-dBQ) for bovine Complex II (31Lee 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, 32Shenoy 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 Complex I (33Gong X. Xie T. Yu L. Hesterberg M. Scheide D. Friedrich T. Yu C.-A. J. Biol. Chem. 2003; 278: 25731-25737Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Here we report the azido-Q2H2-labeled site in cytochrome bd, determined by mass spectrometry (MS). Reduced forms of 2-azido- and 3-azido-Q2 serve as efficient electron donors to cytochrome bd, and UV illumination in the presence of azido-Q2H2 inactivated the quinol oxidase activity. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS identified subunit I as the azido-Q2-labeled site. In-gel digestion followed by electrospray ionization tandem mass spectrometry (ESI MS/MS) revealed the cross-linked amino acid residue for the first time. The oxidized form of a photoproduct of azido-Q2 (azido-Q *2) was covalently linked to the carboxylic side chain of I-Glu280 in the hexapeptide 278EEETNK283, that is present in the Q-loop. This study demonstrated directly that the N-terminal region of periplasmic loop VI/VII (Q-loop) is a part of the quinol oxidation site and indicates that the 2- and 3-methoxy groups of the quinone ring are in the close vicinity of I-Glu280 in loop VI/VII. Purification of Cytochrome bd and Quinol Oxidase Assay—The enzyme was isolated from the cytochrome bd-overproducing strain GR84N/pNG2 (34Green G.N. Kranz R.G. Lorence R.M. Gennis R.B. J. Biol. Chem. 1984; 259: 7994-7997Abstract Full Text PDF PubMed Google Scholar), as described previously (14Hirota S. Mogi T. Ogura T. Anraku Y. Gennis R.B. Kitagawa T. Biospectroscopy. 1995; 1: 305-311Crossref Scopus (20) Google Scholar). The concentration of the enzyme was calculated from the Soret absorption of the air-oxidized form by using an extinction coefficient of 223,000 m–1 cm–1 (35Tsubaki M. Hori H. Mogi T. Anraku Y. J. Biol. Chem. 1995; 270: 28565-28569Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The purified enzyme in 50 mm potassium phosphate (pH 6.8) containing 0.1% sucrose monolaurate (Mitsubishi-Kagaku Foods Co., Tokyo) was stored at –80 °C until use. The enzyme activity was determined at 25 °C with a JASCO V-550 UV-visible spectrophotometer, as described previously (36Sakamoto K. Miyoshi H. Takegami K. Mogi T. Anraku Y. Iwamura H. J. Biol. Chem. 1996; 271: 29897-29902Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The reaction mixture (1 ml) contained 50 mm Tris-HCl (pH 7.3), 0.1% sucrose monolaurate, and 40 nm cytochrome bd. The reaction was started by the addition of a reduced form of Q1, a kind gift from Eisai Co. (Tokyo, Japan), at a final concentration of 0.14 mm. Photoaffinity Labeling of Cytochrome bd—2-Azido-Q2 and 3-azido-Q2 were synthesized as previously described (26Sakamoto K. Nomura K. Miyoshi H. J. Pesticide Sci. 2002; 27: 147-149Crossref Scopus (10) Google Scholar). Cytochrome bd (10 μm in 0.5 ml of 50 mm potassium phosphate (pH 7.4) containing 0.1% sucrose monolaurate) was placed in a quartz cuvette and incubated on ice for 10 min. A 4-fold molar excess of reduced azido-Q2 was added to the enzyme solution and subjected to illumination on ice with long wavelength UV light (Black-Ray model B-100 A; UVP, Upland, CA; 365 nm) at a distance of 5 cm from the light source. For the competition with azido-Q2H2, 2-n-heptyl-hydroxyquinoline-N-oxide (HQNO; Sigma-Aldrich) was added at a final concentration of 1 mm. Q1H2 oxidase activity was measured before and after the illumination. In-gel Digestion of Subunit I—Samples containing 20 μg of proteins were subjected to 10% SDS-PAGE as described by Laemmli (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar), and protein bands were stained with Coomassie Brilliant Blue R-250. In-gel digestion was performed by the method of Hellman et al. (38Hellman U. Wernstedt C. Gonez J. Heldin C.H. Anal. Biochem. 1995; 224: 451-455Crossref PubMed Scopus (686) Google Scholar). Gel pieces containing subunit I band were washed three times with 1 ml of 70% (v/v) acetonitrile, dried in a vacuum centrifuge, and rehydrated with 0.l ml of the digestion buffer. The enzymatic cleavage was allowed to proceed at 37 °C for 18 h at a protease-to-protein ratio of 1:200. The digestion buffers used for lysyl endopeptidase (Lep; Wako Pure Chemical Industries, Osaka) and endoproteinase Asp-N (Roche Applied Science) were 50 mm Tris-HCl (pH 9.0) and 20 mm sodium borate (pH 7.0), respectively. Digestion was terminated by the addition of 1 μl of acetic acid. In-gel CNBr cleavage was performed in 0.1 ml of 70% trifluoroacetic acid at a CNBr-to-protein ratio of 1: 10. The gel pieces were incubated in the dark at room temperature for 18 h. For the double digestion, the gel pieces were first incubated with Lep, washed three times with 1 ml of 70% (v/v) acetonitrile, and then treated with Asp-N or CNBr as described above. The peptides were extracted twice from the gel pieces with 100 μl of 60% (v/v) acetonitrile and concentrated with ZipTip μ-C18 (Millipore) to 20 μl in 60% acetonitrile containing 0.1% trifluoroacetic acid. MALDI-TOF MS—The samples were analyzed on an AXIMA-CFR plus (Shimadzu Co., Kyoto) TOF mass spectrometer in a linear (for proteins) or reflection (for peptides) mode with positive ion detection. The matrix solution was 50% acetonitrile saturated with α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich). One-μl of desalted proteins or peptides were mixed with an equal amount of the matrix solution on the target plate and dried. A nitrogen laser at 337 nm was used to desorb solute molecules from the sample plate. The coarse laser energy was set to 50% with fine adjustment for each sample, and 1–100 laser shots were accumulated for each spectrum. A voltage of 20 kV was established in the source region, and the microchannel detector was set at 2.8 kV. MS spectra were calibrated externally with rabbit muscle aldolase and bovine serum albumin (molecular masses were 39,212 and 66,430 Da, respectively) for proteins and human bradykinin fragment, human angiotensin II, P14R, human ACTH fragment 18–39, and bovine insulin oxidized B chain (molecular masses were 757.40, 1,046.54, 1,533.86, 2,465.20, and 3,494.65 Da, respectively) for peptides. Data analysis was carried out with Kompact (Shimadzu Co.). Reverse-phase HPLC—Subunit I peptides (100 μg) produced by double digestion with Lep and Asp-N were separated by reverse-phase HPLC on a Develosil 300C8-HG-5 column (4.6-mm inner diameter × 15 cm; Nomura-Kagaku Co.) using a gradient formed from 0.1% acetic acid and 30% acetonitrile containing 0.1% acetic acid with a flow rate of 1 ml/min. Elution profiles of peptides were monitored at 212 nm with a SPD-M10AVP Shimadzu photodiode array detector, and 0.5-ml fractions were collected. The fractions were dried up with a vacuum centrifuge, and the peptides were dissolved in 30 μl of 0.1% formic acid. ESI MS—The samples were applied to a C18 column (0.1-mm inner diameter × 25 cm; GL Science) coupled to ESI source and analyzed on a Q-TOF2 (Micromass) quadrupole TOF mass spectrometer. The analysis was carried out in the positive ion detection mode with a capillary voltage of 2.8 kV and a sampling cone voltage of 40 V. For MS experiments, the quadrupole analyzer was used in wide band pass mode, and the microchannel plate detector was set at 2.7 kV. For MS/MS experiments the quadrupole (first) analyzer was used to select sequentially the peaks of interest in the m/z spectrum, allowing one particular precursor or parent mass to proceed through the collision cell into the TOF (second) analyzer. Argon gas was admitted into the collision cell so that the pressure in that region increased by a factor of 10, and the collision energy was 10 eV. The resulting fragment ions were analyzed, and MS/MS spectra acquired over the appropriate m/z range and were deconvoluted with MaxEnt3 (Micromass). Data processing was achieved manually with the aid of the PepSeq program of MassLynx package (Micromass). The nomenclature used for fragment ions was N-terminal C=O-containing fragments (b type) and C-terminal N-H-containing fragments (y type), which were derived by the cleavage at the middle of peptide bonds (39Biemann K. Methods Enzymol. 1990; 193: 886-887Crossref PubMed Scopus (425) Google Scholar). Photo Inactivation of Cytochrome bd with Azido-Q2H2—Electron-donating activities of 2-azido- and 3-azido-Q2H2 to cytochrome bd are 97 and 94%, respectively, of that of Q2H2 at 0.04 mm, and thus they are suitable for probing the structure of the quinol oxidation site. After 15 min of illumination with UV light in the presence and absence of 0.04 mm 3-azido-Q2H2, Q1H2 oxidase activity was decreased to 28 and 68%, respectively, of the original activity without illumination (Fig. 1). Monophasic decay of the oxidase activity indicates the labeling of the quinol oxidation site with a single azido-Q2H2 molecule (data not shown). UV illumination in the presence of 0.04 mm 2-azido-Q2H2 resulted in a 40% loss of the quinol oxidase activity. Inhibition levels are comparable with 35–50% reported for reactions of azidoquinones with cytochrome bd (22Yang F.D. Yu L. Yu C.A. Lorence R.M. Gennis R.B. J. Biol. Chem. 1986; 261: 14987-14990Abstract Full Text PDF PubMed Google Scholar), cytochrome bo (28Welter R. Gu L.Q. Yu L. Yu C.A. Rumbley J. Gennis R.B. J. Biol. Chem. 1994; 269: 28834-28838Abstract Full Text PDF PubMed Google Scholar), succinate dehydrogenase (30Yang X. Yu L. He D. Yu C.-A. J. Biol. Chem. 1998; 273: 31916-31923Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and DsbB (56Xie T. Yu L. Bader M.W. Barwell J.C.A. Yu C.-A. J. Biol. Chem. 2002; 277: 1649-1652Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). A difference in the inactivation levels between 2-azido- and 3-azido-Q2H2 may be related to interactions of their photoproducts with the protein. The incomplete inactivation with azido-Q2H2 may be partly due to the intramolecular reaction with the nearby methoxy group on the quinone ring. These results indicate that the photoaffinity labeling of azido-Q2H2 to the quinol-binding site inactivates the quinol oxidase activity of cytochrome bd. Our azido-Q2 derivatives are not radioactive; thus labeling profiles were examined by mass spectrometry. In MALDI-TOF mass spectra of the purified cytochrome bd, subunits I and II were identified as singly protonated ions at m/z 58,160 ± 60 and 42,410 ± 70 (n = 5) (Fig. 2), respectively, which are comparable with the calculated molecular masses of 58,205 3Because of a sequencing error (I-Phe213 to Leu) found in pNG2, the mass of subunit I was corrected to 58,205 from 58,171 (40Miller M.J. Hermodson J.G. Gennis R.B. J. Biol. Chem. 1986; 263: 5235-5240Abstract Full Text PDF Google Scholar). and 42,460 Da, respectively, from the deduced amino acid sequence (40Miller M.J. Hermodson J.G. Gennis R.B. J. Biol. Chem. 1986; 263: 5235-5240Abstract Full Text PDF Google Scholar). Upon UV illumination in the presence of 2-azido- or 3-azido-Q2H2, a part of subunit I molecules increased molecular mass, indicated by the presence of a shoulder peak at a higher m/z region (Fig. 2). In contrast, subunit II remained as a sharp peak, and nonspecific binding to subunit II (22Yang F.D. Yu L. Yu C.A. Lorence R.M. Gennis R.B. J. Biol. Chem. 1986; 261: 14987-14990Abstract Full Text PDF PubMed Google Scholar) did not occur for our azido-Q2H2 derivatives. These observations indicate that the quinol-binding site of cytochrome bd is located within subunit I and that the labeling of subunit I with azido-Q2H2 resulted in the inactivation of the quinol oxidase activity. Identification of the Azido-Q2-cross-linked Peptide—For the identification of the azido-Q2H2-cross-linked site by MS, subunit I was separated from subunit II and free substrates and its photoproducts by 10% SDS-PAGE and subjected to in-gel digestion with endoproteinase Lep and/or Asp-N or in-gel cleavage with CNBr. In combination with proteolytic and chemical cleavage, peptides corresponding to 89.1% of the total sequence and 99.4% of the Q-loop were recovered and identified (supplemental Table S1). The coverage was comparable with 79% for subunit II of the E. coli cytochrome bo by MALDI MS (29Tsatsos P.H. Reynolds K. Nickels E.F. He D.-Y. Yu C.-A. Gennis R.B. Biochemistry. 1998; 37: 9884-9888Crossref PubMed Scopus (34) Google Scholar), 83% for the E. coli H+/galactose symporter GalP by ESI MS (41Venter H. Ashcroft A.E. Keen J.F. Henderson P.J.F. Herbert R.B. Biochem. J. 2002; 363: 243-252Crossref PubMed Scopus (30) Google Scholar), and 97% for the E. coli H+/lactose symporter LacY by ESI MS (42Weinglass A.B. Whitelegge J.P. Hu Y. Verner G.E. Faull K.F. Kaback H.R. EMBO J. 2003; 22: 1467-1477Crossref PubMed Scopus (52) Google Scholar). In the Lep/Asp-N double digest of subunit I labeled with 2-azido- or 3-azido-Q2H2, we reproducibly identified a new peak at 29.5 min in reverse-phase HPLC (Fig. 3, B and C) and at m/z 1047.5 in MALDI-TOF mass spectra (Fig. 4B). Photoaffinity labeling with 3-azido-Q2H2 in the presence of 1 mm HQNO, a competitive inhibitor (I50,7 μm), eliminated the m/z 1047.5 peak (Fig. 4C), confirming the attachment of the photoproduct to this peptide.FIGURE 4MALDI-TOF mass spectra of in-gel double-digested subunit I with Lep and Asp-N. A digest of subunit I without (A) and with UV illumination in the presence of 40μm 3-azido-Q2H2 (B) or 40 μm 3-azido-Q2H2 plus 1 mm HQNO (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Identification of Azido-Q2-labeled Amino Acid Residue—The Lep/Asp-N double-digested peptide with m/z 1047.5 was subjected to ESI-Q-TOF tandem mass spectrometry (MS/MS). Doubly charged N-terminal fragment ions (b-series) and C-terminal fragment ions (y-series) thus yielded identified the amino acid sequence of this peptide to be the hexapeptide 278EEETNK283 (monoisotopic mass of 748.32) and demonstrated that a photoproduct (300.36 Da) of 2-azido-(data not shown) and 3-azido-Q2H2 (Fig. 5) is covalently linked to the carboxylic side chain of I-Glu280 (m/z 128.03) via the COO-NH linkage (Fig. 6D). The b′ series (b3′,b4′, and b5′), which lost the cross-linked Azido-Q2*, confirmed this assignment. Our result demonstrated that the 2- and 3-substituents of the quinone ring are in the vicinity of I-Glu280 in the periplasmic loop VI/VII of subunit I. The observed mass for the photoproduct indicates a loss of four hydrogen from the expected structure (304.39 Da) (Fig. 6C), because of the auto-oxidation of the reduced photoproduct during the preparation and isolation of the peptide.FIGURE 6Possible mechanism for the photoaffinity labeling with azido-Q2H2. A–D, reactions of cytochrome bd with 3-azido-Q2H2; E–H, reactions of free 3-azido-Q2 in methanol or acetic acid. Structures of products 1 (G) and 2 (H) are tentatively assigned and may correspond to compounds 3 and 4, respectively, of Leyva et al. (63Leyva E. Munoz D. Platz M.S. J. Org. Chem. 1989; 54: 5938-5945Crossref Scopus (76) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cytochrome bd has been isolated as ubiquinol oxidase from γ-proteobacteria including E. coli (5Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 43Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 260: 9159-9165Abstract Full Text PDF Google Scholar, 44Sturr M.G. Krulwich T.A. Hicks D.B. J. Bacteriol. 1996; 176: 1742-1749Crossref Google Scholar, 45Kolonay Jr., J.F. Moshiri F. Gennis R.B. Kaysser T.M. Maier R.J. J. Bacteriol. 1994; 176: 4177-4181Crossref PubMed Google Scholar, 46Jünemann S. Wrigglesworth J.M. J. Biol. Chem. 1995; 270: 16213-16220Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 47Smith A. Hill S. Anthony C. J. Gen. Microbiol. 1990; 136: 171-180Crossref PubMed Scopus (33) Google Scholar, 48Konishi K. Ouchi M. Kita K. Horikoshi I. J. Biochem. 1986; 99: 1227-1236Crossref PubMed Scopus (18) Google Scholar) and as menaquinol oxidase from Gram-positive bacteria (49Gilmore R. Krulwich T.A. J. Bacteriol. 1997; 179: 863-870Crossref PubMed Google Scholar, 50Sakamoto J. Koga E. Mizuta T. Sato C. Noguchi S. Sone N. Biochim. Biophys. Acta. 1999; 1411: 147-158Crossref PubMed Scopus (45) Google Scholar, 51Kusumoto K. Sakiyama M. Sakamoto J. Noguchi S. Sone N. Arch. Microbiol. 2000; 173: 390-397Crossref PubMed Scopus (58) Google Scholar). Steady state kinetics in ubiquinol oxidases has been interpreted as a simple Michaelis-Menten type. The Km values estimated for Q1H2 are usually ∼0.1–0.2 mm (5Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 22Yang F.D. Yu L. Yu C.A. Lorence R.M. Gennis R.B. J. Biol. Chem. 1986; 261: 14987-14990Abstract Full Text PDF PubMed Google Scholar, 43Miller M.J. Gennis R.B. J. Biol. Chem. 1983; 260: 9159-9165Abstract Full Text PDF Google Scholar, 45Kolonay Jr., J.F. Moshiri F. Gennis R.B. Kaysser T.M. Maier R.J. J. Bacteriol. 1994; 176: 4177-4181Crossref PubMed Google Scholar, 47Smith A. Hill S. Anthony C. J. Gen. Microbiol. 1990; 136: 171-180Crossref PubMed Scopus (33) Google Scholar, 48Konishi K. Ouchi M. Kita K. Horikoshi I. J. Biochem. 1986; 99: 1227-1236Crossref PubMed Scopus (18) Google Scholar, 52Lorence R.M. Miller M.J. Borochov A. Faiman-Weinberg R. Gennis R.B. Biochim. Biophys. Acta. 1984; 790: 148-153Crossref PubMed Scopus (33) Google Scholar), and the Km value for Q2H2 has been reported to be 0.05 mm for the E. coli enzyme (22Yang F.D. Yu L. Yu C.A. Lorence R.M. Gennis R.B. J. Biol. Chem. 1986; 261: 14987-14990Abstract Full Text PDF PubMed Google Scholar). Because of auto-oxidation of low potential quinols, the Km values are not reported for menaquinol oxidases. Kinetic studies, electron paramagnetic resonance studies on semiquinone anion (53Hasting S.F. Kaysser T.M. Jiang F. Salerno J.C. Gennis R.B. Ingledew W.J. Eur. J. Biochem. 1998; 255: 317-323Crossref PubMed Scopus (38) Google Scholar), and inhibitor binding studies (54Meunier B. Madgwick S.A. Reil E. Oettemeier W. Rich P.R. Biochemistry. 1995; 34: 1076-10" @default.
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• W2049632955 title "Mass Spectrometric Analysis of the Ubiquinol-binding Site in Cytochrome bd from Escherichia coli" @default.
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