Mass Spectrometric Analysis of the Ubiquinol-binding Site in Cytochrome bd from Escherichia coli*

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.

nol-8 (Q 8 H 2 ), 2 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 (4). During dioxygen reduction, cytochrome bd generates an electrochemical proton gradient (⌬pH and membrane potential) across the membrane through apparent transmembrane movement of four chemical protons (5)(6)(7). 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 b 558 , heme b 595 , and heme d (see Ref. 8 for a review). Unlike cytochrome bo, cytochrome bd does not contain a tightly bound Q 8 . Heme b 558 is a low spin protoheme IX and is ligated by I-His 186 (helix V) and I-Met 393 (helix VII) of subunit I (CydA) (9). Heme b 595 is a high spin protoheme IX bound to I-His 19 (helix I) of subunit I (9) and mediates electron transfer from heme b 558 to heme d, where dioxygen is reduced to water (10 -13). Heme d is a high spin chlorin bound to an unidentified nitrogenous ligand (14 -16) and forms a di-heme binuclear center with heme b 595 (16,17). Topological analysis suggests that all of the hemes are located at the periplasmic end of transmembrane helices (4).
Here we report the azido-Q 2 H 2 -labeled site in cytochrome bd, determined by mass spectrometry (MS). Reduced forms of 2-azido-and 3-azido-Q 2 serve as efficient electron donors to cytochrome bd, and UV illumination in the presence of azido-Q 2 H 2 inactivated the quinol oxidase activity. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS identified subunit I as the azido-Q 2 -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-Q 2 (azido-Q 2 * ) was covalently linked to the carboxylic side chain of I-Glu 280 in the hexapeptide 278 EEETNK 283 , 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-Glu 280 in loop VI/VII.

EXPERIMENTAL PROCEDURES
Purification of Cytochrome bd and Quinol Oxidase Assay-The enzyme was isolated from the cytochrome bd-overproducing strain GR84N/pNG2 (34), as described previously (14). 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 Ϫ1 cm Ϫ1 (35). The purified enzyme in 50 mM potassium phosphate (pH 6.8) containing 0.1% sucrose monolaurate (Mitsubishi-Kagaku Foods Co., Tokyo) was stored at Ϫ80°C until use.
The enzyme activity was determined at 25°C with a JASCO V-550 UV-visible spectrophotometer, as described previously (36). 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 Q 1 , a kind gift from Eisai Co. (Tokyo, Japan), at a final concentration of 0.14 mM.
Photoaffinity Labeling of Cytochrome bd-2-Azido-Q 2 and 3-azido-Q 2 were synthesized as previously described (26). Cytochrome bd (10 M 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-Q 2 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-Q 2 H 2 , 2-n-heptyl-hydroxyquinoline-N-oxide (HQNO; Sigma-Aldrich) was added at a final concentration of 1 mM. Q 1 H 2 oxidase activity was measured before and after the illumination.
In-gel Digestion of Subunit I-Samples containing 20 g of proteins were subjected to 10% SDS-PAGE as described by Laemmli (37), and protein bands were stained with Coomassie Brilliant Blue R-250. In-gel digestion was performed by the method of Hellman et al. (38). 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 l of acetic acid.

FIGURE 2. MALDI-TOF mass spectra of subunits I and II of cytochrome bd without (A) and with UV illumination in the presence of 2-azido-Q 2 H 2 (B) and 3-azido-Q 2 H 2 (C).
The insets show the mass range for subunit I. 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 l of 60% (v/v) acetonitrile and concentrated with ZipTip -C18 (Millipore) to 20 l 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 ␣-cyano-4hydroxycinnamic acid (Sigma-Aldrich). One-l 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, P 14 R, 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 g) 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 ϫ 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-M10A VP 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 l of 0.1% formic acid.
ESI MS-The samples were applied to a C 18 column (0.1-mm inner diameter ϫ 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 experi-ments, 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 CϭO-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 (39).

RESULTS
Photo Inactivation of Cytochrome bd with Azido-Q 2 H 2 -Electrondonating activities of 2-azido-and 3-azido-Q 2 H 2 to cytochrome bd are 97 and 94%, respectively, of that of Q 2 H 2 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-Q 2 H 2 , Q 1 H 2 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-Q 2 H 2 molecule (data not shown). UV illumination in the presence of 0.04 mM 2-azido-Q 2 H 2 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 (22), cytochrome bo (28), succinate dehydrogenase (30), and DsbB (56). A difference in the inactivation levels between 2-azidoand 3-azido-Q 2 H 2 may be related to interactions of their photoproducts with the protein. The incomplete inactivation with azido-Q 2 H 2 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-Q 2 H 2 to the quinol-binding site inactivates the quinol oxidase activity of cytochrome bd.
Our azido-Q 2 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 Ϯ 60 and 42,410 Ϯ 70 (n ϭ 5) (Fig. 2), respectively, which are comparable with the calculated molecular masses of 58,205 3 and 42,460 Da, respectively, from the deduced amino acid sequence (40). Upon UV illumination in the presence of 2-azido-or 3-azido-Q 2 H 2 , 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 (22) did not occur for our azido-Q 2 H 2 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-Q 2 H 2 resulted in the inactivation of the quinol oxidase activity.
Identification of the Azido-Q 2 -cross-linked Peptide-For the identification of the azido-Q 2 H 2 -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 (29), 83% for the E. coli H ϩ /galactose symporter GalP by ESI MS (41), and 97% for the E. coli H ϩ /lactose symporter LacY by ESI MS (42).
In the Lep/Asp-N double digest of subunit I labeled with 2-azido-or 3-azido-Q 2 H 2 , 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-Q 2 H 2 in the presence of 1 mM HQNO, a competitive inhibitor (I 50 , 7 M), eliminated the m/z 1047.5 peak (Fig. 4C), confirming the attachment of the photoproduct to this peptide.
Identification of Azido-Q 2 -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 278 EEETNK 283 (monoisotopic mass of 748.32) and demonstrated that a photoproduct (300.36 Da) of 2-azido-(data not shown) and 3-azido-Q 2 H 2 (Fig. 5) is covalently linked to the carboxylic side chain of I-Glu 280 (m/z 128.03) via the COO-NH linkage (Fig. 6D). The bЈ series (b3Ј, b4Ј, and b5Ј), 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-Glu 280 in the  Table S1) are circled, and strictly conserved residues are highlighted. The epitope of monoclonal antibody and putative internal repeats are boxed. The proteolytic cleavage sites and azido-Q 2 cross-linked site are indicated by arrows. His 19 serves as an axial ligand for heme b 595 , and His 186 and Met 393 serve as axial ligands for heme b 558 , which accepts electrons from quinols.
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.

DISCUSSION
Cytochrome bd has been isolated as ubiquinol oxidase from ␥-proteobacteria including E. coli (5,(43)(44)(45)(46)(47)(48) and as menaquinol oxidase from Gram-positive bacteria (49 -51). Steady state kinetics in ubiquinol oxidases has been interpreted as a simple Michaelis-Menten type. The K m values estimated for Q 1 H 2 are usually ϳ0.1-0.2 mM (5,22,43,45,47,48,52), and the K m value for Q 2 H 2 has been reported to be 0.05 mM for the E. coli enzyme (22). Because of auto-oxidation of low potential quinols, the K m values are not reported for menaquinol oxidases. Kinetic studies, electron paramagnetic resonance studies on semiquinone anion (53), and inhibitor binding studies (54) are consistent with the presence of a single quinol/quinone-binding site in cytochrome bd.
Identification of Quinone/Quinol-binding Site with Azidoquinones-To identify the quinol oxidation site in cytochrome bd, we have improved the method for synthesis of 2-azido-and 3-azido-Q 2 (26) and carried out photoaffinity cross-linking studies with the reduced forms. MALDI-TOF MS showed that photo inactivation of the Q 1 H 2 oxidase activity in the presence of azido-Q 2 H 2 was accompanied with the labeling of subunit I. Nonspecific labeling of subunit II reported for 3-azido-2-methyl-5-methoxy-BQ 2s , which has only 8% of the electron donating activity of Q 1 H 2 (22), was not observed. We identified a candidate for the azido-Q 2 -labeled peptide(s) by reverse-phase HPLC and MALDI-TOF MS. ESI quadrupole TOF MS/MS analysis determined the amino acid sequence of the peptide (m/z 1047.5) to be 278 EEETNK 283 where the air-oxidized photoproduct (m/z 300.36) was covalently linked to the carboxylic side chain of I-Glu 280 . This is the first report that identified the amino acid residue cross-linked with azidoquinones.
Structure of the Quinol Oxidation Site in Cytochrome bd-In the N-terminal half of periplasmic loop VI/VII (Q loop) of subunit I (Fig. 7), proteolytic cleavage at I-Tyr 290 with trypsin or at I-Arg 298 with chymotrypsin (18,19) and binding of the monoclonal antibody to the epitope 252 KLAAIEAEWET 262 (20,21) inhibit the quinol oxidase activity. The azido-Q 2 -cross-linked site (I-Glu 280 ) was mapped in the same domain, where the internal repeat PX 9 -10 Q(E/H)EEX 2 (K/R)X 3 (Q/K) can be recognized. Proteolytic cleavage at either I-Tyr 290 or I-Arg 298 would lose the interactions between the internal repeats (i.e. I-Glu 280 in the first repeat and the quinone binding motif in the second repeat). The sequence 310 LMVQHEERI 318 in the second repeat may be related to the proposed quinone-binding motif (LX 3 HX 3 (T/S/L) (55)). Similar motifs can be found in the azidoquinone-binding peptides of Complex I ( 192 LVFVHVNAT 200 in NuoM (23)), Complex II ( 116 ISQLHQSGV 124 in bovine QPs1 (31), 26 ASILHRVSG 34 in the E. coli SdhC (30)), and periplasmic disulfide bond protein ( 87 LTYEHTMLQ 95 in DsbB (56)). The atomic structure of the E. coli Complex II (57) revealed that C-Ile 28 and the C␤ atom of C-Ser 27 participate in the hydrophobic quinonebinding pocket and that the C-Arg 31 side chain is within 4 Å of the 2-methoxy group. Substitutions of C-Ser 27 and -Arg 31 abolished the succinate-ubiquinone reductase activity and the labeling with 3-azido-2-methyl-5-methoxy-dBQ 2 (30). Thus photoaffinity cross-linking with azidoquinones has proved to be useful for probing the quinol/quinonebinding site in the respiratory complexes.
Cytochrome bd is widely distributed as the terminal oxidase from Archaea to Eubacteria and can be divided into two groups with or without the C-terminal third of Q-loop (58). Alternatively, there are three groups with different substrate specificity: ubiquinol (␣-, ␤-, and ␥-proteobacteria), menaquinol (other bacteria), or plastoquinol (cyanobacteria). Amino acid residues, which define the substrate specificity, remain unknown. I-Glu 280 is not conserved even in ␥-proteobacteria, but we can find acidic residues at approximately the equivalent position, ϳ10 amino acid residues upstream of the conserved I-Pro 289 (Fig. 8). Our photoaffinity cross-linking studies with azido-Q 2 H 2 directly demonstrated that I-Glu 280 in the first internal repeat is a part of the quinolbinding pocket of the E. coli cytochrome bd. A hydrophilic segment containing I-Glu 280 can be predicted to be coil by Jpred (Pro 275 -Thr 281 ) (59), PROF (Ile 274 -Glu 280 ) (60), and the New Joint method (Gly 273 -Asn 282 ) (61) between two short ␤-strands, allowing the accommodation of both a bulky azido or methoxy group and a reactive nitrene group on the quinone ring. In the periplasmic region of subunit I, I-Gln 249 , I-Lys 252 , and I-Glu 257 at the N terminus of loop VI/VII and I-Arg 448 at the N terminus of loop VIII/IX are conserved charged and/or hydrophilic residues and could directly provide hydrogen bonds to the hydroxy and/or oxo group of the quinone ring. Our recent kinetic studies on the loop VI/VII mutants provided the supporting evidence for the involvement of I-Gln 249 , I-Lys 252 , I-Glu 257 , and I-Glu 280 in the binding and oxidation of ubiquinols (62). In the E. coli cytochrome bo (25), a member of the heme-copper terminal quinol oxidases, a ubiquinonebinding motif (RX 3 D plus HX 2 Q) has been proposed for the high affinity quinone-binding site, where Asp 75 in helix I and His 98 in helix II in subunit I serve as ligands for the quinone carbonyl groups. Thus quinol/ quinone-binding motifs in bacterial quinol oxidases are different from LX 3 HX 3 (T/S/L) proposed by Fischer and Rich (55).
Mechanism of Photoaffinity Labeling with Azidoquinones-Upon photolysis of an oxidized form of 3-azido-Q 2 (329.4 Da; Fig. 6E) in organic solvents (methanol and acetic acid), ESI MS analysis identified a principle product [MϩH] ϩ with m/z 302.4 (Fig. 6, G or H) (data not shown), indicating the release of molecular nitrogen from 3-azido-Q 2 to form a nitrene derivative (Fig. 6F). 1 H NMR spectra showed resonance signals of the methoxy and methyl groups similar to be attached to quinone ring, although their chemical sifts slightly differ from those of 3-azido-Q 2 , and no signal was assigned to solvent insertion adduct. 1 H NMR spectra also indicated that the number of total protons is identical before and after the photolysis. 4 By MALDI-TOF MS studies on the E. coli cytochrome bo, Tsatsos et al. (29) identified the tryptic fragment at m/z 1931.9 as the cross-linked product of Val 165 -Arg 178 (1597.7 Da) of subunit II with 3-azido-2methyl-5-methoxy-BQ 2 (329.4 Da). Because of the release of N 2 from azido-BQ 2 , a mass of the cross-linked peptide would be 1900.1 Da. Gong et al. (33) labeled the E. coli Complex I with 3-azido-2-methyl-5-methoxy-dBQ (341.5 Da) and identified the cross-linked peptide at m/z 2929.4 to be Val 184 -Asn 206 (2593.1 Da) of NuoM. However, the observed mass was 22 Da larger than that for the peptide plus the protonated photoproduct (2907.6 Da). The cause of such large discrepancies in assignments is not known.
In photoaffinity labeling of cytochrome bd with 3-azido-Q 2 H 2 , the protonated photoproduct covalently linked to I-Glu 280 showed m/z 300.4 (Fig. 5), which is m/z 4.0 smaller than that expected (Fig. 6C). Auto-oxidation of the primary photoproduct during the isolation of peptides would yield a loss of two hydrogens from both the quinone ring and the isoprene moiety (Fig. 6D). The presence of [MϩH] ϩ ions for the cleaved azido-Q 2 * and its oxidized isoprene unit at m/z 301.19 (301.37 expected) and 136.09 (136.23 expected), respectively, in the ESI-Q-TOF MS/MS spectrum (Fig. 5) supports the proposed structure for the crosslinked photoproduct (Fig. 6D).
In conclusion, photoaffinity labeling of the E. coli cytochrome bd, followed by MS analysis of peptides, revealed a new feature of the quinol oxidation site in loop VI/VII of subunit I. Although distal to the strictly conserved I-Gln 249 , I-Lys 252 , and I-Glu 257 at the N terminus of loop VI/VII, I-Glu 280 in the first internal repeat has been demonstrated to be a part of the binding site for the 2-and 3-methoxy groups of the quinone ring. Mutational (62) and x-ray crystallographic studies would provide further insights into the understanding of the catalytic mechanism for the quinol oxidation in bd-type terminal oxidases.