Location of the Substrate Binding Site of the Cytochrome bo3

Site-directed mutagenesis confirmed that residues within this cleft (R71, D75, H98, and Q101) are important for enzyme activity(3, 25) and for stabili...
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Location of the Substrate Binding Site of the Cytochrome bo3 Ubiquinol Oxidase from Escherichia coli Sylvia K. Choi,†,‡,§ Lici Schurig-Briccio,‡ Ziqiao Ding,‡ Sangjin Hong,‡ Chang Sun,‡ and Robert B. Gennis*,†,‡ †

Center for Biophysics and Quantitative Biology, University of Illinois, Urbana, Illinois 61801, United States Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, United States

‡

ABSTRACT: Cytochrome bo3 is a respiratory proton-pumping oxygen reductase that is a member of the heme-copper superfamily that utilizes ubiquinol-8 (Q8H2) as a substrate. The current consensus model has Q8H2 oxidized at a low affinity site (QL), passing electrons to a tightly bound quinone cofactor at a high affinity site (QH site) that stabilizes the one-electron reduced ubisemiquinone, facilitating the transfer of electrons to the redox active metal centers where O2 is reduced to water. The current work shows that the Q8 bound to the QH site is more dynamic than previously thought. In addition, mutations of residues at the QH site that do not abolish activity have been re-examined and shown to have properties expected of mutations at the substrate binding site (QL): an increase in the KM of the substrate ubiquinol-1 (up to 4-fold) and an increase in the apparent Ki of the inhibitor HQNO (up to 8-fold). The data suggest that there is only one binding site for ubiquinol in cyt bo3 and that site corresponds to the QH site.

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INTRODUCTION Cytochrome bo3 (cyt bo3) is a ubiquinol-8 (Q8H2) oxidase and one of three respiratory oxygen reductases in Escherichia coli.1 The enzyme catalyzes the following reaction.

dioxygen come from the sequential oxidation of four reduced cytochrome c molecules. The cytochrome c docking site is on subunit II of cytochrome c oxidase,5 and the immediate electron acceptor is the CuA redox center. A series of oneelectron transfers delivers each electron to the active site: cyt c → CuA → heme a → heme a3/CuB.6−10 Although cyt bo3 has a homologue of subunit II, the CuA site is not present nor is the docking site of cyt c.11,12 Rather, the enzyme contains an active site where Q8H2 is oxidized in two one-electron steps: [Q8H2 or Q8H·] → heme b → heme o3/ CuB. Heme O was first discovered in cyt bo3 and replaces heme A at the heme/Cu active site.13 The oxygen chemistry at the heme/Cu active site is the same for both the quinol and cyt c oxidases, as are the mechanisms for proton pumping.14,15 Questions remain about the oxidation of Q8H2 by cyt bo3, including the question of whether the enzyme contains one or two quinone binding sites.3 This is the question addressed in the current work. The consensus has been that there are two quinone binding sites in cyt bo3:3 (1) A low affinity site (QL) where the bound quinone readily exchanges with the quinone pool in the lipid bilayer; and (2) a high affinity site (QH) which does not exchange readily with the quinone in the membrane. The twosite model proposes that the Q8H2 binds transiently at the QL site and transfers both electrons to the quinone bound at the QH site. The quinone bound to the QH site acts as a cofactor and passes the electrons to heme b in two one-electron steps. The QH site stabilizes the one-electron reduced semiquinone species to facilitate this switch from a two-electron transfer

2Q 8H 2 + 8H+in + O2 ⇄ 2Q 8 + 2H 2O + 8H+out

For each dioxygen reduced to water, four cytoplasmic protons are pumped across the E. coli cytoplasmic membrane to the periplasm (“pumped” protons) and four protons from the cytoplasm are consumed to generate water (“chemical” protons). The four pumped protons are released to the periplasm concomitant with the oxidation of the two reduced quinol molecules. As a consequence of charges crossing the membrane, the enzyme generates a transmembrane voltage that contributes to the proton motive force across the E. coli membrane. Cyt bo3 is a member of the large heme-copper oxidoreductase superfamily2 which includes both respiratory oxygen reductases as well as nitric oxide reductases. Whereas most of the heme-copper respiratory oxygen reductases utilize cyt c as a substrate, cyt bo3 is part of a subfamily that oxidizes quinol within the membrane bilayer.3 Cyt bo3 consists of one copy each of four subunits, and subunits I, II, and III are homologues of the mitochondrialencoded subunits of cytochrome c oxidase.4 Subunit I of cyt bo3 contains a low spin heme b as well as a bimetallic active site consisting of a high spin heme o3 and CuB. Oxygen binds to heme o3 and is reduced to water in a series of one-electron transfers.1 The three redox-active metal centers (heme b, heme o3, and CuB) are equivalent to the metal redox centers in subunit I of cytochrome c oxidase (heme a, heme a3, and CuB). In the cytochrome c oxidases, the electrons used to reduce © 2017 American Chemical Society

Received: April 17, 2017 Published: May 24, 2017 8346

DOI: 10.1021/jacs.7b03883 J. Am. Chem. Soc. 2017, 139, 8346−8354

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Journal of the American Chemical Society

selectively perturb the QL site will eliminate activity but that it should also be possible to obtain nonlethal mutants at this site that alter the KM of Q1H2 and/or alter the apparent Ki of inhibitors that bind to that site, in particular HQNO. Early studies implicated subunit II (CyoA) as the location of the QL site, based upon covalent attachment of photoreactive quinol analogues35,36 and on the location of mutations selected for resistance to inhibitors.37 However, the residues implicated in these studies are not clustered so as to define a binding site nor are the locations of these residues plausible as being at or near a substrate binding site. Site-directed mutagenesis of conserved residues in either subunit I or subunit II also failed to locate a residue convincingly at the QL site.3,38 A tryptophan (W136) in subunit II was initially identified as a candidate,38 but these results were subsequently shown to be incorrect.3 A recent bioinformatics and computational study identified a promising QL sequence motif in subunit I,34 but site-directed mutagenesis to verify this proposal demonstrated that residues within this sequence motif are not required for function and do not have the phenotypes expected for a perturbation of the QL site.39 The chronic failure to locate the QL site in cyt bo3 has motivated the current work. Our results strongly suggest that the QH and QL sites are in fact coincident. It is likely that at least some of the results previously used to support the two-site model may be explained by the poor solubility of the long chain ubiquinone species in the detergent DDM, leading to differences in the apparent binding affinity of Q8 to the enzyme in detergent compared to the enzyme embedded in a phospholipid bilayer. In addition, the binding of Q8 to the enzyme appears to be partially mediated through the polyisoprene side chain beyond the first isoprene unit, allowing the head group of Q8 to be displaced by a competing ligand without removing Q8 from the protein.

reaction to a one-electron transfer reaction. The evidence supporting this model includes the fact that the enzyme can be isolated in the detergent dodecyl maltoside (DDM) associated with one equivalent of ubiquinone-8 (Q8) and that this bound quinone can form a stable semiquinone species (approximately 20% occupancy by the semiquinone at pH 8 when poised at 0 mV).16−18 The quinone associated with the DDM-solubilized enzyme does not appear to readily dissociate, though washing the enzyme with Triton X-100 effectively strips the Q8 from the enzyme.19 By changing the preparative protocol, cyt bo3 can be isolated with no bound quinone or copurified with more than one equivalent of quinone.3,19,20 These data have been reasonably explained in terms of two distinct quinone binding sites, one (Q H) which binds a slowly exchanging or nonexchanging quinone that acts as a cofactor in the reaction, and the second (QL) which exchanges rapidly with the pool of ubiquinol-8 in the lipid bilayer.3,19−22 Due to insolubility of the natural Q8H2 substrate, routine enzyme assays are performed using the more water-soluble ubiquinol-1 (Q1H2) which has a side chain with only a single isoprene unit. Enzyme from which the Q8 has been removed retains full activity with the substrate Q1H2 in the presence of the detergent DDM. Steady state kinetics studies with inhibitors have been interpreted in terms of a model in which there are two quinone binding sites, each which binds to a subset of inhibitors.23,24 Additional evidence in support of two Q-binding sites comes from using purified enzyme containing one equivalent of bound Q8 and analyzing the quinone content of the enzyme after multiple turnovers with the water-soluble Q1H2 substrate.3,20 If there were a single site, the expectation is that reaction with Q1H2 would by necessity require the displacement of the bound Q8H2. However, this does not occur and the enzyme retains the stoichiometric bound Q 8. Furthermore, cyt bo3 binds to one equivalent of the inhibitor HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide), and it has been shown that the enzyme can bind to both HQNO and Q8 simultaneously.3,20 The bound Q8 is not displaced either by HQNO or by another high affinity inhibitor aurachin C1−10.3 Furthermore, the binding of HQNO eliminates formation of the ubisemiquinone-8 EPR signal from the quinone bound at the QH site, suggesting that the inhibitor, presumably binding at the QL site, must be close enough to perturb the semiquinone at the QH site.3,18 The determination of the X-ray structure of cyt bo3 provided the first indication of the location of the quinone binding sites, despite the fact that the bound quinone had been stripped out of the protein by the octyl glucoside detergent and 25% of the protein is not seen in the structure.25 A cleft at the level that would presumably be submerged in the lipid bilayer was identified within subunit I (CyoB) that contains a set of residues uniquely conserved in the family of ubiquinol oxidases. Site-directed mutagenesis confirmed that residues within this cleft (R71, D75, H98, and Q101) are important for enzyme activity3,25 and for stabilizing the ubisemiquinone at the QH site.16 Subsequent EPR studies of the ubisemiquinone species have provided considerable detail about the hydrogen bond network between these residues and the bound ubisemi-qui none at this site.3,26−33 The residues at this site are also within a sequence identified recently by computational methods as being a QH-site motif in the family of quinol oxidases including cyt bo3 from E. coli.34 In contrast to the QH site, efforts to locate the QL site have all failed. The expectation has been that some mutations that

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MATERIALS AND METHODS

Protein Production and Purification of Mutants. All mutants were grown under conditions of high aeration in shaker flasks using M63 medium with the addition of thiamine, MgSO4, CuSO4, glucose, ampicillin, and kanamycin at 37 °C. The enzymes, each with a His-tag at the N-terminus of subunit II, were isolated by affinity chromatography at 4 °C using a Ni-NTA resin as previously described.3,30 Site-Directed Mutagenesis. All mutants were expressed in a C43(DE3) strain from which the cyoABCDE operon has been deleted. The cyoABCDE operon encoding cyt bo3 containing a 6-histidine tag at the N-terminus of CyoA (subunit II) was cloned into the expression plasmid pET17b and mutations were generated using the QuikChange kit from Agilent (Santa Clara, CA) as described.3 DNA oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). Sequence verifications of the mutagenesis products were performed at the University of Illinois at Urbana−Champaign (UIUC) Biotechnology Center. Enzyme Assays. Steady state ubiquinol-1 (Q1H2) oxidase activity was measured for each mutant using purified protein at 25 °C by monitoring the depletion of O2 with YSI model 53 oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH) equipped with a 1.8 mL reaction chamber or Unisense Oxygen MicroRespiration sensor (Aarhus, Denmark) with a 1 mL reaction chamber. The reaction mixture contained 50 mM potassium phosphate, pH 7.0, 0.05% DDM with 2 mM dithiothreitol and the indicated concentration of ubiquinone-1 (Q1, Sigma-Aldrich, St. Louis, MO). Oxygen saturated buffer at 25 °C is estimated to be 250 μM. The reaction was initiated by the addition of enzyme (pM concentrations). The kcat and KM values for Q1H2 were obtained as previously described3 from the initial velocity as a function of the concentration of Q1H2. Turnover numbers (e−1/s/enzyme) of the enzyme were calculated based on the 8347

DOI: 10.1021/jacs.7b03883 J. Am. Chem. Soc. 2017, 139, 8346−8354

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Journal of the American Chemical Society consumption of oxygen, subtracting the background prior to the addition of the enzyme. The Q2H2 oxidase assay was measured for most of the mutants with 250 μM Q2 (Sigma-Aldrich, St. Louis, MO), using the same protocol described for Q1. The wild type enzyme has a turnover (kcat) of approximately 1500 e−1/s with Q1H2 and about 480 e−1/s with Q2H2. Inhibition of Q1H2 oxidase activity was determined using either Noxo-2-heptyl-4-hydroxyquinoline (HQNO) from Enzo Life Sciences (Farmingdale, NY) or aurachin C1-10 which was kindly provided by Dr. Hanlin Ouyang and synthesized using the synthetic protocol40 modified as described previously.3 Inhibition of activity was determined as a function of the concentration of the inhibitor using a fixed concentration of Q1H2 of 300 μM. The concentration at which activity is inhibited by 50% is the apparent Ki or Kapp i . Extraction and Analysis of Ubiquinone. The amount and type of ubiquinone present in purified samples of cyt bo3 was determined as previously described.3 For the extraction, the purified enzyme was within 1 mL of a buffered solution containing 0.05% DDM in a 15 mL glass tube. To this was added 3 mL of a 3:2 mixture of petroleum ether:methanol. The top layer of solvent was carefully removed with a glass pipet and dried under a stream of nitrogen gas. This was repeated twice more for a total of three times, and the extracts were combined and analyzed by HPLC chromatography.3 Reconstitution of Cyt bo3 into Proteoliposomes. Cyt bo3 was incorporated into proteoliposomes using 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) either with or without ubiquinone-10 (Q10) (Sigma-Aldrich, St. Louis, MO). Typically, 10 mg of DOPC, dissolved in a small amount of chloroform, was placed in a round-bottom flask and thoroughly dried under a stream of nitrogen while rotating the flask.41 When Q10 was included, it was cosolubilized in chloroform at about 9 mol %. The dried lipids were resuspended in 10 mL of 20 mM HEPES-NaOH buffer, pH 7.5. The lipid suspension was subjected to several cycles of freeze−thaw and then passed through an extruder (Avanti Inc., Alabaster, AL) to make unilamillar 200 nm diameter vesicles. Extrusion was performed 17 times to ensure a uniform size distribution of the vesicles. The vesicles were frozen at −20 °C for further use. The vesicle suspension (470 μL containing about 470 μg of phospholipid) was combined with 17 μL of a solution of 20% Nacholate, and cyt bo3 was added to a final concentration of 1 μM. The purified cyt bo3 stock solution was highly concentrated (300 μM in 0.05% DDM) to minimize the amount of DDM in the final mixture.42 The solution (about 0.5 mL) was incubated at 25 °C for 30 min with occasional gentle mixing and then loaded onto a Sephadex PD10 column and eluted with buffer without detergent. The proteoliposomes containing the reconstituted cyt bo3 was collected with final volume of about 1.5 mL, and used without storage. Enzyme Assays of Cyt bo3 in Liposomes. The Q10H2 oxidase activity of cyt bo3 was measured in Q10-containing liposomes in the presence of 1 mM NADH, 1.3 μM FAD plus 6.7 μg/mL of a preparation of E. coli NDH-243 to reduce the Q10 within the liposomes. Activity was measured as described previously by monitoring oxygen utilization. Reisolation of Cyt bo3 from Reconstituted Proteoliposomes. Although cyt bo3 could be readily solubilized from E. coli membranes using 1% DDM, this was found not to be the case for cyt bo3 that was reconstituted into proteoliposomes using either asolectin or DOPC regardless of whether Q10 was also present. This observation is consistent with previous studies on the effectiveness of different detergents to solubilize phospholipid vesicles.44 For this purpose DDM is very inefficient, and Triton X-100 is the most efficient detergent. However, Triton X-100 could not be used because this strips the quinone that is bound to the QH site.19 The protocol that was successful to solubilize cyt bo3 from reconstituted proteoliposomes required a 2 h incubation in the presence of 10% DDM at room temperature. The liposome suspension became translucent by this treatment and the cyt bo3 was isolated using the Ni-NTA affinity column, washing the column with 0.05% DDM, 50 mM potassium phosphate buffer at pH 8.3, followed by elution using 150 mM imidazole in the same buffer. Following elution, the imidazole was

removed using 100-kDa cutoff Amicon (Millipore) by several rounds of concentration and dilution into buffer without imidazole. Displacement of Bound Q8 in DDM-Purified Cyt bo3 by Exogenous Q2 or Q4. The exchange of the endogenous Q8 in purified cyt bo3 solubilized in DDM was carried out using two different procedures, with identical results. In one protocol, the Q2 was added to the enzyme solution prior to binding the cyt bo3 to the Ni-NTA affinity column and, in the other protocol, the enzyme was bound to the Ni-NTA column and then a solution containing either Q2 or Q4 was passed through the column prior to elution. Following elution of the enzyme, the quinone was extracted and quantified as described above. All quinones were purchased from Sigma-Aldrich, St. Louis, MO. Protocol 1: Exchange Followed by Binding to Ni-NTA Resin. Purified cyt bo3 (5 nmol) was added to 1.8 mL of 1% DDM, 50 mM potassium phosphate buffer, pH 7. To this was added indicated amounts of Q2 (Sigma-Aldrich, St. Louis, MO), and the mixture was incubated with agitation for the stated times. Adding the reductant DTT to reduce Q2 and initiate enzyme activity made no difference to the final result. After incubation, the mixture was loaded onto the NiNTA column and washed thoroughly using 0.05% DDM, 50 mM potassium phosphate buffer at pH 8.3. The enzyme was eluted using 150 mM imidazole, 0.05% DDM, 50 mM potassium phosphate buffer at pH 8.3. Following elution, imidazole was removed using 100 kDa cutoff Amicon (Millipore) by several rounds of concentration and dilution into buffer without imidazole. Protocol 2: Exchange of Quinones on the Ni-NTA Resin. Purified cyt bo3 (5 nmol) was added to 0.5 mL of Ni-NTA resin which was then washed with 2 mL of 1% DDM, 50 mM potassium phosphate buffer, pH 7, which contained the indicated concentration of Q2 or Q4. The resin was then immediately washed with 15 mL of 0.05% DDM, 50 mM potassium phosphate buffer, pH 8.3. The enzyme was eluted using 150 mM imidazole, 0.05% DDM, 50 mM potassium phosphate buffer, pH 8.3. Following elution, imidazole was removed using 100kDa cutoff Amicon (Millipore) by several rounds of concentration and dilution into buffer without imidazole. EPR Spectroscopy. Samples of purified cyt bo3 in DDM for EPR spectroscopy and the spectra were obtained as previously described.29,30

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RESULTS Characterization of Mutants at the QH Site that Retain Residual Activity. The basic strategy was to examine mutations at the previously defined QH site of cyt bo316,25 that have sufficient residual steady state activity to permit an evaluation of characteristics expected due to perturbations of the QL site: an increased KM for Q1H2 and an increase in the IC50 or apparent Ki (Kapp i ) for the inhibitor HQNO. For most QH-site mutants kinetics studies are not possible because typically mutations at the QH site eliminate quinol oxidase activity. However, the initial identification of the four residues at the QH site (R71, D75, H98, and Q101) showed one exception, the Q101N mutant, which was reported to have 24% residual activity and a KM of 209 μM (vs 18 μM for the wild type).25 Two-dimensional pulsed EPR experiments subsequently demonstrated that the interaction between the ubisemiquinone bound at the QH site with Q101, though measurable, is very weak.26,29 Therefore, a set of mutants were generated at this site: Q101N, Q101A, Q101T, Q101L, Q101E, and Q101M. In addition, the D75E mutation was previously shown to alter the interactions of the ubisemiquinone bound to the QH site, but retained about half of the catalytic activity.29 The D75E mutant, as well as the set of Q101 mutants, were purified in DDM and the Q1H2 oxidase activity of each was examined as a function of the concentration of Q1H2 and analyzed using Michaelis−Menten kinetics. The kcat and KM values were determined and compared to those of the wild 8348

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Journal of the American Chemical Society Table 1. Properties of Mutants of E. coli cyt bo3 within or near the QH Site Kapp i relative Q1H2 activity (%) WT D75E

100 48 ± 3

Q101N Q101A Q101T Q101L Q101E

10 26 27 11 55

Q101M

51 ± 9

± ± ± ± ±

3 2 6 1 1

Q1H2 KM (μM)

relative Q2H2 activity (%)a

quinone content (Q8/enzyme)

50 16 ± 9

100b 88 ± 1b

1.7 1.7

72 ± 1 30 ± 4 200 ± 3 141 ± 24 124 ± 3

61 82 62 58

± ± ± ±

1 1b 1 1

1.9 1.8 1.8 1.3 1.3

24 ± 3

72 ± 1b

1.5

c

R71Q H98N H98T F93A F93Y

3 3 4 37 ± 6 81 ± 5

91 ± 11 72 ± 15

Q82A N157V L160W

88 23 ± 1 58 ± 7

45 100 ± 6 30 ± 4

8 10c 9±1 102 ± 1b 107 ± 1b

1.1 1.4d 1.5d 1.4 1.3

1.4 1.3

semiquinone formation YES YES small perturbationse NO YES perturbed YES very low amplitude YES very low amplitude YES very large amplitude YES very large amplitude NOd NOd NOd YES large amplitude YES very large amplitude YES large amplitude YES very low amplitude

HQNO (μM) 0.7 5.8 ± 0.4 5.4 0.9 4.8 4.0 3.0

± ± ± ± ±

0.7 0.2 0.3 0.8 0.4

3.4 ± 0.5

AC1-10 (nM) 5 ± 0.6 67 ± 8 287 ± 59 13 ± 2 53 ± 16 227 ± 21 72 ± 4

2.2 ± 0.5 2.7 ± 0.6 9.7 ± 0.4 2.8 ± 0.8

134 ± 22

a Activity was measured with 250 μM Q2H2 bOxygen consumption traces vs time were biphasic and the values apply to the slow phase. cMeasured once. dPreviously published data.3 eData shown in ref 29.

Figure 1. Location of the residues mutated in the current work. (A) Structure of cyt bo3 showing the eight residues that were mutated as part of this study. The structure is pdb 1FFT from ref 25. (B) Another view of the eight residues examined in this study as part of a membrane-buried portion of subunit I, residues 70−162.

24%,25 but similar to the value of 5% in a previous report from this laboratory.3 Three mutations have significantly larger KM values for Q1H2: Q101T (200 μM), Q101L (141 μM), and Q101E (124 μM). Although the previously reported 12-fold increase in the KM for Q101N25 was not replicated, the conclusion that mutations at Q101 can increase the KM for Q1H2 is confirmed. The Kapp for the inhibitor HQNO was also determined at a i fixed concentration of the substrate (250 μM Q1H2). The wild ≈ 0.7 μM as previously type enzyme is inhibited with a Kapp i 3 app reported. In contrast, the Ki of the D75E mutation is 5.8 μM,

type. In addition, the Kapp values for inhibition by HQNO and i by aurachin C1-10 were determined for the wild type and mutants. The results, summarized in Table 1, show that several of these mutations substantially perturb these steady state kinetic parameters. Figure 1 shows the locations of each of the mutated residues. Under the conditions used for these assays, the KM for Q1H2 is 50 μM and kcat is about 1500 e−1/s (750 quinol/s) for the wild type enzyme. All of the mutations (D75E and the Q101 mutants) result in a lower kcat, with the lowest activities observed with Q101N (10%) and Q101L (11%). The 10% activity of Q101N is lower than the previously reported value of 8349

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Journal of the American Chemical Society

Figure 2. Continuous wave EPR spectra of redox-poised samples of DDM-solubilized cyt bo3 and indicated mutants in or near the QH site. The shapes and intensities of the signals are altered by several of the mutations. The signal intensities have been adjusted to account for differences in the concentration of cyt bo3 in each sample, and the same protocol was used in each case to generate the semiquinone species. (A) Mutations at position Q101. (B) Mutations at other positions at or near the QH site.

Q1H2, but the Kapp of HQNO is significantly increased from 0.7 i μM to 2.2 μM and 2.7 μM, respectively (aurachin C1−10 was not examined) (Table 1). Q82 is located in the same loop as is F93 but is more distant from the four QH-site residues. The Q82A mutation has no significant effect on either the kcat or KM of the Q1H2 oxidase activity (Table 1). N157 and L160 are located about 7 Å from R71 on the cytoplasmic side of the QH site and are within transmembrane helix 4 (TM4) that runs parallel to TM2 (Figure 1). The effects of both the N157 V and L160W on the quinol oxidase steady state parameters are also modest (Table 1), but the interactions with both inhibitors HQNO and aurachin C1−10 are significant. The largest effect was observed with N157 V which shows a 14-fold increase of the Kapp of HQNO and 27-fold for aurachin C1−10. i Figure 2B shows the EPR spectra of the ubisemiquinones stabilized by the L160W, N157 V, F93A and F93Y mutants. In each case, there is an EPR-detectable ubisemiquinone but, as with the mutants at position Q101, there are clearly perturbations. The most important point is that most of these mutations have phenotypes that are expected of both QH site (EPR perturbations) and the QL site (altered KM and Kapp i ). Re-Examination of Quinone Exchange at the QH Site. The simplest explanation for the increased KM and Kapp values i by the QH-site mutations is that the QH and QL sites are the same, i.e., there is only one quinone binding site. However, this appears to be inconsistent with several observations which underlie the argument for cyt bo3 having two separate quinone binding sites: (1) Q8 copurifies with the enzyme in DDM and does not dissociate to any measurable extent; (2) HQNO binds stoichiometrically to the enzyme and does not displace Q8, nor is the binding of HQNO perturbed by the presence/absence of Q8;3 (3) Multiple catalytic turnovers using the water-soluble substrate analogue Q1H2 do not displace the bound Q8 at the QH site of the DDM-solubilized enzyme,3 nor does the presence of 500 μM Q1 displace the bound Q8 in the presence of sucrose monolaurate.20 To explain the paradoxical observations that argue either for one or two quinone binding sites, two postulates were experimentally tested: (A) The lack of exchange of Q8 is

an 8-fold increase. Similarly, most of the Q101 mutants also show a substantial increase in the Kapp for HQNO (Table 1). i A second high affinity inhibitor of cyt bo3 is aurachin C1103,40,45 which, under the conditions used in the current work, app has a Kapp i of 5 nM. The Ki for aurachin C1-10 is increased 13fold (67 nM) for the D75E mutant and is also increased significantly by all the Q101 mutants except Q101A. The largest increase was observed with the Q101N mutant (57fold). These results indicate that the binding sites of both HQNO and for aurachin C1-10 overlap with the QH site. The QH site is occupied by one molecule of Q8 which can form a stable ubisemiquinone species under appropriate redox conditions. Using the detergent DDM for the enzyme purification, cyt bo3 copurifies with between one and two equivalents of ubiquinone-8,3 with one equivalent presumably being that which is occupying the QH site. Previously, it was shown that mutations of D75 (H, N or R), R71 (K, Q), H98 (N, S or T), or Q101N completely or nearly eliminate Q1H2 oxidase activity (0−5%), but do not result in eliminating the Q8 that copurifies with the enzyme.3 The data in Table 1 show that the mutations examined in the current work also do not result in eliminating the copurified Q8. In addition, each mutant was examined under the conditions which stabilize the EPR-visible ubisemiquinone at the QH site. With the exception of Q101N, all of the Q101 mutants exhibit the ubisemiquinone EPR signal although the magnitude and spectral shape are perturbed. The EPR signal is much smaller than that of the wild type in Q101T and Q101L but is much larger for Q101E and Q101 M (Figure 2A). The EPR signal for D75E has been previously examined and has minor perturbations compared to the wild type.29 Four additional residues, Q82, F93, N157, and L160, were also selected for mutagenesis (Figure 1). F93 is part of the sequence motif identified by Bossis et al.34 as defining the QH site. This residue (F93) is located in the periplasmic loop connecting the two transmembrane helices of subunit I which contain the four residues known to interact with the ubisemiquinone (R71 and D75 in TM2; H98 and Q101 in TM3). F93 is about 5 Å away from H98 on the periplasmic side of the QH site. Both F93A and F93Y have modest effects on the kcat and KM values with 8350

DOI: 10.1021/jacs.7b03883 J. Am. Chem. Soc. 2017, 139, 8346−8354

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Journal of the American Chemical Society

Table 2. Percentage of Remaining Ubiquinone-8 after Incubating Cyt bo3 with Different Concentrations of Ubiquinone-4, Ubiquinone-2, Ubiquinone-1, or HQNO UQ4 2 min UQ2 2 min UQ2 30 min UQ1 30 min HQNO 30 min

0 μM

7 μM

33 μM

50 μM

67 μM

100 μM

100% 100% 100%

58%

53%

25% 66%

33%

32%

125 μM

150 μM

250 μM

375 μM

500 μM

24% 20%

20% 26% 13%

18%

25% 30%

100% 100%

7 μM Q4 (Table 2). Repeating the same experiment with 500 μM Q1 or with 10 μM HQNO had no influence on the content of Q8 in cyt bo3.

detergent-specific and is at least partially due to the low solubility of Q8 in the DDM micelle and slow exchange between micelles. (B) A significant portion of the binding between Q8 and the protein involves the isoprene side chain, allowing short chain analogues such as Q1 and HQNO to displace the head group of Q8, but not displace the entire molecule from the QH site. The following experiments were performed. (1) Removal of the endogenous ubiquinone-8 in proteoliposomes: Purified cyt bo3 (containing approximately 1.5 equiv of Q8) was reconstituted into phospholipid liposomes. The proteoliposomes were subsequently solubilized and the protein was reisolated using Ni-NTA column chromatography. The repurified cyt bo3 retained only about 10% of the initial Q8. Over the time scale (several hours) of this experiment, the endogenous Q8 has dissociated from the enzyme and not copurified. This shows that when the enzyme is embedded in a phospholipid bilayer, the endogenous Q8 dissociates into the bilayer. This is in contrast to the observations of the enzyme in DDM, where incubation of the enzyme for several hours, or days, does not result in dissociation of Q8 to any extent. (2) Exchange of the endogenous ubiquinone-8 by ubiquinone-10 in proteoliposomes: Purified cyt bo3 with endogenous Q8 was reconstituted into phospholipid vesicles containing 9 mol percent of Q10. The catalytic turnover of reconstituted wild type cyt bo3 using Q10H2 as the substrate was measured using NADH and the enzyme NDH-2 to reduce the membraneembedded Q10 (see Materials and Methods) to be about 500 e−1/s, similar to that reported previously using Q8-containing vesicles and pyruvate oxidase to reduce the Q8.46 The reconstituted enzyme was resolubilized in DDM, repurified by affinity chromatography and the Q8 and Q10 content of the preparation determined. Following this treatment, all of the endogenous Q8 was replaced by Q10. This experiment suggests that when the enzyme is embedded in a phospholipid bilayer, exchange of the endogenous Q8 at the QH site does occur at least on the time scale of a few hours. (3) Displacement of endogenous ubiquinone-8 in DDM in the presence of ubiquinone-2 or ubiquinone-4: Purified detergentsolubilized cyt bo3 (0.05% DDM) containing endogenous Q8 was bound to the Ni-NTA resin, washed with more than 10 column volumes of phosphate buffer with 1% DDM containing various concentrations of Q2, and then eluted and the content of Q8 was quantified. The time of exposure to Q2 was less than 2 min. The data in Table 2 show that at the lowest concentration of Q2 tested, 50 μM, the content of Q8 was reduced by one-third (66%) and that only 30% of the endogenous Q8 remained if the experiment was performed with 125 μM Q2. Longer times of exposure to Q2 prior to elution or high concentrations of Q2 reduced the amount of retained Q8 to as little as 13% (Table 2). Using Q4, with a longer isoprene side chain, the endogenous Q8 is displaced at lower concentrations, e.g., 58% of Q8 remains afterexposure to

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DISCUSSION It has long been recognized that cyt bo3 has a distinct quinone binding site to which one equivalent of Q8 remains bound following purification using either the detergent sucrose monolaurate20 or DDM.3,19 The Q8 bound to the enzyme solubilized in these detergents has not been observed to dissociate nor is it displaced either by the protein binding quinone-analogue inhibitors (e.g., HQNO) or by the watersoluble substrate analogue ubiquinol-1.3,20 In contrast, the enzyme isolated using Triton X-100 does not contain ubiquinone-8,19 although it catalyzes steady state Q1H2 oxidase activity as well as enzyme isolated with the bound Q8. Nevertheless, the absence of the bound quinone alters the intramolecular electron transfer in single-turnover experiments. 19,47 The functional significance of the bound ubiquinone-8 is demonstrated by the fact that this endogenous quinone can be stabilized by the protein as an EPR-detectable ubisemiquinone species3,17,18,26−31,33,48 that has been shown to be a reaction intermediate during quinol oxidation.49,50 Pulsed EPR techniques have shown that the ubisemiquinone stabilized by cyt bo3 forms hydrogen bonds with four residues in subunit I, R71, D75, H98 and Q101, which define the QH site.26,28−30 The Q8 bound to the QH site has been considered to be a firmly bound cofactor that serves to facilitate electron transfer from the ubiquinol substrate, transiently bound at the QL site, to the heme o3/CuB active site where oxygen is reduced to water. If there are two binding sites, as in this model, then the ubiquinol substrate (Q1H2 or Q8H2) must turnover at the QL site at a rate of about 103 s−1, and this same QL site is also the binding site for high-affinity inhibitors such as HQNO. The search for QL site has been extensive,34−39 but no experimental data have convincingly defined where this site is located. The current work strongly indicates that the site of substrate oxidation (QL) and the site that stabilizes the ubisemiquinone (QH) are the same. This conclusion is based on the effects of mutations of residues at the QH site that do not completely abolish steady state quinol oxidase activity and, therefore, allow a determination of the KM for the substrate ubiquinol-1 (Q1H2). The largest effects are observed for Q101T, Q101L and Q101E (Table 1). This suggests that at least Q101 is shared between the QH and QL sites. The proposed “QL sequence motif”34 is adjacent to Q101, but recent site-directed mutagenesis data39 show that residues within this sequence are not critical for either catalysis or for binding to the substrate Q1H2. The simplest explanation is that the same residues that have been shown to interact with the ubisemiquinone intermediate26,28−30 are also important in the interaction with the ubiquinol substrate. 8351

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experiments the time-traces of substrate oxidation were biphasic. Another source of complex steady state kinetics might be heterogeneity of the enzyme preparation with regards to heme content53 or the presence of residual Q8. The heme stoichiometry and quinone content of all the preparations of cyt bo3 (wild type and mutants) were measured in the current work to avoid these issues. The postulated binding of the endogenous Q8 to the protein by interactions with the isoprenoid side chain may provide an explanation why the steady state inhibition pattern by HQNO with Q1H2 or Q2H2 is not competitive, since these substrates may also be able to bind to the enzyme by interactions with their isoprenoid chains. Hence, simultaneous binding by the inhibitor and substrate is possible, which would perturb the concentration dependence of the inhibition. Although the current proposal is that there is only one quinone binding site, the binding of the quinone through the isoprenoid side chain as well as the head group could make it appear as if the inhibitor and substrate were binding at separate sites, i.e., not competitive inhibition. (4) The apparent lack of exchange of Q8 from the QH site of DDM-solubilized cyt bo3 has been interpreted to mean that this cannot be the site where Q8H2 is oxidized at rates of about 1000 s−1 under physiological conditions.20 In the current work, however, it is shown that whereas the endogenous Q8 has never been observed to dissociate in the presence of DDM, it can diffuse away from the enzyme embedded in a phospholipid bilayer, at least on the time scale of hours. This is far from a demonstration of exchange on the millisecond time scale in a phospholipid bilayer, but suggests the possibility of much more rapid exchange at the QH site under physiological conditions. The slow exchange of Q8 between detergent micelles54 has previously been recognized as a potential artifact in studies of cyt bo3.23 This issue has been previously observed in other systems. The kinetics observed with detergent-solubilized photosynthetic reaction centers is quite different than the kinetics measured in situ with chromatophores. This difference has been analyzed to quantify the slow exchange of Q8 between detergent micelles.54 Another example is the bc1 complex, in which the substrate at the quinone reductase site (Qi or QN) remains bound in the detergent-solubilized enzyme and is resolved in many of the reported crystal structures.55,56 Like the quinone bound to cyt bo3 discussed in the current work, the quinone at the QN site of the bc1 complex must also exchange rapidly during catalytic turnover and this site also stabilizes a semiquinone state. (5) Simultaneous binding to cyt bo3 at the QH site of Q8 and inhibitors such as HQNO or piericidin A, or by the watersoluble substrate Q1H23,20 has also been used to argue that the QH site cannot be the substrate binding site. No displacement of Q8 is observed in the DDM-solubilized cyt bo3 in the presence of a vast excess of Q1 (Table 2),3 and the same has been reported for cyt bo3 solubilized by sucrose monolaurate.20 In contrast, Q2 or Q4 are effective at displacing Q8 from the QH site (Table 2). Presumably, the presence of the longer polyisoprene side chain (2 or 4 isoprenes in Q2 and Q4) allow Q2 and Q4 to compete effectively with Q8 for binding to the enzyme. These data argue that Q8 can remain bound to the DDM-solubilized enzyme through interactions with the isoprenoid tail even when the head group has been displaced by a competing ligand. Previous data have demonstrated that each of the two isoprene units present in Q2 contribute to the

The data in Table 1 also show that mutations of residues at of both HQNO and or near the QH site increase the Kapp i aurachin C1-10. HQNO has been characterized as either an uncompetitive23,24 or noncompetitive3 inhibitor of Q1H2 oxidation by cyt bo3, whereas aurachin C1-10 is a competitive inhibitor.3 While the conservative D75E mutant, at the heart of the QH site, has subtle effects on the interaction between cyt bo3 and the ubisemiquinone29 and decreases the KM of Q1H2 (Table 1), this mutation increases the Kapp of both HQNO and i aurachin C1−10 (Table 1). Mutations of Q101 that result in the largest increases in the KM of Q1H2 also have large effects on the Kapp aurachin C1−10 and HQNO (Table 1). One i complicating factor is that uncompetitive or noncompetitive inhibition by HQNO suggests that this inhibitor binds at a separate site from Q1H2. Perhaps relevant is that the N157 V HQNO but mutation results in a very large increase in the Kapp i only a modest increase in the KM of Q1H2 (Table 1). It is possible that HQNO binds in a separate pocket in the protein that is adjacent to the Q1H2 binding site but interacts with some of the same residues. This would also be consistent with the observation that aurachin C1−10 and HQNO can bind simultaneously to cyt bo3.3 If the site of quinol oxidation is the same as the site that stabilizes the semiquinone intermediate, then a large body of experimental data previously used in support of two quinone binding sites must be reinterpreted. (1) Affinity labeling with a radiolabeled azido-ubiquinone derivatives identified peptides in subunit II as being part of the QL site.35,51 However, in view of the subsequent X-ray structure,25 it is not plausible to consider these peptides as part of a physiologically relevant quinone binding site. The data must be ascribed to nonspecific binding of the probe. (2) Spontaneous point mutations resistant to “QL site” inhibitors were sequenced and shown to all be located in the hydrophilic domain of subunit II: I129T, N198T, Q233H, M248I, S258N, F281S, and H284Q.37 However, the X-ray structure25 shows these residues to be distributed widely within the hydrophilic domain of subunit II and they do not define any plausible quinone binding site. The mutations have substantial effects on the Kiapp of the inhibitors (2,6-dimethyl-1,4 benzoquinone; 2,6-dichloro-4-nitrophenol; 2,6-dichloro-4-dicyanovinylphenol), but not on the KM of Q1H2.37 The large effects on the inhibitor binding remain unexplained, but may be due to conformational changes resulting from the mutations. (3) Steady state kinetics of cyt bo3 have been interpreted in terms of two quinone binding sites.23,24 The inhibition of cyt bo3 (using either Q1H2 or Q2H2 as the substrate) by HQNO (and closely related NQNO) is uncompetitive23,24,52 or noncompetitive3 suggesting that HQNO binds to a separate site from where Q1H2 is oxidized. Since these inhibitors are analogues of the semiquinone state, the logical interpretation is that the semiquinone state is formed at a site on the protein that is distinct from the site of quinone oxidation (QL vs QH). The most extensive kinetics study23 showed substrate inhibition and concluded that there must be two Q binding sites that are both in rapid equilibrium with the quinol pool and which are allosterically coupled. The question is how can the noncompetitive or uncompetitive inhibition pattern of HQNO be explained if there is a single quinone binding site. It is possible that substrate aggregation as well as distribution within detergent micelles, particularly for Q2H2,23 contributes to complex (i.e., not competitive) kinetics patterns. In our studies, kinetics studies with Q2H2 were not pursued because in many 8352

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binding affinity to the enzyme.57 When studies are performed in detergents such as DDM, solubility and micelle exchange issues of Q8 and the competing ligands may also contribute to the observations of displacement or its lack.54 It is likely that the binding sites for the inhibitors and the ubiquinol head group partially overlap, but the binding sites of different inhibitors need not overlap with each other. This appears to be the case for the inhibitors HQNO and aurachin C1−10, which each bind with 1:1 stoichiometry to the enzyme,3,45 and which can bind simultaneously to cyt bo3.3 It is also noted that although the binding of HQNO does not displace the bound Q8 from the enzyme, it does eliminate the formation of the ubisemiquinone species,3 consistent with the postulate that the hydrogen binding of the semiquinone to the protein at the QH site has been perturbed by HQNO binding at the same site. (6) The enzyme can be isolated with more than one equivalent of “bound” Q8, and this has been interpreted to mean there is more than one Q binding site. The stoichiometry of co-purified Q8 depends on the detergent as well as the details of the preparative protocol.19,20 It is clear that one equivalent of copurified Q8 is functionally relevant and is bound to a physiologically significant binding site, because this site stabilizes a ubisemiquinone when the enzyme is appropriately redox poised.16 However, beyond this, additional Q8 associated with the enzyme in DDM or sucrose monolaurate detergents is likely adventitious and not bound to a physiologically meaningful site on the protein. Functional relevance can be distinct from the apparent “tight binding” in DDM or sucrose monolaurate. The data presented in the current work do not absolutely prove that substrate oxidation occurs at the same site where the semiquinone is bound, but the data demonstrate that the singlesite model is plausible and needs to be seriously reconsidered. If, as we propose, the single-site model is correct, the following is a reasonable scheme for quinol oxidation by cyt bo3. In a phospholipid bilayer or in the native membrane, the native quinone Q8/Q8H2 is proposed to rapidly diffuse into and out of this site, a phenomenon that is not observed in detergents in which the endogenous Q8 is retained (DDM or sucrose monolaurate). The single Q8H2 binding site (equivalent to the QH site) is close to the low spin heme b25 which acts as a oneelectron acceptor, leaving the one-electron reduced ubisemiquinone species stabilized at the binding site by its hydrogen bond interactions with R71, D75, and to a lesser extent, H98 and Q101. Electron transfer from heme b to the heme o3/CuB active site allows the second electron transfer from the ubisemiquinone to heme b. The oxidation of the substrate at the QH site is also consistent with the concomitant release of chemical protons to the periplasm.58 The oxidized Q8 leaves the active site and is rapidly replaced by fresh Q8H2. In detergents DDM or sucrose monolaurate, inhibitors such as HQNO or the soluble substrate Q1H2 displace the head group of the endogenous Q8, but not the entire molecule, which has an affinity to the protein through the long polyisoprene tail. Although this is consistent with observations of the enzyme in DDM and sucrose monolaurate, it may also be the case in the native membrane or in a phospholipid bilayer, depending on the concentration of Q8. Further quantitative studies are needed to confirm this single-site model, including further structural work with both the endogenous Q8 as well as bound inhibitors.

Article

AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Chang Sun: 0000-0001-6070-5545 Robert B. Gennis: 0000-0002-3805-6945 Present Address §

S.K.C.: Lawrence Berkeley National Laboratory, Molecular Biophysics and Integrated Bioimaging, Berkeley, CA 94720, USA. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Dr. Christoph von Ballmoos (Universität Bern, Switzerland) for helping with the reconstitution of proteoliposomes. We are also grateful to Dr. Yi Lu as well as the Mining Microbial Genomes group (Dr. William van der Donk, UIUC) for kindly allowing us to use their HPLCs to measure quinone content. We further thank Dr. Ranjani Murali, Dr. Padmaja Venkatakrishnan, and Mariana Lencina for insightful discussions and suggestions. Funding for this work was provided by Grant DE-FG02-87ER13716 (R.B.G.) from Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Sciences, US DOE.

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REFERENCES

(1) Borisov, V. B.; Verkhovsky, M. I. EcoSal Plus 2015, DOI: 10.1128/ecosalplus.ESP-0008-2013. (2) Sousa, F. L.; Alves, R. J.; Ribeiro, M. A.; Pereira-Leal, J. B.; Teixeira, M.; Pereira, M. M. Biochim. Biophys. Acta, Bioenerg. 2012, 1817, 629. (3) Yap, L. L.; Lin, M. T.; Ouyang, H.; Samoilova, R. I.; Dikanov, S. A.; Gennis, R. B. Biochim. Biophys. Acta, Bioenerg. 2010, 1797, 1924. (4) Chepuri, V.; Lemieux, L. J.; Au, D. C.-T.; Gennis, R. B. J. Biol. Chem. 1990, 265, 11185. (5) Kadenbach, B.; Hüttemann, M. Mitochondrion 2015, 24, 64. (6) Yoshikawa, S.; Shimada, A. Chem. Rev. 2015, 115, 1936. (7) Babcock, G. T.; Varotsis, C. J. J. Bioenerg. Biomembr. 1993, 25, 71. (8) Ferguson-Miller, S.; Babcock, G. T. Chem. Rev. 1996, 96, 2889. (9) Babcock, G. T. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 12971. (10) Verkhovsky, M. I.; Belevich, I.; Bloch, D. A.; Wikstrom, M. Biochim. Biophys. Acta, Bioenerg. 2006, 1757, 401. (11) van der Oost, J.; Pappalainen, P.; Musacchio, A.; Warne, A.; Lemieux, L.; Rumbley, J.; Gennis, R. B.; Aasa, R.; Pascher, T.; Malmström, B. G.; Saraste, M. EMBO J. 1992, 11, 3209. (12) Saraste, M.; Raitio, M.; Jalli, T.; Chepuri, V.; Lemieux, L.; Gennis, R. B. Ann. N. Y. Acad. Sci. 1988, 550, 314. (13) Wu, W.; Chang, C. K.; Varotsis, C.; Babcock, G. T.; Puustinen, A.; Wikström, M. J. Am. Chem. Soc. 1992, 114, 1182. (14) Belevich, I.; Verkhovsky, M. I. Antioxid. Redox Signaling 2008, 10, 1. (15) Wikstrom, M.; Verkhovsky, M. I. Biochim. Biophys. Acta, Bioenerg. 2007, 1767, 1200. (16) Hellwig, P.; Yano, T.; Ohnishi, T.; Gennis, R. B. Biochemistry 2002, 41, 10675. (17) Ingledew, W. J.; Ohnishi, T.; Salerno, J. C. Eur. J. Biochem. 1995, 227, 903. (18) Sato-Watanabe, M.; Itoh, S.; Mogi, T.; Matsuura, K.; Miyoshi, H.; Anraku, Y. FEBS Lett. 1995, 374, 265. (19) Puustinen, A.; Verkhovsky, M. I.; Morgan, J. E.; Belevich, N. P.; Wikström, M. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 1545. (20) Sato-Watanabe, M.; Mogi, T.; Ogura, T.; Kitagawa, T.; Miyoshi, H.; Iwamura, H.; Anraku, Y. J. Biol. Chem. 1994, 269, 28908. 8353

DOI: 10.1021/jacs.7b03883 J. Am. Chem. Soc. 2017, 139, 8346−8354

Article

Journal of the American Chemical Society (21) Mogi, T.; Sato-Watanabe, M.; Miyoshi, H.; Orii, Y. FEBS Lett. 1999, 457, 61. (22) Sato-Watanabe, M.; Mogi, T.; Miyoshi, H.; Anraku, Y. Biochemistry 1998, 37, 5356. (23) Musser, S. M.; Stowell, M. H. B.; Lee, H. K.; Rumbley, J. N.; Chan, S. I. Biochemistry 1997, 36, 894. (24) Sato-Watanabe, M.; Mogi, T.; Miyoshi, H.; Iwamura, H.; Matsushita, K.; Adachi, O.; Anraku, Y. J. Biol. Chem. 1994, 269, 28899. (25) Abramson, J.; Riistama, S.; Larsson, G.; Jasaitis, A.; Svensson-Ek, M.; Laakkonen, L.; Puustinen, A.; Iwata, S.; Wikstrom, M. Nat. Struct. Biol. 2000, 7, 910. (26) Lin, M. T.; Baldansuren, A.; Hart, R.; Samoilova, R. I.; Narasimhulu, K. V.; Yap, L. L.; Choi, S. K.; O’Malley, P. J.; Gennis, R. B.; Dikanov, S. A. Biochemistry 2012, 51, 3827. (27) Lin, M. T.; Shubin, A. A.; Samoilova, R. I.; Narasimhulu, K. V.; Baldansuren, A.; Gennis, R. B.; Dikanov, S. A. J. Biol. Chem. 2011, 286, 10105. (28) Lin, M. T.; Samoilova, R. I.; Gennis, R. B.; Dikanov, S. A. J. Am. Chem. Soc. 2008, 130, 15768. (29) Yap, L. L.; Samoilova, R. I.; Gennis, R. B.; Dikanov, S. A. J. Biol. Chem. 2007, 282, 8777. (30) Yap, L. L.; Samoilova, R. I.; Gennis, R. B.; Dikanov, S. A. J. Biol. Chem. 2006, 281, 16879. (31) Grimaldi, S.; Ostermann, T.; Weiden, N.; Mogi, T.; Miyoshi, H.; Ludwig, B.; Michel, H.; Prisner, T.; MacMillan, F. Biochemistry 2003, 42, 5632. (32) Grimaldi, S.; MacMillan, F.; Ostermann, T.; Ludwig, B.; Michel, H.; Prisner, T. Biochemistry 2001, 40, 1037. (33) Sun, C.; Taguchi, A. T.; Vermaas, J. V.; Beal, N. J.; O’Malley, P. J.; Tajkhorshid, E.; Gennis, R. B.; Dikanov, S. A. Biochemistry 2016, 55, 5714. (34) Bossis, F.; De Grassi, A.; Palese, L. L.; Pierri, C. L. Biochem. J. 2014, 461, 305. (35) Tsatsos, P. H.; Reynolds, K.; Nickels, E. F.; He, D.-Y.; Yu, C.-A.; Gennis, R. B. Biochemistry 1998, 37, 9884. (36) Welter, R.; Gu, L.-Q.; Yu, L.; Yu, C.-A.; Rumbley, J.; Gennis, R. B. J. Biol. Chem. 1994, 269, 28834. (37) Sato-Watanabe, M.; Mogi, T.; Sakamoto, K.; Miyoshi, H.; Anraku, Y. Biochemistry 1998, 37, 12744. (38) Ma, J.; Puustinen, A.; Wikström, M.; Gennis, R. B. Biochemistry 1998, 37, 11806. (39) Choi, S. K.; Lin, M. T.; Ouyang, H.; Gennis, R. B. Biochim. Biophys. Acta, Bioenerg. 2017, 1858, 366. (40) Miyoshi, H.; Takegami, K.; Sakamoto, K.; Mogi, T.; Iwamura, H. J. Biochem. 1999, 125, 138. (41) Ingolfsson, H. I.; Sanford, R. L.; Kapoor, R.; Andersen, O. S. J. Visualized Exp. 2010, 44, e2131. (42) von Ballmoos, C.; Biner, O.; Nilsson, T.; Brzezinski, P. Biochim. Biophys. Acta, Bioenerg. 2016, 1857, 321. (43) Villegas, J. M.; Volentini, S. I.; Rintoul, M. R.; Rapisarda, V. A. Arch. Biochem. Biophys. 2011, 505, 155. (44) Kragh-Hansen, U.; le Maire, M.; Moller, J. V. Biophys. J. 1998, 75, 2932. (45) Meunier, B.; Madgwick, S. A.; Reil, E.; Oettmeier, W.; Rich, P. R. Biochemistry 1995, 34, 1076. (46) Carter, K.; Gennis, R. B. J. Biol. Chem. 1985, 260, 10986. (47) Orii, Y.; Mogi, T.; Kawasaki, M.; Anraku, Y. FEBS Lett. 1994, 352, 151. (48) Taguchi, A. T.; O’Malley, P. J.; Wraight, C. A.; Dikanov, S. A. J. Phys. Chem. B 2015, 119, 5805. (49) Kobayashi, K.; Tagawa, S.; Mogi, T. Biochemistry 2000, 39, 15620. (50) Osborne, J. P.; Musser, S. M.; Schultz, B. E.; Edmondson, D. E.; Chan, S. I.; Gennis, R. B. In Oxygen Homeostasis and Its Dynamics; Ishimura, Y., Shimada, H., Suematsu, M., Eds.; Springer-Verlag: Tokyo, 1998; p 33. (51) Ghaim, J. B.; Tsatsos, P. H.; Katsonouri, A.; Mitchell, D. M.; SalcedoHernandez, R.; Gennis, R. B. Biochim. Biophys. Acta, Biomembr. 1997, 1330, 113.

(52) Matsushita, K.; Patel, L.; Kaback, H. R. Biochemistry 1984, 23, 4703. (53) Puustinen, A.; Morgan, J. E.; Verkhovskii, M.; Thomas, J. W.; Gennis, R. B.; Wikström, M. Biochemistry 1992, 31, 10363. (54) Shinkarev, V. P.; Wraight, C. A. Biophys. J. 1997, 72, 2304. (55) Gao, X.; Wen, X.; Esser, L.; Quinn, B.; Yu, L.; Yu, C. A.; Xia, D. Biochemistry 2003, 42, 9067. (56) Xia, D.; Esser, L.; Tang, W.-K.; Zhou, F.; Zhou, Y.; Yu, L.; Yu, C.-A. Biochim. Biophys. Acta, Bioenerg. 2013, 1827, 1278. (57) Sakamoto, K.; Miyoshi, H.; Ohshima, M.; Kuwabara, K.; Kano, B.; Akagi, T.; Mogi, T.; Iwamura, H. Biochemistry 1998, 37, 15106. (58) Puustinen, A.; Finel, M.; Virkki, M.; Wikström, M. FEBS Lett. 1989, 249, 163.

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