Identification of a novel quinone-binding site in the cytochrome bo ...

THE

JOURNALOF BIOLCGICAL CHEMISTRY

Vol. 269, No. 46, Issue of November 18,pp. 28908-28912, 1994 Printed in U.S.A.

0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Identification of a Novel Quinone-bindingSite in the Cytochrome bo Complex from Escherichia coli* (Received forpublication, April 12, 1994, and in revised form, August 11, 1994)

Mariko Sato-WatanabeS, TatsushiMagi$, Takashi OguraO, Teizo KitagawaP, Hideto Miyoshin, Hajime Iwamuran, and Yasuhiro AnrakuSII From the $Department of Plant Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, the §Institute for Molecular Science, Okazaki National Research Institute /Graduate University forAdvanced Studies, Myodaiji, Okazaki 444, and the %Department of Agricultural Chemistry, Kyoto University, Sakyo-ku,Kyoto 606, Japan

The cytochrome bo complex is a heme BO-type heme- low-spin heme B. These important questions must be answered copper quinol oxidase in the aerobic respiratory chain t o increase the understanding of the electron transfer mechaof Escherichia coli and functionsas an electron transfer-nism of the quinol oxidases. linked proton pump. To study the protein-mediated elec- In contrast to theredox metal centers in subunitI (low-spin tron transfer from substrates to metal centers, we car- heme B, high-spin heme 0, and the Cu, center) (3, 51, little is riedoutquantitativeandqualitativeanalysesofa known about thelocation and structureof the quinol oxidation bound quinonein the purified oxidase and found that site it (the QL site)’ of the cytochrome bo complex. Isolation and has a novel high affinity ubiquinone-binding site disanalysis of quinone analogue-resistant mutants are potential tinct from the quinol oxidation site. Enzymatic andspec- approaches for the localization and characterization of the Q, troscopic studies suggest that the quinone-binding site site; however, only a few inhibitors havebeen reported so far (6, is located close to both the quinol oxidation site in sub7), and no analogue-resistant mutants have been isolated. In unit I1 and low-spin hemeB in subunit I. The quinonethe preceding paper (81, the asymmetricmolecular recognition binding siteof a bound ubiquinone-free oxidase was reat the QL site was suggested to account for the sequential constituted with the potent quinol oxidation site in the quinol oxidases, and a crossoxidation of substrates inhibitor 2,6-dichloro-4-nitrophenol, whichdecreased linking study usingan azidoubiquinone derivative showed the the V,, value of the ubiquinol-1 oxidase activity to onepresence of the substrateoxidation site in subunit I1 (9). fourth of the control activity. These results indicate that The aim of this study was t o determine the number of quithe quinone-binding site is essential for the catalytic functions of the cytochrome bo complex and mediates none/quinol-binding site(s) in the cytochrome bo complex and t o electron transfer from the quinol oxidation site to the characterize their functionalrole(s) in substrate oxidation and t o understanding the electron transfer. These issues are central low-spin heme. coupling of quinoloxidation withthereduction of molecular oxygens and alsoprotonpumping. We found that the cytochrome bo complex contains a novel high affinity quinoneThe cytochrome bo complex is a heme BO-type quinol oxidase binding site (designatedas the QH site) besides the low affinity in the aerobic respiratory chain of Escherichia coli (1)and QL site for substrate oxidation and that theQN site is close to belongs to theheme-copper respiratory oxidase superfamily, as both the QLsite and thelow-spin heme. These results suggest does cytochrome c oxidase (2). However, the electron-donating that the QHsite mediates electron transfer from ubiquinols to substrates of these two oxidases are quite different: ubiquinol low-spin heme B as a l-electron transfer gate. (a lipid-soluble 2-electron, 2-proton redox component) and ferrous cytochrome c (a water-soluble l-electron carrier) (1). In EXPERIMENTAL.PROCEDURES addition, the Cu, center, which is the fourth metal center in Chemicals cytochrome c oxidase and isinvolved in electron transfer from The sources and synthesisof p-benzoquinones, substituted phenols ferrocytochrome c to low-spin heme A, does not exist in the (PC), 2-heptyl-4-hydroxyquinolineN-oxide,piericidin A, andubiquicytochrome bo complex (1,3).Since the oxygen reduction mech- none-1 (Q1), -6 (QJ, -7 (Q7), and -8 (QJ were as described (8). Other anism is thought to be identical in bothenzymes (41, ubiquinol chemicals were commercial productsof analytical grade. t o the low-spin heme. Howmust sequentially donate electrons Bacterial Strains ever, it is still unknown whether there is,as in the Cu, center Strain G0103/pMF02 (cyo’ Acyd-Km’lcyo’)was used for isolationof of cytochrome c oxidase or in the primaryquinone-binding site the &,-bound cytochrome bo complex (designatedas the wild-type oxi(QAsite) of the photosyntheticreaction center, any mediation in dase)(10). A ubiquinonebiosynthesis mutant, MU1227 (cyo’cyd’ the electron transfer pathway from ubiquinol t o the low-spin AubiA-Km‘), harboring pMFO2 (cyo’) was used for isolation of the Q8heme or whether ubiquinol can donate electrons directly t o free cytochrome bo complex (the AUbiA oxidase). Strain MU1227 was a kind gift from Dr. M. Kawamukai(ShimaneUniversity,Matsu-e, Japan). * This work was supported in part by a grant-in-aid for scientific research on priority areas from the Ministry of Education,Science, and Reverse-phase HPLC Analysis of Bound Qsin the Purified Culture, Japan (to T. M., T. O., and T. K.),grants from the Asahi Glass Oxidase Foundation and the Ciba-Geigy Foundation for the Promotion of SciThe cytochrome bo complex was purified by anion-exchange HPLC ence (to T. M.), and a grant from the International Human Frontier Science Program Organization (to Y.A,). This is Paper X in the series, (10)and was stored in 50 l l l ~“is-HC1 (pH7.4) containing 0.1% sucrose “Structure-Function Studies on the E. coli Cytochrome bo Complex.” The abbreviations used are: QL site, quinol oxidation site; QH site, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked high affinity quinone-binding site; PC, substituted phenol; Ql, ubiqui“advertisement” in accordancewith 18 U.S.C.Section1734 solely to none-1; HPLC, high performance liquid chromatography; CHAPS,3[(3-cholamidopropyl)dimethylammoniol-l-propanesulfonicacid Q,H,, indicate this fact. 11 To whom correspondence shouldbe addressed. Fax: 81-3-3812-4929. ubiquinol-1. ~~~~~~

28908

,

28909

Quinone-bindingSite in the Cytochrome bo Complex monolaurate (bufferA) (Mitsubishi Kasei Food Corp., Tokyo). Quinones were extracted from 30 nmolof the purified oxidase (5.1 mg of protein) with 2 ml of ethanol in the presence of 30 nmol of Q7 and then transferred into 5 ml of n-hexane. The upper phase was dried under a stream of N, gas, and the oily residue was dissolved in ethanol. The quinones were separated by reverse-phase HPLC using an Altex Ultrasphere ODS column (4.6 mm, inner diameter, x 25 cm) and 3 ModelLC-SA HPLC system equipped with an SPD-M6A photodiode array detector (Shimadzu Corp., Kyoto, Japan). The elution profile was monitored at 278 nm, and the Q, content was estimated from a ratioof the peak area of Qato that of Q7.The solvent was ethanoVmethanoYacetonitrile(4:3:3, : v/v), and the flow rate was 0.8 mumin. Removal of Loosely Associated Quinones Loosely associated quinones were removed by precipitation of the oxidase with ammonium sulfate orpolyethylene glycol or by sizeexclusion chromatography. Precipitation Method-To 2 mlof buffer, A containing 8 mg of the oxidase was added ammonium sulfate or polyethylene glycol 4000 (gas chromatography-gradefrom Merck)at final concentrations of 50%(w/v) and 40% (w/v), respectively. The mixture was kept on icefor 30 min and centrifuged at 100,000 x g and 4 "C for 1 h. The resultant precipitate was solubilizedin 0.5 ml of buffer A and thendialyzed against 1 liter of buffer A for 2 days. Size-exclusionHPLC-One ml of the concentrated oxidase (-200 w) was applied to a G3000SW column (7.8 mm, inner diameter, x 30 cm; Tosoh Corp., Tokyo) and was eluted at a flow rate of 0.5 mumin. The solvent was 50 mM Tris-HC1(pH 7.4) containing 1%CHAPS (Sigma),0.1 m phenylmethylsulfonylfluoride (Sigma), and 0.5 M NaCl.

e A. Wild4ype

E m

B. AUblA

h

E

$

, C. AUblNUQG

0!

10

20

30

Retention Time (mln)

FIG.1. Elution profile of quinones extracted from the wildtype oxidase (A), the AUbiA oxidase ( B ) , and the &-reconstituted A U b i A oxidase (C). Reverse-phase HPLC analyses of quinones were performed as described under "Experimental Procedures." The flow rate was 0.8 mumin. UQ8, Q8; UQ6,Q6.

determined tobe 1.3-2.2 mol of &$mol of the oxidase preparations. However, we noticed that the purified oxidase that had been extensively washed with the buffer reproducibly showed only a stoichiometric amount of bound Q8(Fig. 1and Table I). Competition of Q8 Binding with Exogenous Compounds We then tried toremove loosely associated quinones and phosTwo ml of the wild-type oxidasewas incubated at 4 mg of proteidml in bufferA for 10 min with 0.5 m &,Hzor 20II~Msodium ascorbateplus pholipids present in detergent micelles bound to the oxidase. 10 nm phenazine methosulfate at room temperature or with 0.5 m Q1 After precipitation of the oxidase with ammonium sulfate or or inhibitors on ice. Unbound quinones or inhibitors were removed by polyethylene glycol 4000 or size-exclusion HPLC, the Q8/ polyethylene glycol precipitation, followed by size-exclusion HPLC. oxidase ratio decreased to nearly 1:l (Table I), suggesting that one molecule of Q, is tightly bound to a specific binding site (the Measurements of Absorption a n d Resonance Raman Spectra QH site) in thecytochrome bo complex. A previous study, howAbsolute absorption spectra were measured in buffer A at 5 p and at room temperature in thepresence or absence of 1mM Q1as described (6, ever, failed to identify the presence of a bound form of either Q, 8). Resonance Raman spectra were measured in buffer A at 25 p~ in the or menaquinone-8 in the purified two-subunit oxidase, probpresence or absence of 1 m QI. Raman scattering was excited with a ably due to the small scale of the experimentor to a difference 406.7-nm line of a krypton laser (Spectra-Physics, Model 2016) whose in subunit composition (6). power was at 2 milliwatts at the sample point. The scattered light was Next, the functional properties of the QHsite were examined dispersed with a double monochromator (Spex 1404) and detected with it identical to the Q, site. Since the K, an intensified photodiode array apparatus (PAR 1421Q). Digital to determine whether is oxidation reaction is relatively outputs wereprocessed and analyzed by using the software "Igor" value for Qo and Q6 in theQ1H2 high (70 PM) (6,8), theQ, bound to the Q, site canbe removed (WaveMetrics, Lake Oswego, OR). by incubation with an excess amount of Q,H, or potent Q, site Reconstitution of the QHSite with Exogenous Compounds inhibitors such as 2,6-dichloro-4-nitrophenol(PC15) and 2,6At roomtemperature, 150 pl of 250 PMAUbiAoxidase in buffer A was dichloro-4-dicyanovinylphenol(PC16) (8). However, the bound incubated for 20 min with Q6 or 2,6-dichloro-4-nitrophenol(PC15) at a final concentration of 5 nm and then at 4 "C overnight. Unbound qui- Q8 did not releasefrom its binding siteeven in thepresence of none or inhibitor was removedfrom the oxidase by size-exclusion a 1000-fold excess of exogenous ubiquinone-related compounds HPLC. The reconstituted oxidase was concentrated to -100 1.1~by (Table I). Thus, we concluded that the cytochrome bo complex ultrafiltration using a Centriprep 100 apparatus (Amicon, Inc.). contains a novel highaffinityquinone-binding sitedistinct from the QL site and that the QH site seems not to be in a Miscellaneous The quinol oxidase activity was determined spectrophotometrically dynamic equilibrium with the quinone pool in the cytoplasmic using 50 J ~ MQIH, (8). Other analytical procedures were as described membrane. (8, 10).

Kinetic Analysisof the Quinol OxidaseActivity of the &,-free

Oxidase-The effect of unbound Q, on the quinol oxidase activRESULTS ity was studied using the &,-free oxidase ( A U b i A oxidase) isoQuantitativeAnalysis of the Qs ContentinthePurified lated from the ubiquinonebiosynthesis mutant MU1227/ Oxidase-For a mechanisticconsideration of the electron pMFO2. We confirmed that the A U b i A oxidase (Fig. 1) and transfer reactionsof the quinol oxidases, quantitative determi- the cytoplasmic membrane vesicles (data not shown) isolated nations of the quinone/quinol-binding site(s) are essential. Qui- from strain MU1227/pMF02 did not contain any ubiquinones. nones were ethanol-extracted from several preparations of the Kinetic analysis of the quinol oxidase activity demonstrated purified cytochrome bo complex andseparated by reverse- that theAUbiA oxidase exhibited a higherK, value (75w)for phase HPLC. Ubiquinones were identified by running the au- both Q1H2 (Fig. 2) and ubiquinol-6 (data not shown) than the thentic samples separately (Q, with a retention timeat 9.2 min, wild-type oxidase (50 m) from strain G0103/pMF02. FurtherQ7at 14.5 min, andQ, at 22.5 min) andby simultaneous meas- more, the A U b i A oxidase showed much higher K, values for the urement of their absorption spectra using a photodiode array potent Q, site inhibitors, which were 1.2-7.4-fold (3.1-fold on detector. average) higher than those of the wild-type oxidase (Table 11). The Q, content of the purified oxidase was calculated from These results suggest that some structural alterations take the peak area after correction for the extraction yield and place in the QL site of the A U b i A oxidase.

Quinone-binding Site

28910

in the Cytochrome bo Complex

TABLE I Qx content in the purified wild-type oxidasebefore and after removal of unbound quinones (A) and after treatment with theQL site inhibitors or &,Hz (B) The Qs content in thecytochrome bo complex was determined by the HPLC method described under “Experimental Procedures.”

(A)

Treatment

Q,/oxidase

Wild-type control 50% (NH,),SO, precipitation 40% PEG” 4000 precipitation Size-exclusion chromatography

1.2 1.1 1.0 1.0

‘i ( 6 ) ,i

50 PM2.6-dimethyl-BQ 0.97 500 PM PC16 0.98 500 V M piericidin A 0.76 500 PMHHQNO 0.73 500 PM QP, 0.83 PEG, polyethylene glycol; BQ, p-benzoquinone; HHQNO, 2-heptyl4-hydroxyquinoline N-oxide.

AUbiA+UQl

(B)

D )(1-

1

(c’wT

i

(D) WT+UQl

AUbiNPC15

(C) AUbiNUQ6 (B)AUbiA

500

400

460

5b0

FIG.3. Absolute absorption spectra of the AUbiA oxidase (A and B)and the wild-type (WT)oxidase (C and D ) in the absence or presence of 1 m~ Q1 (VQI).Spectra were taken at 5 PM under air-oxidized ( O X ) ,dithionite-reduced (RD), and dithionite-reducedCObound (CO) conditions.

b IQ1H2l”

450

Wavelength (nm)

(A) WT

-0.05

rl 400

0:05

chiometric amount of Q, was incorporated into the AUbiA oxi-

(pM)-1

FIG.2. Kinetic analyses of the Q,H, oxidase activities of the dase (Fig. 1C). However, the K, value for Q,H, of the Q,-reconwild-type (WT) oxidase (line A), the AUbiAoxidase (CineB),the stituted oxidase remained unchanged (75 VM) (Fig. 2).These &,-reconstitutedAUbiA oxidase (line C ) ,and the PC15-reconsti- results suggest that exogenous ubiquinones are incorporated tuted AUbiA oxidase (Cine D ) . into theQ, site of the AUbiA oxidase during thequinol oxidase TABLEI1 Effect of the absence of bound QB at theQH site on the Q,H, oxidase activity of the cytochrome bo complex The assay was performed in the presence of the QL site inhibitors (piericidin A, 2-heptyl-4-hydroxyquinoline N-oxide (HHQNO) and substituted phenols) at 100 PM or of the p-benzoquinones (BQ) a t 10 PM. Compound

K,

WT

AubiA

AUbiAiWT

PM

Piericidin A HHQNO Substituted phenols PC13 PC15 PC16 PC35 PC42 PC53 p-Benzoquinones 2-Methyl-BQ 2,6-Dimethyl-BQ Tetramethyl-BQ

2.2 2.3

4.3 4.4

2.0 1.9

6.2 4.3 3.0 19 24 29

10 32 15 43 29 79

1.6 7.4 5.0 2.3 1.2 2.7

6.7 0.7 100

43 3.3 125

6.4 4.7 1.3

a WT, wild-type oxidase, PC13, PC15, and PC16, 4-cyano-, 4-nitro-, and 4-dicyanovinyl-2,6-dichlorophenols, resDectively: PC35, 2-chloro-4PC53, cyano-6-iodophenol; PC42, 2,6-dibromo-4-dicyano~inylphenol; 2,4-dinitro-6-sec-butylphenol.

To test the functional role of the QHsite, thepurified A U b i A oxidase was reconstituted with eitherQ, or the potent QL site inhibitor 2,6-dichloro-4-nitrophenol (PC15). Reverse-phase HPLC analysis of the reconstituted oxidase showed that a stoi-

assay, but that themodes of binding of exogenous quinones to the QH site are not the same as that of Qs in the wild-type oxidase. Upon reconstitution with PC15 in the air-oxidized state, a red shiftof the Soretpeak of the AUbiA oxidase was completely restored, indicating thatone PC15 molecule similarly binds to the QH site (data notshown).Kinetic analysis of the Q,H, oxidation reaction showed that the apparentV,, value of the PC15-reconstituted AUbiA oxidase decreased t o one-fourth of that of the wild-type or unreconstituted A U b i A oxidase, with no ) 2).This impliesthat the change in theK, value (i.e. 75 p ~ (Fig. bound PC15 at the QHsite perturbs the intramolecular electron transfer in the AUbiA oxidase. Taken together, these results indicate that the QH site is close t o the QL site and seems essential for the electron transfer reactions in the cytochrome bo complex. Perturbations of the Low-spin Heme in the AUbiA Oxidase under Air-oxidized Conditions-Absolute absorption spectra of the wild-type and AUbiA oxidases were recorded at room temperature underair-oxidized, dithionite-reduced, and dithionitereduced CO-bound conditions (Fig. 3). We found that the Soret absorption peak of the A U b i A oxidase in theair-oxidized state was shifted t o 412 nm from 409 nm for the wild-type oxidase (Fig. 3). This spectral shiftwas completely restored by incubation with exogenous Q,, with a Kd of 2.5 VM, while the wild-type oxidase showed no spectral change upon addition of excess Q1 (Fig. 3). Resonance Raman spectra of the wild-type and AUbiA oxidases were recorded at room temperature under air-oxidized

28911

Quinone-binding Site in the Cytochrome bo Complex 2H+

4

Raman Shifi (cm-1)

FIG.4. Resonance Raman spectra of the wild-type (WT)oxidase (A and C ) and the AUbiA oxidase ( B and D ) in the absence or presence of 1 n m Q, (UQZ). Spectra were taken at 25 w under air-oxidized conditions.

FIG.5. Schematic model for interactions at the QHsite between the QL site and low-spin heme B.Two possible pathways for electron transfer from ubiquinols to heme B are proposed as described under "Discussion." UQ, ubiquinone; UQH,, ubiquinol.

between the QHsite and hemeB is likely to be electronic. Since low-spin heme B is sandwiched between transmembrane helices I1 and X of subunit I (3, 5 ) and theQHsite aswell as theQL site is accessible from the external aqueous medium, the loca(Fig. 4), dithionite-reduced, and dithionite-reduced CO-bound tion of the QH site is probably in the periplasmic region of conditions (data notshown). We found that the Raman featuressubunit I or at theinterface betweensubunit I and theperiplasof the A U b i A oxidase were perturbed only in the air-oxidized mic hydrophilic domain of subunit 11. state (Fig. 4). Raman linesassociated with low-spin heme B of Kinetic studies on quinol oxidation by the PC15-reconstithe wild-type oxidase (redox state marker a t 1372 (v,) cm", tuted AUbiA oxidase provide insight into thefunctional role of spin state marker at1504 (v,) cm", and other featuresat 1125 the QH site (Fig. 2). Spectroscopic evidence indicates that the (v,) cm-l, 1575 (v,) cm", and 1625 ( u ~ ~cm") ~ , ) were shifted to bound PC15 molecule occupies the QHsite; however, the overall 2-5cm" higher wave numbers in the AUbiA oxidase. Upon enzyme turnover was reduced to one-fourth of the wild-type addition of 1 mM Q,, the original spectrum was almost com- andthe Q,-reconstituted A U b i A oxidase activities(Fig. 2). pletely restored. In contrast, the Feg-CO stretching frequency Since the K, values for substrates of the QLsite were the same (521 cm") was unaffected by either the absence of bound Q8 or as thatof the Q,-reconstituted AUbiA oxidase, the effect of the the addition of an excess amount of Q1 (data not shown), indi- bound PC15 molecule is attributable toa decrease in the eleccating that the binuclear centerof the oxidase is unperturbed tron transfer rate(s) between the QL site and the metal cenunder dithionite-reduced conditions. ter(s). Recent kinetic studies on the QL site suggest that seIn addition, quantitation of the heme composition and copper quential oxidation of ubiquinols occurs at the QLsite and that content of the purified AUbiA oxidase indicated that the ab- there may be separate electron transfer pathways correspondsence of bound Qs at the QH site did not affect the affinity or ing to the two spatially separatedhydroxyl groups on ubiquinol specificity ofthe metal-binding sites (datanot shown). Based on (8).The present studypoints to thepossibility that theQHsite these observations, we conclude that the QHsite is close to the provides a branch in theelectron transfer pathwaybetween the low-spin heme B center and thusaffects the electronic state of QLsite and heme B. In other words, the QH site facilitates the the metal center. electron transfer reaction within the oxidase molecule, as does the Cu, site of cytochrome c oxidase. However, it isalso possible DISCUSSION that the QH site may not be involved in electron transfer, but Previous enzymatic studies (6-8) and a recent cross-linking may play some structural role in the oxidase complex since the study using an azidoubiquinone derivative(9) suggest thatsub- bound PC15 molecule perturbed the metal center. unit I1 ofthe E. coli cytochrome bo complex contains theQLsite, Generally, many redox proteins that use quinone as anelecwhich binds one ubiquinol molecule. The present quantitative troncarrierhave two quinone/quinol-binding sites, even analysis of bound Qs in purified oxidase revealed that the though their quinone redox mechanisms are different. ReE. coli cytochrome bo complex contains a novel high affinity cently, fumarate reductase (complex 11) from E. coli was proquinone-binding site (theQHsite), which has a Kd of 2.5 PM for posed t o have separate sites for interaction with reduced and Ql, and that the QHsite isdistinct from the low affinity QLsite, oxidized quinones, and anelectron transfer mechanism similar with a K, of 70 for Qo and Q6 in the &,Hz oxidase reaction to that in the photosynthetic reaction center appeared to be to (Fig. 5)(8).It should be noted that thisis the first study show operative at these quinone redox sites (11). Musser et al. (12) the presence of two quinonelquinol-binding sites in the heme- recently postulated that a proton-motive Q-loop mechanism of copper respiratory quinol oxidases. During the catalyticcycle, the cytochrome bc, complex operates in the cytochrome bo comQLsite to plex. However, the binding affinity for oxidized quinones and the relatively highK, value for substrate enables the quinone/quinol molecules the topological location of the QLand QHsites of the cytochrome be in a dynamic equilibrium with free in thequinone pool of the cytoplasmic membrane. In contrast, bo complex (this study andRef. 8) suggest that thelocations of one Q8 molecule is incorporated into the QH site during the two quinonefquinol-binding sites (theQHand QLsites) aresimbiosynthesis of the oxidase complex in a manner different from ilar to those in the photosynthetic reaction center (13). The that of exogenous quinone-related compounds incorporated primary quinone, QH, is always tightly bound and acts as a into the ALJbiA oxidase during in vitro reconstitution. 1-electron gate, while the secondary quinone, doubly reduced Enzymatic and spectroscopic studies on the native and re- QL,is a 2-electron, 2-protonredox component and can leavethe constituted AUbiAoxidases have demonstrated that the QHsite quinol oxidase after oxidation to QL. This mechanism is very is structurally close t o both the QL site and low-spin heme B. compatible with the dioxygen reduction chemistry in the hemePerturbation of the metal center in the A U b i A oxidase was copper respiratory oxidases, which is commonly catalyzed by a found only in the air-oxidized state; therefore, the interaction single electron transfer system (i.e. heme irons).

28912

Quinone-binding Site in the Cytochrome bo Complex

In conclusion, this study demonstrates that the cytochrome bo complex contains a novel high affinity quinone-binding site (the QH site) distinct from the quinol oxidation site (the QLsite) and provides insight into electron transfer from the quinol oxidation site to the metal center in theheme-copper respiratory quinol oxidases. Future kinetic studies, which will simultaneously monitor quinol oxidation and reduction of heme centers, will aim to elucidate the functional role of the Q, site and the mechanisms of quinol oxidation and protein-mediated electron transfer in this oxidase superfamily. Acknowledgments-We thank K. Kita (University of Tokyo), S. Itoh (National Institute for Basic Biology), T. Kubota (Osaka University), and T.Kishi (Kobe Gakuin University) for invaluable comments and M. Kawamukai for strain MU1227. REFERENCES 1. Anraku, Y., and Gennis, R. B. (1987)Bends Biochem. Sci. 12,262-266 2. Saraste, M., Holm, L., Lemieux, L., Liibben, M., and van der Oost, J. (1991)

Biochem. SOC. Bans.19,608-612 3. Mogi, T., Nakamura, H., and Anraku, Y. (1994)J. Biochem. (Tokyo)116,471477 4. Babcock, G. T.,and Wikstram, M. (1992)Nature 366,301309 5. Hosler, J. P., Ferguson-Miller, S., Calhoun, M.W., Thomas, J. W., Hill, J., Lemieux, L., Ma, J.,Georgiou, C.,Fetter, J.,Shapleigh, J., Tecklenburg, M. M. J., Babcock, G. T., and Gennis, R. B. (1993)J . Bioenerg. Biomembr. 26, 121-136 6. Kita, K., Konishi, K , and Anraku, Y. (1984)J . BioZ. Chem. 269, 3368-3374 I. Matsushita, K., Patel, L., and Kaback, H. R. (1984)Biochemistry 23, 47034714 8. Sato-Watanabe, M., Mogi, T.,Miyoshi, H., Iwamura, H., Matsushita, K., Adachi, O.,and Anraku, Y. (1994)J . BioZ. Chem. 269,28899-28907 9. Welter, R., Gu, L.-Q., Yu, C.-A,, Rumbley,J., and Gennis, R. B. (1994)Biophys. J . 66, 367 (abstr.) 10. Tsubaki, M., Mogi, T., Anraku, Y., and Hori, H. (1993)Biochemistry 32,60656072 11. Westenberg, D. J., Gunsalus, R. P., Ackrell,B. A. C., Sices, H., and Cecchini, G. (1993)J. B i d . Chem. 268,815-822 12. Musser, S. M., Stowell, M. H. B., and Chan, S. I. (1993)FEBS Lett. 327, 131-136 13. McPherson, P. H., Okamura, M. Y., and Feher, G . (1990)Biochim. Biophys. Acta 1016,289-292