JOURNAL OF BACTERIOLOGY, Jan. 1987, p. 205-209 Vol. 169, No. 1 0021-9193/87/010205-05$02.00/0 Copyright © 1987, American Society for Microbiology Reconstitution of Pyrroloquinoline Quinone-Dependent D-Glucose Oxidase Respiratory Chain of with o Oxidase KAZUNOBU MATSUSHITA, MASATSUGU NONOBE, EMIKO SHINAGAWA, OSAO ADACHI, AND MINORU AMEYAMA* Department ofAgricultural Chemistry, Faculty ofAgriculture, Yamaguchi University, Yamaguchi 753, Japan Received 23 June 1986/Accepted 24 September 1986

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent primary dehydrogenase linked to the respiratory chain of a wide variety of bacteria. The exists in the membranes ofEscherichia coli, mainly as an apoenzyme which can be activated by the addition of pyrroloquinoline quinone and magnesium. Thus, membrane vesicles of E. coli can oxidize D-glucose to gluconate and generate an electrochemical proton gradient in the presence of pyrroloquinoline quinone. The D-glucose oxidase-respiratory chain was reconsti- tuted into proteoliposomes, which consisted of two proteins purified from E. coli membranes, D-glucose dehydrogenase and cytochrome o oxidase, and E. coli phospholipids containing ubiquinone 8. The electron transfer rate during D-glucose oxidation and the membrane potential generation in the reconstituted proteoliposomes were almost the same as those observed in the membrane vesicles when pyrroloquinoline quinone was added. The results demonstrate that the quinoprotein, D-glucose dehydrogenase, can reduce ubiquinone 8 directly within phospholipid bilayer and that the D-glucose oxidase system of E. coli has a relatively simple respiratory chain consisting of primary dehydrogenase, ubiquinone 8, and a terminal oxidase.

D-Glucose dehydrogenase (GDH) is a unique membrane- between the dehydrogenase and cytochrome o oxidase bound dehydrogenase which was shown recently to have within a phospholipid bilayer. The reconstituted pro- pyrroloquinoline quinone (PQQ) as the prosthetic group (2, teoliposomes appear to function as effectively as native 9). The enzyme catalyzes the direct oxidation of D-glucose to membrane vesicles with respect to turnover and AjIH+ gluconate; it is known to be located on the cytoplasmic generation. membrane and linked to the respiratory chain in certain bacteria, especially in so-called oxidative bacteria, such as MATERIALS AND METHODS Gluconobacter (6), Pseudomonas (21), and Acinetobacter (9) species. Recently, this enzyme has been detected as an Materials. PQQ was prepared from a culture broth of a apo form lacking PQQ in a wide variety of bacteria including methylotrophic bacterium as described previously (1). Escherichia coli (3, 12). The apo-GDH can be activated by Phospholipids were extracted from E. coli K-12 with the addition of PQQ in the presence of Mg2+ (4, 12). isopropanol-hexane, washed with acetone, dissolved in Recently, in our laboratory, GDH has been purified from the ether, and stored as described previously (P. Viitanen, M. J. membranes of E. coli (5). The enzyme can reduce Newman, D. L. Foster, T. H. Wilson, and H. R. Kaback, ubiquinone 1 (Qj) like the Pseudomonas enzyme, which is Methods Enzymol., in press). Octyl-p-D-glucopyranoside able to reduce long-chain homologs as well as Qi (17). Thus, (octylglucoside), valinomycin, and nigericin were purchased the question is whether the native ubiquinone of E. coli, Q8, from Calbiochem-Behring. 3,3'-Diisopropylthiodicarbocya- is able to accept electrons from the quinoprotein, GDH, nine [diS-C3-(5)] was from Molecular Probes. Q, and Q8 within the phospholipid bilayer. were kindly supplied by Eizai Co. and Nissin-seifun Co. The aerobic respiratory chain of E. coli is relatively Q1H2 was prepared from Qi as described by Rieske (24). simple, with respect to its components and its sequence, and DEAE-Toyopearl, which can be used as a naedium- has a number of primary dehydrogenases coupled to the performance anion exchanger, was purchased from Toyo respiratory chain, including flavoproteins such as D-lactate, Soda Co. All other materials were of reagent grade and succinate, and NADH dehydrogenases (13) and the obtained from commercial sources. quinoprotein GDH (25). The E. coli aerobic respiratory Bacterial strains and preparation of membrane vesicles. E. chain is also branched at the end with cytochrome o and coli GR19N (cyd) (11) was grown aerobically into the late cytochrome d as terminal oxidases. Both of these logarithmic phase in lactate medium (16). Right-side-out have been purified to homogeneity. Reconstitution experi- (RSO) membrane vesicles were prepared by osmotic lysis ments have shown that both terminal oxidases act as Q8H2 (14) and suspended in 50 mM potassium phosphate (KP,), pH oxidases (7, 15, 16) that generate an electrochemical proton 6.5. gradient (A,LH+) during turnover (19, 22). Enzyme preparation. GDH was purified from the mem- In this paper, we report the reconstitution of the PQQ- branes of E. coli K-12 in the presence of Triton X-100 as dependent D-glucose oxidase system from defined compo- described previously (5), and cytochrome o oxidase was nents. The results show clearly that a quinoprotein, GDH, purified from the membranes of E. coli GR19N in the donates electrons to Q8, which mediates electron transfer presence of octylglucoside as described previously (20). For the reconstitution experiment, Triton X-100 in GDH solution was replaced by octylglucoside as follows. A 1-ml sarnple of * Corresponding author. GDH solution dissolved in 5 mM KP, (pH 6.8) containing 0.2 205 206 MATSUSHITA ET AL. J. BACTERIOL.

activity. Phospholipids containing Q8 were prepared as de- scribed previously (16). Sonicated E. coli phospholipids containing Q8 (35 mg of lipids and 500 nmol of Q8) were a mixed with purified cytochrome o oxidase (0.59 mg of protein) and purified GDH (0.2 mg of protein), and octylglu- 0 0 coside was added to a final concentration of 1.25%; the final volume was brought to 4 ml with 50 mM KPi (pH 7.5). The 0 mixture was incubated on ice for 20 min and then diluted into 150 ml of 50 mM KPi (pH 7.5) that had been equilibrated to 25°C. Proteoliposomes were collected by centrifugation at 110,000 x g for 3 h and then suspended in 50 mM KPi (pH 0 6.5). Enzyme assays. GDH activity was assayed with phenazine 0 methosulfate (PMS) and dichlorophenol indophenol (DCIP) as electron acceptors (17) in the presence of 0.4 ,uM PQQ and 5 mM Mg2+. Alternatively, GDH activity was measured 0 with Qi as an electron acceptor by monitoring the decrease 0 in absorbance at 275 nm. The reaction mixture (total volume, 2.5 ml) contained 50 mM KPi (pH 6.0), 5 mM MgSO4, 100 ,uM Ql, 10 mM D-glucose, 0.4 ,uM PQQ, and the enzyme. D-Glucose oxidase activity was measured with an oxygen electrode at 25°C. Reaction mixtures (total volume, 3 ml) contained 50 mM KP, (pH 6.0), 5 mM MgSO4, 10 mM D-glucose, 0.4 ,uM PQQ, and the enzyme. Q1H2 oxidase 6 7 8 3 4 5 activity was measured spectrophotometrically as described pH previously (18). Other Membrane was FIG. 1. pH profiles of enzyme activities in the respiratory chain analytical procedures. potential (Al) D-glucose oxidase system of E. coli GR19N RSO membrane vesi- measured by following fluorescence quenching of diS-C3-(5) cles. RSO membrane vesicles were suspended in 50 mM KP, (pH by using valinomycin-induced potassium diffusion potentials 6.5) containing 5 mM MgSO4 and 7.9 ,uM PQQ. QjH2 oxidase (A) for quantification (18). Protein content was determined by and D-glucose oxidase (0) activities were measured as described in the modified Lowry method, in which sodium dodecyl Materials and Methods. D-Glucose-PMS-DCIP (C) and D- sulfate was included in the alkali solution (10). glucose-Q1 (O) oxidoreductase activities were measured in the RESULTS presence of 8 and 24 mM NaN3, respectively. The enzyme assay and of oxi- was done in 50 mM KPi (pH 6.0 through 8.0) or 0.1 M sodium acetate Enzyme activities AILH+ generation D-glucose buffer (pH 3.5 through 5.0). dase system in E. coli membrane vesicles. E. coli has been shown to contain GDH, mainly as an apoenzyme, in the cytoplasmic membranes (3, 12). The enzyme also has been M KC1 and 0.1% Triton X-100 was diluted with 4 ml of water shown to be linked to the respiratory chain of E. coli, and then applied onto a DEAE-Toyopearl column (bed showing D-glucose oxidase activity in the presence of PQQ volume, 1 ml) equilibrated with 10 mM KPi (pH 7.0) con- (25). Although E. coli intact cells and even membranes taining 1% octylglucoside. After the column was washed contain about 10% holo-GDH (3, 25), D-glucose oxidase with 10 ml of the same buffer, which removes almost all of activity is almost completely dependent on the addition of the Triton X-100 from the column, GDH was eluted with 0.1 PQQ in RSO membrane vesicles prepared in the presence of M KP, (pH 7.0) containing 1% octylglucoside. The GDH EDTA, which depletes PQQ from the membranes. In RSO thus prepared had a specific activity of 220 p.mol of Qi membrane vesicles from E. coli GR19N, D-glucose oxidase reduced per min per mg at pH 6.0 in the presence of PQQ. activity was saturated with around 50 nM PQQ (data not The cytochrome o oxidase that was used in this experiment shown). The D-glucose oxidase activity measured in the had a specific activity of 30 ,umol of Q1H2 oxidized per min presence of PQQ showed the maximum at pH 6.0 (Fig. 1), per mg at pH 7.5. which is considerably lower than the optimum pH (pH 7.5) Reconstitution of proteoliposomes with D-glucose oxidase for cytochrome o oxidase. The lower pH optimum of the

TABLE 1. Activities of enzymes of the D-glucose oxidase system in E. coli GR19N RSO membrane vesicles and reconstituted proteoliposomesa Enzyme activityb (,umol/min per mg) Prepn D-Glucose oxidase D-Glucose-PMS-DCIP D-Glucose-Q1 Q1H2 oxidase Turnover no.c pH 6.0 pH 7.5 Membrane vesicles 0.158 0.241 0.102 0.569 0.984 573 Proteoliposomes 1 5.89 10.0 6.70 7.60 13.2 517 Proteoliposomes 2 1.92 18.6 13.7 1.62 4.03 89 a Proteoliposomes containing GDH, Q8, and cytochrome o oxidase were prepared essentially as described in Materials and Methods. Proteoliposomes 1 were prepared from 0.59 mg of cytochrome o and 0.2 mg of GDH, and proteoliposomes 2 were prepared from 0.59 mg of cytochrome o and 0.51 mg of GDH. b The activities of D-glucose-PMS-DCIP and D-glucose-Q1 oxidoreductases were measured as described in Materials and Methods in the presence of 8 mM NaN3. D-Glucose and Q1H2 oxidase activities were also measured as described in Materials and Methods. Values are in micromoles of D-glucose or Q1H2 oxidized per minute per milligram at pH 6.0. c Values were calculated by using 88,000 g/mol and 300 PMS-DCIP reductase units per mg of purified GDH (5) and are expressed as e-/s per mol of GDH. VOL. 169, 1987 RECONSTITUTION OF GLUCOSE OXIDASE SYSTEM 207

01H2 D-glucose oxidase system may be due to the nature of D-Lac primary dehydrogenase, since D-glucose-Q1 oxidoreductase A showed maximum activity at pH 5.0. Since E. coli GR19N membranes contain cytochrome o oxidase as a sole terminal oxidase, the overall reaction of D-glucose oxidation, i.e., D-glucose oxidase activity, can be assayed individually; the activities of D-glucose-PMS-DCIP and D-glucose-Q1 oxidoreductases reflect GDH activity, whereas Q1I{2 oxi- dase activity monitors cytochrome o oxidase activity. The activity of GDH was much less than the activity of cyto- chrome o oxidase (Table 1, Fig. 1), implying that GDH is rate limiting in the D-glucose oxidation. van Schie et al. (25) showed that D-glucose oxidation generated A4i in E. coli membrane vesicles by using a tetraphenylphosphonium-electrode. Generation of A* was 2min 1 confirmed here by fluorescence quenching of diS-C3-(5) in Va' RSO membrane vesicles of E. coli GR19N (Fig. 2A). The F:10% extent of fluorescence quenching observed with D-glucose in the presence of PQQ and Mg2, was comparable to that observed with Q1H2, indicating that the D-glucose oxidase system is able to generate a A4 (inside negative) of the same Q 1H2 Glu magnitude as that generated by Q1H2 oxidase. It should also B be noted that this quenching with D-glucose is dependent on the concentration of PQQ added (data not shown). The extent of fluorescence quenching with D-lactate was signifi- - -PQQ cantly less than that with D-glucose or Q1H2. This is proba- bly due to low D-lactate oxidase activity at pH 6.5. At pH 7.5, the extent of the quenching with D-lactate was compa- rable to that with Q1H2 (16). Reconstitution of D-glucose oxidase system in proteolipo- purified from somes with purified components. GDH has been Nig the membrahe ofE. coli K-12 (5), and cytochrome o oxidase has been purified from the membrane of E. coli GR19N and Nig reconstituted into proteoliposomes by octylglucoside dilu- . ~~~~~~~~I tion (19). Therefore, if conditions can be found where GDH is incorporated into liposomes, it would be possible to obtain 2m1n Val #POQ proteoliposomes with both GDH and cytochrome o oxidase. VaI In previous studies, D-lactate dehydrogenase (16) and pyru- F: 10% vate oxidase flavoprotein (7, 15) have been shown to bind to proteoliposomes by direct addition or by dilution of the FIG. 2. Fluorescence quenching of diS-C3-(5) in E. coli GR19N enzymes in the presence of proteoliposomes. However, this RSO membrane vesicles (A) and the reconstituted proteoliposomes procedure is not useful for GDH, since only 10% of the (B). The reaction mixture (total volume, 1 ml) contained 50 mM KPi dissolved in Triton (pH 6.5), 5 mM MgSO4, 1 ,uM diS-C3-(5), and membrane vesicles-(10 added GDH was bound when the enzyme ,ug of protein) or proteoliposomes (3.3 ,ug of protein). Membrane X-100, cholate, or octylglucoside was diluted in the presence vesicles and proteoliposomes containing GDH, Q8, and cytochrome of liposomes. Instead, reconstitution was performed by the o oxidase were prepared as described in Materials and Methods. octylglucoside dilution method, in which GDH and lipo- Reactions were initiated by adding 10 mM D-lactate (D-Lac), 2.5 somes were mixed with 1.25% octylglucoside, followed by mM dithiothreitol and 25 puM Qi (QjH2), or 10 mM D-glucose (Glu), the dilution of the mixture into a 30-fold volume of buffer. By followed by the addition of 0.025 ,uM nigericin (Nig) and 1 ,uM this means, about 50% of the added GDH is bound stably to valinomycin (Val), as indicated. For the assay with D-glucose, the the liposomes. reaction mnixture was supplemented with 0.4 ,uM PQQ. The A4is Reconstitution of the D-glucose oxidase system was car- obtained with Q1H2 and D-glucose were -118 and -124 mV, ried out by octylglucoside dilution with purified GDH, respectively, in the case of the proteoliposomes. cytochrome o oxidase, and E. coli phospholipids supple- mented with Q8, which was essential for the reconstitution (interior negative) during D-glucose oxidation in the pres- (data not shown). Proteoliposomes thus prepared showed ence of nigericin; this value was comparable to that pro- significant D-glucose oxidase activity in the presence of PQQ duced with Q1H2 (-118 mV). The data also suggest that the and Mg2+ (Fig. 3). The D-glucose oxidase activity in the systems can generate a ApH in addition to A* because the proteoliposomes was saturated with around 50 nM PQQ quenching is largely stimulated with nigericin, an agent (Fig. 4). The activity was inhibited with KCN, which is an known to dissipate ApH with a concomitant increase in A+i. inhibitor for cytochrome o oxidase, and stimulated with Reconstitution was done with different ratios of GDH and valinomycin and nigericin, suggesting that the proteolipo- cytochrome o oxidase (Table 1). When the amount of the somes generate A1iH' during D-glucose oxidation. The oxidase was limited in the system, the reconstituted activity 4i,H' generation was demonstrated more directly by fluo- was restricted by the oxidase present in the proteolipo- rescence quenching studies with diS-C3-(5) (Fig. 2B). With a somes. When the amount of the oxidase was saturating, saturating concentration of PQQ (about 20 nM) (Fig. 4), the however, glucose oxidase activity was limited by the amount proteoliposomes generated a Al of -120 to -124 mV of GDfI present in the proteoliposomes. In this case, the 208 MATSUSHITA ET AL. J. BACTERIOL.

&.6nu Glu Glu able thermodynamically, since the potentials (E0) of PQQ-PQQH2 and Q-QH2 are 90 mV (8) and 65 mV, respec- 13.2n*f tively. I',' 1 130nll POO Our results also indicate that the D-glucose oxidase system 19.8nM of E. coli consists of only three components, GDH, Q8, and a terminal oxidase, cytochrome o in this particular case, and that additional components are not required for electron transfer from D-glucose to oxygen. The same conclusion has 39.5nM been obtained in reconstitution studies with the pyruvate 2mmn oxidase system of E. coli which contains a primary dehydro- 176 natom 0 genase, Q8, and a terminal oxidase (7, 15). In the case of the D-lactate oxidase system, however, an additional thiol- sensitive component appears to be required for maximum rates of electron flow from D-lactate dehydrogenase to Q8 2 mMIKCN (16), as mentioned above. The data presented also show that native and reconsti- FIG. 3. D-Glucose oxida* activity in proteoliposomes reconsti- tuted with GDH, Q8, and cytochrome o oxidase. Proteoliposomes tuted D-glucose oxidase systems are able to generate AjlH'. preparation and D-glucose Goxidase assay were done as described in In the D-lactate and pyruvate oxidase systems, APNH' is Materials and Methods. The reaction mixture for the right-hand generated during electron flow through the terminal oxidase curve contained 50 mM KPi (pH 6.5), 5 mM MgSO4 and pro- only (i.e., the terminal oxidase is the only site at which teoUposomes (26.4 ,ug of protein). The reaction was started by Ai\H' is generated) (7, 16). Since D-glucose oxidation gen- adding 10 mM D-glucose (Glu), followed by 130 nM PQQ, 1 ,uM erates a APiH' comparable to that generated with Q1H2, valinomycin-0.025 ,uM nigericin (Val/Nig), and 2 mM KCN, as which donates electrons directly to the terminal oxidase, indicated. The actual values were 3.13, 4.60, and 0.11 ,ug-atoms of cytochrome o oxidase may also be the only site involved in 0 per min per mg, respectively, after the addition of PQQ, Ai\H' generation in the D-glucose oxidase system of E. coli valinomycin-nigericin, and KCN. The reaction mixture for the GR19N. Cytochrome o oxidase has been shown to generate left-hand curve contained 50 mM KPi (pH 6.0), 5 mM MgSO4, 1 ,uM valinomycin-0.025 ,uM nigericin, and proteoliposomes (26.4 ,ug of A1iH' by catalyzing the scalar release of protons from protein). The reaction was started with 10 mM D-glucose (Glu), on the outer surface of the membrane, vectorial followed by each final concentrations of PQQ, as indicated. The transfer of electrons from the outer to the inner surface, and actual values were 1.03, 2.12, 3.30, and 5.01 ,ug atoms of 0 per min scalar utilization of protons on the inner surface to reduce per mg, respectively. oxygen (16, 19). The results that the proteoliposomes generate AjIH+ (in- apparent turnover of D-glucose oxidase in the proteolipo- side negative) suggest that a relatively high percentage of the somes was about 90% of that in RSO membrane vesicles in which cytochrome o oxidase is saturating.

DISCUSSION The main purpose of this paper is to determine the physiological electron acceptor of PQQ-dependent GDH in 0 the respiratory chain of E. coli. GDH has a novel prosthetic C %C.- group, PQQ, which is unique relative to the flavin prosthetic 0c group present in many primary dehydrogenases in the mem- o brane-bound respiratory chain. We have demonstrated that a 0c. single peptide GDH purified from Pseudomonas sp. can a react with ubiquinone, including long chain homologs, but the reactivity with Q6 or Qg is very low when GDH and ubiquinone are not reconstituted into a phospholipid bilayer 0 (17). In this study, we demonstrated that GDH purified from E. coli reduces Q8, the native ubiquinone in the E. coli respiratory chain, when Q8 is reconstituted into the phos- P Q 0 (nM) pholipid bilayer. Since E. coli cytochrome o oxidase is a FIG. 4. D-Glucose oxidase activity and D-glucose-dependent Q8H2 oxidase (7, 16), reconstitution of high D-glucose oxi- membrane potential generation in the reconstituted proteolipo- dase activity with cytochrome o oxidase indicates that GDH somes. Proteoliposomes containing GDH, Q8, and cytochrome o is able to reduce Q8 to produce Q8H2, which is then oxidized oxidase were prepared as described in Materials and Methods. rapidly by the oxidase in the proteoliposomes. D-Lactate D-Glucose oxidase activity (0) was measured in a reaction mixture dehydrogenase has been shown to require an additional that contained 50 mM KPi (pH 6.0), 5 mM MgSO4, and comnponent to reduce Q8 at maximum rates in the respiratory proteoliposomes (26.4 ,ug of protein). Reaction was initiated with chain of E. coli (16). In E. coli succinate dehydrogenase, D-glucose, and PQQ was added as shown in Fig. 3. Atp generation iron-sulfur protein and even may be required (0) was measured as described in Materials and Methods. The to reduce ubiquinone (13). Although pyruvate oxidase reaction mixture (total volume, 1 ml) contained 50 mM KPi (pH 6.0), flavoprotein is able to reduce Q8 (7, 15), the enzyme is 5 mM MgSO4, 1 ,uM diS-C3-(5), different concentrations of PQQ, and proteoliposomes (3.3 ,ug of protein). The A* generated in each different from flavoproteins because it requires an additional case after the addition of nigericin was determined from a calibration , thiamine pyrophosphate. The quinoprotein GDH curve derived from experiments with the same amounts of the appears to be capable of direct donation of electrons to the proteoliposomes, in which potassium diffusion potentials of known respiratory chain at the level of ubiquinone. This is reason- magnitude were imposed in the presence of valinomycin. VOL. 169, 1987 RECONSTITUTION OF GLUCOSE OXIDASE SYSTEM 209

1981. D-glucose dehydrogenase of Gluconobacter suboxydans: Glucose Gluconate 2Hs Solubilization, purification and characterization. Agric. Biol. Chem. 45:851-861. 7. Carter, K., and R. B. Gennis. 1985. Reconstitution of the ubiquinone-dependent pyruvate oxidase system of Escherichia coli with the cytochrome o terminal oxidase complex. J. 13iol. Chem. 260:10986-10990. 8. Duine, J. A., and J. F. Jzn. 1981. Quinoproteins: a novel class of dehydrogenases. Trends Biochem. Sci. 6:278-280. 9. Duine, J. A., J. F. Jzn, and J. K. Zeeland. 1979. Glucose dehydrogenase from Acinetobacter calcoaceticus: a quinopro- tein. FEBS Lett. 108:443-446. 10. Dulley, J. R., and P. A. Grieve. 1975. A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Anal. Biochem. 64:136-141. D.Lactate 11. Green, G. N., and R. B. Gennis. 1983. Isolation and character- FIG. 5. Postulated D-glucose and D-lactate oxidase systems in E. ization of an Escherichia coli mutant lacking cytochrome d coli GR19N aerobic respiratory chain. GDH, which is located on the terminal oxidase. J. Bacteriol. 154:1269-1275. outer surface, oxidizes glucose to gluconate and donates electrons 12. Hommes, R. W. J., P. W. Postma, 0. M. Nelissel, D. W. directly to Q8. The Q8H2 thus produced is oxidized with cytochrome Tempest, P. Dokter, and J. A. Duine. 1984. Evidence of a o oxidase, which functions to release protons outside and quinoprotein glucose dehydrogenase apo-enzyme in several translocate electrons inside to reduce oxygen, generating a AIH+ strains of Escherichia coli. FEMS Microbiol. Lett. 24:329-333. (16, 19). On the other hand, D-lactate dehydrogenase (D-LDH), 13. Ingledew, W. J., and R. K. Poole. 1984. The respiratory chains which is located on the inner surface, may transfer electrons to Q8 of Escherichia coli. Microbiol. Rev. 48:222-271. via a thiol-sensitive component (SH) (16). GDH and D-LDH contain 14. Kaback, H. R. 1971. Bacterial membranes. Methods Enzymnol. PQQ and FAD, respectively, as a prosthetic group, and cytochrome 22:99-120. o (Cyt o) oxidase contains both cytochrome b (Cyt b) and cyto- 15. Koland, J. G., M. J. Miller, and R. B. Gennis. 1984. Reconsti- chrome o (16). tution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with cytochrome d terminal oxidase. Biochemistry 23:445-453. cytochrome o molecules have the RSO orientation as de- 16. Matsushita, K., and H. R. Kaback. 1986. D-lactate oxidation and scribed previously (19). Although there is no evidence on the generation of the proton electrochemical gradient in membrane orientation of GDH in the proteoliposomes, it is conceivable vesicles from Escherichia coli GR19N and in proteoliposomes reconstituted with purified D-lactate dehydrogenase and cyto- that only GDH located on the outer surface of the proteo- chrome o oxidase. Biochemistry 25:2321-2327. liposomes can react with D-glucose, which may be imper- 17. Matsushita, K., Y. Ohno, E. Shinagewa, 0. Adachi, and M. meable. Furthermore, GDH has been shown to be located on Ameyama. 1982. Membrane-bound, electron transport-linked, the outer surface of the cytoplasmic membrane of E. coli D-glucose dehydrogenase of Pseudomonas fluorescens. Inter- GR19N by using Western blotting (20a), whereas D-lactate action of the purified enzyme with ubiquinone or phospholipid. dehydrogenase is present on the inner surface of the mem- Agric. Biol. Chem. 46:1007-1011. brane (23). 18. Matsushita, K., L. Patel, R. B. Gennis, and H. R. Kaback. 1983. Thus, the respiratory chain of D-glucose oxidase system of Reconstitution of active transport in proteoliposomes contain- E. coli GR19N, which contains cytochrome o oxidase as the ing cytochrome o oxidase and lac carrier protein purified from sole terminal can be as in 5. The Escherichia coli. Proc. Nati. Acad. Sci. USA 80:4889-4893. oxidase (11), depicted Fig. 19. Matsushita, K., L. Patel, and H. R. Kaback. 1984. Cytochrome respiratory chain is simple, generates APiHE at only one site, o type oxidase from Escherichia coli. Characterization of the cytochrome o oxidase, and is remarkably similar to the enzyme and mechanism of electrochemical proton gradient D-lactate oxidase system (16), with the exception of the thiol generation. Biochemistry 23:4703-4714. component and the orientation of primary dehydrogenase. 20. Matsushita, K., L. Patel, and H. R. Kaback. 1986. Purification and reconstitution of the cytochrome o-type oxidase from ACKNOWLEDGMENT Escherichia coli. Methods Enzymol. 126:113-122. We thank H. R. Kaback for critical reading of the manuscript. 20a.Matsushita, K., E. Shinagawa, T. Inoue, 0. Adachi, and M. Ameyama. 1986. Immunological evidence for two types of LITERATURE CITED PQQ-dependent D-glucose dehydrogenase in bacterial mem- branes and the location of the enzyme in Escherichia coli. 1. Ameyama, M., M. Hayashi, K. Matsushita, E. Shinagawa, and FEMS Microbiol. Lett. 37:141-144. 0. Adachi. 1984. Microbial production of pyrroloquinoline 21. Midgley, M., and E. A. Dawes. 1973. The regulation of transport quinone. Agric. Biol. Chem. 48:561-565. of glucose and methyl-a-glucoside in Pseudomonas aeruginosa. 2. Ameyama, M., K. Matsushita, Y. Ohno, E. Shinagawa, and 0. Biochem. J. 132:141-154. Adachi. 1981. Existence of a novel prosthetic group, PQQ, in 22. Miller, M. J., and R. B. Gennis. 1985. The cytochrome d oxidase membrane-bound, -linked, primary complex is a coupling site in the aerobic respiratory chain of dehydrogenases of oxidative bacteria. FEBS Lett. 130:179-183. Escherichia coli. J. Biol. Chem. 260:14003-14008. 3. Ameyama, M., M. Nonobe, M. Hayashi, E. Shinagawa, K. 23. Owen, P., and H. R. Kaback. 1979. Antigenic architecture of Matsushita, and 0. Adachi. 1985. Mode of binding of pyr- membrane vesicles from Escherichia coli. Biochemistry 18: roloquinoline quinone to apo-glucose dehydrogenase. Agric. 1422-1426. Biol. Chem. 49:1227-1231. 24. Rieske, J. S. 1967. Preparation and properties of reduced 4. Ameyama, M., M. Nonobe, E. Shinagawa, K. Matsushita, and 0. coenzyme Q-cytochrome c reductase (complex III of the respi- Adachi. 1985. Method of enzymatic determination of pyr- ratory chain). Methods Enzymol. 10:239-245. roloquinoline quinone. Anal. Biochem. 151:263-267. 25. van Schie, B. J., K. J. Hellingwerf, J. P. van Dijken, M. G. L. 5. Ameyama, M., M. Nonobe, E. Shinagawa, K. Matsushita, K. Elferink, J. M. van DOI, J. G. Kuenen, and W. N. Konings. 1985. Takimoto, and 0. Adachi. 1986. Purification and characteriza- Energy transduction by electron transfer via a pyrroloquinoline tion of quinoprotein apo-D-glucose dehydrogenase from Esch- quinone-dependent glucose dehydrogenase in Escherichia coli,, erichia coli. Agric. Biol. Chem. 50:49-57. Pseudomonas aeruginosa, and Acinetobacter calcoaceticus 6. Ameyama, M., E. Shinagawa, K. Matsushita, and 0. Adachi. (var. lwoffi). J. Bacteriol. 163:493-499.