J. Biochem. 107, 863-867 (1990)
Evidence for Electron Transfer via Ubiquinone between Quinoproteins D-Glucose Dehydrogenase and Alcohol Dehydrogenase of Gluconobacter suboxydans1 Emiko Shinagawa, Kazunobu Matsushita, Osao Adachi, and Minoru Ameyama Department of Agricultural Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi 753 Received for publication, January 9, 1990
Gluconobacter suboxydans contains membrane-bound D-glucose and alcohol dehydrogenases (GDH and ADH) as the primary dehydrogenases in the respiratory chain. These enzymes are known to be quinoproteins having pyrroloquinoline quinone as the prosthetic group. GDH reduces an artificial electron acceptor, ferricyanide, in the membrane, but not after solubilization with Triton X-100, while ADH can react with the electron acceptor even after solubilization and further purification. In this study, it has been shown that the ferricyanide reductase activity of GDH is restored by adding the supernatant solubilized with Triton X-100 to the residue, and also by incorporation of purified ADH into the membranes of an ADH-deficient strain, G. suboxydans var. a. In addition, the ferricyanide reductase activity of GDH was reconstituted in proteoliposomes from GDH, ADH, and ubiquinone-10. Thus, the results indicated that the electron transfer from GDH to ferricyanide was mediated by ubiquinone and ADH. The data also suggest that GDH and ADH transfer electrons mutually via ubiquinone in the respiratory chain.
It is well known that the oxidative bacteria such as acetic acid bacteria oxidize D-glucose to D-gluconic acid, or ethanol to acetic acid. The incomplete oxidation is carried out by quinoprotein D-glucose dehydrogenase or alcohol dehydrogenase (GDH or ADH) located on the outer surface of the cytoplasmic membrane and linked to the respiratory chain. GDH has been purified from the membranes of many bacteria {1-4) and shown to be a hydrophobic single polypeptide of 83 to 87 kDa, which contains pyrroloquinoline quinone as the prosthetic group (5, 6). Furthermore, GDH of enteric bacteria such as Escherichia coli has been shown to exist as the apo-form (7, 8). The purified enzymes are capable of reacting with several artificial dyes, although ubiquinone has now been shown to be a physiological electron acceptor for the enzyme (9, 10). During studies on GDH of Gluconobacter, a peculiar phenomenon was observed; although GDH exhibits an activity for ferricyanide reduction in the membranes, the purified enzyme loses the ferricyanide reductase activity at around neutral pH, the optimum pH observed in the membranes (2), while ADH retains the ferricyanide reductase activity after purification (11). Since the purified enzyme has normal GDH activity with other electron acceptors (2, 10), the membranes may contain a mediator transferring electrons from GDH to ferricyanide. There are some exceptional strains of Gluconobacter having no ferricyanide reductase activity of GDH in the membranes, 1 This work was supported by a Grant-in-Aid (No. 62440014) for Scientific Research from the Ministry of Education, Science and Culture of Japan. Abbreviations: GDH, D-glucose dehydrogenase [EC 1.1.99.17]; ADH, alcohol dehydrogenase; Qi0, ubiquinone-10; HQNO, 2-heptyl4-hydroxyquinoline iV-oxide; octylglucoside, /3-octyl-D-glucopyranoside; PMS, phenazine methosulfate; DCIP, dichlorophenol indophenol.
Vol. 107, No. 6, 1990
which are 5-keto-D-gluconate-producing G. suboxydans var. a strains. Since the strains contain a normal level of GDH which seems to oxidize D-glucose to produce Dgluconate, the precursor for 5-keto-D-gluconate, G. suboxydans var. a seems to lack the mediator described above. In this study, thus, the mediator between GDH and ferricyanide was investigated, and the results obtained here demonstrate that both ADH and ubiquinone are required for the electron transfer. Concomitantly, the data also suggested that ubiquinone mediates the direct electron transfer between the two quinoproteins, GDH and ADH. MATERIALS AND METHODS Materials—Ubiquinone-10 (Qio), Amphitol, and yeast extract were kindly supplied by Eisai, Kao, and Oriental Yeast Industry, respectively. /?-Octyl-D-glucopyranoside (octylglucoside) and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) were obtained from Calbiochem and Sigma, respectively. All other materials used in this study were commercial products of analytical grade. Phospholipids were extracted from G. suboxydans cells with isopropanol/ hexane, washed with acetone, dissolved in diethyl ether, and stored as described (12). GDH was purified from the membranes of G. suboxydans in the presence of 0.1% Triton X-100 and the detergent was replaced by 1% octylglucoside, as described (20). ADH was also purified from the membranes of the organisms essentially by the same methods as described previously (11). Triton X-100 in the enzyme solution was depleted by precipitating with 20% (w/v) polyethylene glycol 6000 and resuspending in 10 mM potassium phosphate buffer, pH 6.0, or was exchanged with octylglucoside by using hydroxyapatite as in the case of GDH.
Bacterial Strains, Growth Conditions, and Membrane 863
864 Preparation—G. suboxydans EFO 12528 and G. suboxydans var. a IFO 3254 were used throughout this work. The organisms were grown in a sugar-rich medium with rotary shaking or in a 50-liter jar fermentor at 30"C (23). Cells were harvested at the late exponential phase by centrifugation and then washed twice with distilled water. The cell paste was suspended in 10 mM potassium phosphate buffer, pH 6.0 at a concentration of 1 g wet weight of cells per 5 ml, and then passed through a French pressure cell press at 16,000 psi. After centrifugation to remove intact cells, the supernatant was centrifuged at 80,000 x g for 60 min. The resultant precipitates were homogenized in 10 mM potassium phosphate buffer, pH 6.0, at a protein concentration of 10 mg/ml to use as membrane fraction. Reconstitution of the Ferricyanide Reductase Activity of GDH from the Solubilized Supernatant and Membrane Residue—The membrane fraction prepared from G. suboxydans as described above was treated with 0.3 or 1.0% Triton X-100. After adding Triton X-100 to the membrane fraction, the suspension was incubated for 30 min on ice and then centrifuged at 80,000 Xg for 60 min to obtain the supernatant and the membrane residue. To the supernatant obtained by treatment with 0.3% Triton X-100, polyethylene glycol 6,000 was added to a final concentration of 25% . (w/v). The mixture was stirred for 30 min on ice and centrifuged at 12,000 X g for 10 min. The resultant precipitate was dissolved in 10 mM potassium phosphate buffer, pH 6.0, or in the same buffer containing 1% octylglucoside or 1% Amphitol. The suspension was mixed with a half volume of the membrane residues prepared by treatment with 1% Triton X-100 and suspended in 10 mM potassium phosphate buffer, pH 6.0, at a protein concentration of 10 mg/ml. The mixture was incubated for 60 min at room temperature, or dialyzed overnight against 10 mM potassium phosphate buffer, pH 6.0, below 10'C for reconstitution. Reconstitution of ADH into Membranes Prepared from G. suboxydans var. a—To the membrane fraction prepared from G. suboxydans var. a as described above, octylglycoside was added to a final concentration of 1%. The suspension (1 ml, 10 mg of protein) was allowed to stand for 30 min on ice, and then mixed with 0.5 ml (1 mg of protein) of purified ADH from which Triton X-100 had been depleted by polyethylene glycol-precipitation. After standing on ice for 60 min, the mixture was dialyzed overnight against 10 mM potassium phosphate buffer, pH 6.0, and used for enzyme assay. Reconstitution of Proteoliposomes with Ferricyanide Reductase Activity of GDH—Phospholipids containing Qi0 (10 nmol of Qio per mg of phospholipid) were prepared as described previously (20), and sonicated. The liposomes (1 mg) with or without Q[0 were mixed with GDH (10 ng of protein) in the presence of 1.25% octylglucoside, and the mixture was incubated on ice for 30 min. Thereafter, proteoliposomes were prepared by dialysis and centrifugation, as described {10). The proteoliposomes containing only GDH, or both Q lo and GDH, were mixed with 2 volumes of ADH solution containing 1% octylglucoside; the final octylglucoside concentration was 0.67%. The mixture was incubated on ice for 30 min and then dialyzed overnight against 50 mM potassium phosphate buffer, pH 6.5. Proteoliposomes were collected by centrifugation at 120,000 X g for 3 h and then suspended in the same buffer (0.1 ml per
E. Shinagawa et al. mg of phospholipid). Enzyme Assay—GDH and ADH activities were measured by using two different electron acceptor systems, ferricyanide and phenazine methosulfate (PMS)-dichlorophenol indophenol (DCIP). The ferricyanide reductase activity was determined colorimetrically under essentially the same conditions as described previously (2, 11), while the PMS-DCEP reductase activity was assayed spectrophotometrically (2). Other Analytical Procedures—Protein content was determined by the modified Lowry method, in which sodium dodecyl sulfate was included in the alkali solution {14). RESULTS Effect of Solubilization with Triton X-100 on Enzyme Activity of GDH and ADH in the Membranes of G. suboxydans—When the membranes of G. suboxydans were treated with Triton X-100, only ferricyanide reductase activity of GDH was selectively damaged, while other enzyme activities, i.e. ferricyanide reductase of ADH and PMS-DCIP reductase of GDH, were not affected (Table I). These enzyme activities were also examined after solubilization and subsequent separation into the solubilized supernatant and precipitate (Table I). Ferricyanide reductase activity of ADH was detected only in the supernatant, while PMS-DCIP reductase of GDH was only partially solubilized. At 1.0% Triton X-100, no ferricyanide reductase activity of GDH was detected in either the solubilized or the residual fraction. Although the ferricyanide reductase activity was observed over a rather wide range of pH in the native membrane, as shown in Fig. 1, solubilization of the membrane with Triton X-100 decreased the activity near neutral pH (pH 4-7) but not at acidic pH (below pH3), at which purified GDH exhibits ferricyanide reductase activity (2). Since glucose oxidase activity has been shown to be retained in the residual membrane after solubilizaiton with Triton {13), however, the respiratory chain itself may be not much damaged by the treatment. Reconstitution of Ferricyanide Reductase Activity of GDH from the Supernatant Solubilized with Triton X-100 TABLE I. Effect of solubilization with Triton X-100 on enzyme activities of GDH and ADH in the membranes of G. suboxydans. The membranes were incubated with 0.3 or 1.0% Triton X-100 for 30 min on ice (suspension), and then the supernatants and precipitates were obtained by centrifugation at 80,000 X g for 60 min. Ferricyanide reductase and PMS/DCIP reductase activity of GDH and ferricyanide reductase activity of ADH were measured as described in 'MATERIALS AND METHODS." Enzyme activities are expressed as the relative activity (%) to the native membranes. Specific activities (units/mg protein) of the enzymes in the native membranes are shown in parentheses. Samples Membranes 0.3% Triton X-100 Suspension Supernatant Precipitate 1.0% Triton X-100 Suspension Supernatant Precipitate
GDH
Ferricyanide 100 (0.68) 10 0 13 0 0.5 0
ADH
PMS/DCP 100 (3.68)
Ferricyanide 100 (1.70)
100 11 89
105 101 9
100 20 78
82 100 2 J. Biochem.
Ubiquinone-Dependent Electron Transfer in Quinoproteins and the Precipitate—The data presented above suggest that Triton X-100 solubilizes some mediators transferring electrons from GDH to ferricyanide. To examine whether the solubilized supernatant is able to restore ferricyanide reductase activity of GDH in the membrane residues, reconstitution of the activity was tried using the residual membranes solubilized with 1% Triton X-100 and the supernatant solubilized with 0.3% Triton X-100, which were used for the reasons that the residual membranes show almost no ferricyanide reductase activity of GDH and the supernatant has a relatively low level of solubilized GDH (about 10%). For the reconstitution, the solubilized Fig. 1. Effect of pH on ferricyanide reductase activity of GDH in the membranes of G. suboxydans before and after solnbilization with Triton X-100. The membranes were solubilized with 0.3 or 1.0% Triton X-100 as described in the footnote of Table I and the residual membranes were used for enzyme assay. Ferricyanide reductase activity (units/mg of protein) was measured in 50 mM citratephosphate buffer at various pHs. — • —, native membrane; — •—, residual membranes solubilized with 0.3% Triton X-100; —O—, residual membranes solubilized with 1.0% Triton X-100.
865 supernatant was precipitated with polyethylene glycol to deplete Triton X-100 and dissolved again in buffer with or without dialyzable detergent, Amphitol or octylglucoside. When the Triton-depleted supernatant was mixed with the residual membranes, some ferricyanide reductase activity (at around pH 5) of GDH was restored (Fig. 2A). Furthermore, dialysis of the mixture to remove detergent remarkably improved the restoration of ferricyanide reductase activity of GDH (Fig. 2B). As shown, the reconstitution was more efficient when performed in buffer containing Amphitol or octylglucoside. Although the data are not shown, the solubilized supernatant could also restore the ferricyanide reductase activity in the membranes of G. suboxydans var. a which have recently been shown to exhibit very low ferricyanide reductase activity of GDH in spite of having a normal level of GDH (unpublished). Reconstitution of Ferricyanide Reductase Activity of GDH from Purified ADH and the Membranes of G. suboxydans var. a—The results obtained above have shown that the supernatant from G. suboxydans membranes after treatment with Triton X-100 contains some components able to mediate electron transfer from GDH to ferricyanide, as well as almost all of the ADH activity (Table I). Furthermore, the membranes of G. suboxydans var. a, having a negligible level of ferricyanide reductase activity of GDH, have been shown to lack the second subunit of ADH complex and thus also ADH activity (unpublished). Thus, ADH is a candidate to mediate the electron transfer from GDH to ferricyanide. To examine the possibility of ADH mediating the electron transfer from GDH to ferricyanide, therefore, ADH purified from G. suboxydans
'as c 0.4 = 0.3 -
8
Fig. 3. Reconstitution of ferricyanide reductase activity of GDH from ADH and the membranes of G. suboxydans var. a. Reconstitution was performed as described in "MATERIALS AND METHODS." Ferricyanide reductase activity (units/mg of protein) was measured with the native ( o ) and reconstituted ( • ) membranes.
Fig. 2. Reconstitution of ferricyanide reductase activity of GDH from the supernatant and precipitate obtained by solubilizing the membranes of G. suboxydans with Triton X-100. Reconstitution was performed, as described in "MATERIALS AND METHODS," in buffer without detergent (— • —), with 1% octylglucoside (—O—), or with 1% Amphitol (—•—). Ferricyanide reductase activity (unita/mg of protein) was measured with the solubilized precipitate (— O —) and the reconstituted membranes before (A) and after (B) dialysis. Vol. 107, No. 6, 1990
TABLE II. Effect of HQNO on enzyme activities of GDH and ADH in the membranes of G. suboxydans. Each enzyme activity was measured after pre-incubation of the membranes (26 ^g of protein) with 3 //I of 1 or 10 mM HQNO dissolved in dimethylsulfoxide in the assay mixture (1 ml) at 25'C for 5 min. Remaining enzyme activities are expressed as the relative activity (%) with respect to the activity of the membranes treated with the same volume of dimethylsulfoxide. GDH
3//M HQNO 30 MM HQNO
Ferricyanide 83.5 65.9
ADH
PMS/DCIP 93.8 88.7
Ferricyanide 95.8 100.6
E. Shinagawa et al.
866
PH
Fig. 4. Reconstitution of ferricyanide reductase activity of GDH into proteoliposomes containing GDH, ADH, and ubiquinone. Reconstitution was performed, as described in "MATERIALS AND METHODS," with different combinations of GDH, ADH, andQ.o- Activity (ferricyanide reductase) of GDH is expressed as 100 times the [activity ratio of ferricyanide reductase versus PMS/DCIP reductase measured at pH 6.0]. (A) — o—, native membrane of G. suboxydans (ferricyanide reductase activity at pH 6.0 of GDH, 0.87 unit/mg protein; PMS/DCIP reductase activity at pH 6.0 of GDH, 3.68 units/mg; PMS/DCIP reductase activity at pH 6.0 of ADH, 0.99 unit/mg); — o—, proteoliposomes prepared only with GDH (PMS/ DCIP reductase activity, 147 units/mg; ferricyanide reductase activity at pH 3.0, 2.0 units/mg); — • —, proteoliposomes prepared with Q10 and GDH (PMS/DCIP reductasne activity, 83.7 units/mg; ferricyanide reductase activity at pH 3.0, 2.5 units/mg). (B) — o—, ADH dissolved in 0.1% Triton X-100 was reconstituted into proteoliposomes containing only GDH at 1 : 1 molar ratio of ADH to GDH (PMS/DCIP reductase activities of GDH and ADH, 131 and 16.9 units/mg; ferricyanide reductase activity at pH 3 of GDH, 3.4 units/ mg); — •—, ADH dissolved in 0.1% Triton X-100 was reconstituted into proteoliposomes containing both Q,o and GDH at 1 : 1 molar ratio of ADH to GDH (PMS/DCIP reductase activities of GDH and ADH, 73.3 and 14.3 units/mg; ferricyanide reductase activity at pH 5.0 of GDH, 3.4 units/mg); — O —, ADH dissolved in 1% octylglucoside was reconstituted into proteoliposomes containing both Q,o and GDH at 1 : 1 molar ratio of ADH to GDH (PMS/DCIP reductase activities of GDH and ADH, 106 and 22.8 units/mg; ferricyanide reductase at pH 5.0 of GDH, 9.2 units/mg); —•—, ADH dissolved in 1% octylglucoside was reconstituted into proteoliposomes containing both Q]o and GDH at 5 : 1 molar ratio of ADH to GDH (PMS/DCIP reductase activities of GDH and ADH, 35.0 and 24.6 units/mg; ferricyanide reductase activity at pH 4.0 of GDH, 5.2 units/mg).
was reconstituted into the membranes of G. suboxydans var. a. As shown in Fig. 3, reconstitution of ADH into the membranes generates ferricyanide reductase activity (at around neutral pH) of GDH which is not detected in the native membranes of G. suboxydans var. a. Reconstitution of Ferricyanide Reductase Activity of GDH from Purified GDH and ADH, and Ubiquinone—The data described above show that ADH can mediate the electron transfer reaction from GDH to ferricyanide. Since
no such activity was detected by simple mixing of GDH and ADH (data not shown), it is conceivable that another factor such as lipid environment or another respiratory components) such as ubiquinone may be required for the electron transfer. As shown in Table II, an ubiquinone analogue, HQNO, inhibits ferricyanide reductase of GDH but not of ADH, and only partially inhibits PMS-DCIP reductase of GDH. Although the inhibition is not so marked as in the case of terminal cytochrome o oxidase (15), the results suggest that ubiquinone may be involved in the electron transfer from GDH to ferricyanide. Therefore, we attempted to reconstitute such an electron transfer reaction into an artificial phospholipid bilayer by incorporating ADH into proteoliposomes containing GDH and/or ubiquinone (Fig. 4). When GDH alone was reconstituted into proteoliposomes, GDH exhibited low ferricyanide reductase activity only at pH 3.0 (Fig. 4A), like the membrane treated with Triton X-100 (Fig. 1). Although this situation was not much changed by double reconstitution with GDH and ADH, as shown in Fig. 4B, the ferricyanide reductase activity at neutral pH appeared when both GDH and ADH were reconstituted with phospholipids containing ubiquinone (Qio). In this experiment, Q,o was used at a concentration of 10 nmol per mg of phospholipid because the glucose oxidase respiratory chain consisting of GDH, Q10 and cytochrome o has been reconstituted most efficiently at the concentration of 5-10 nmol per mg of phospholipid (10). Thus, ubiquinone was shown to be indispensable for the reconstitution, but the quinone itself could not be a mediator for ferricyanide because the ferricyanide reductase activity was not reconstituted only by GDH and Qi0 (Fig. 4A). The efficiency of the reconstitution was dependent on the enzymes used; when ADH dissolved in 1% octylglucoside was used instead of the enzyme dissolved in Triton X-100, the ferricyanide reductase activity was increased (Fig. 4B). Furthermore, when ADH was added in five times greater amount, the activity was not much increased but the optimum pH shifted from pH 5 to pH 4 (Fig. 4B), which was different from that of the native membrane (Fig. 4A). Thus, the efficiency to transfer electrons from GDH to ferricyanide is about half in the reconstituted system as compared with the native membrane; the values of the ferricyanide reductase/PMS-DCIP reductase activity ratio were 0.1 and 0.23, respectively (Fig. 4). DISCUSSION Quinoproteins GDH and ADH are the primary dehydrogenases for the glucose and ethanol oxidase respiratory chains of acetic acid bacteria. Both dehydrogenases contain the same prosthetic group, pyrroloquinoline quinone, but exhibit different structural character from each other; GDH is a single polypeptide quinoprotein but ADH is composed of three distinct subunits, quinohemoprotein, cytochrome c, and a small unidentified peptide (for a review, see Ref. 16). Furthermore, although both enzymes can reduce ferricyanide in the membranes of acetic acid bacteria, only ADH can react with the artificial dye after isolation (16). In this study, the identity of the mediators for electron transfer between GDH and ferricyanide in the membranes was addressed. The results presented here provide a clear indication that electron transfer from GDH to ferricyanide is mediated by J. Biochem.
Ubiquinone-Dependent Electron Transfer in Quinoproteins
867
ubiquinone and ADH in the membranes of G. suboxydans. This conclusion is supported by the following observations, (i) Solubilization of the membranes with Triton X-100 diminishes ferricyanide reductase activity of GDH concomitantly with depletion of ADH from the membranes (Table I and Fig. 1). (ii) The solubilized supernatant, including ADH, has a capability of restoring the ferricyanide reductase activity of GDH in the residual membranes (Fig. 2). (iii) ADH can confer ferricyanide reductase activity of GDH upon the membranes of G. suboxydans var. a in which both ferricyanide reductase activities of GDH and ADH are deficient (Fig. 3). (iv) HQNO selectively inhibits ferricyanide reductase activity of GDH (Table II). (v) Finally, the ferricyanide reductase activity can be reconstituted into proteoliposomes from GDH, ADH, and Qlo (Fig. 4). Although the electron transfer reaction to ferricyanide is not physiological and the significance of the conclusions described above seems unsubstantial, the observations presented here give some important clues for understanding the electron transfer reaction in the respiratory chain of G. suboxydans. Reconstitution of ferricyanide reductase activity of GDH was not accomplished by only GDH and Q,o or by only GDH and ADH, and all three components were indispensable, for the reconstitution. Since GDH has been shown to be able to react directly with ubiquinone in a phospholipid bilayer (10), it is suggested that GDH donates electrons to ubiquinone first and then reduced ubiquinone reacts directly with ADH, which subsequently reduces ferricyanide. Recently, we have shown that the ethanol oxidase respiratory chain can be reconstituted into proteoliposomes from ADH, Q lo , and cytochrome o terminal oxidase (unpublished). In conjunction with the other observations described above, the results obtained in this study suggest that both quinoproteins, GDH and ADH, may transfer electrons mutually via ubiquinone in the respiratory membranes of G. suboxydans.
We wish to thank Ryozi Takagaki, Masanori Uchida, Naoko Hirayama, and Shinjirou Sakurai for their technical assistance.
Vol. 107, No. 6, 1990
REFERENCES 1. Matsushita, K., Ohno, Y., Shinagawa, E., Adachi, 0., & Ameyama, M. (1980) Agric. Biol. Chem. 44, 1505-1512 2. Ameyama, M., Shinagawa, E., Matsushita, K., & Adachi, 0. (1981) Agric. Biol. Chem. 45, 851-861 3. Ameyama, M., Nonobe, M., Shinagawa, E., Matsushita, K., Takimoto, K., & Adachi, 0. (1986) Agric. Biol. Chem. 50, 49-57 4. Matsushita, K., Shinagawa, E., Adachi, O., & Ameyama, M. (1989) Biochemistry 28, 6276-6280 5. Duine, J.A., Frank, Jzn. J., & van Zeeland, J.K. (1979) FEBS Lett. 108, 443-446 6. Ameyama, M., Matsushita, K., Ohno, Y., Shinagawa, E., & Adachi, O. (1981) FEBS Lett. 130, 179-183 7. Hommes, R.W.J., Postoma, P.W., Neijssel, O.M., Tempest, D.W., Dokter, P., & Duine, J.A. (1984) FEMS Microbiol. Lett. 24, 329-333 8. Ameyama, M., Nonobe, M., Hayashi, M., Shinagawa, E., Matsushita, K., & Adachi, 0. (1985) Agric. Biol. Chem. 49,12271231 9. Matsushita, K., Nonobe, M., Shinagawa, E., Adachi, O., & Ameyama, M. (1987) J. Bacteriol. 169, 205-209 10. Matsushita, K., Shinagawa, E., Adachi, 0., & Ameyama, M. (1989) J. Biochem. 105, 633-637 11. Adachi, 0., Tayama, K., Shinagawa, E., Matsushita, K., & Ameyama, M. (1978) Agric. Biol. Chem. 42, 2045-2056 12. Viitanen, P., Newman, M.J., Foster, D.L., Wilson, T.H., & Kaback, H.R. (1986) Methods Enzymol. 125, 429-452 13. Ameyama, M., Matsushita, K., Shinagawa, E., & Adachi, 0. (1987) Agric. Biol. Chem. 51, 2943-2950 14. Dulley, J.R. & Grieve, P.A. (1975) Anal. Biochem. 64, 136-141 15. Matsushita, K., Shinagawa, E., & Adachi, 0., & Ameyama, M. (1987) Biochim. Biophys. Acta 894, 304-312 16. Ameyama, M., Matsushita, K., Shinagawa, E., & Adachi, 0. (1990) Vitam. Horm. 46, in press
Evidence for Electron Transfer via Ubiquinone between Quinoproteins D-Glucose Dehydrogenase and Alcohol Dehydrogenase of Gluconobacter suboxydans1 Emiko Shinagawa, Kazunobu Matsushita, Osao Adachi, and Minoru Ameyama Department of Agricultural Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi 753 Received for publication, January 9, 1990
Gluconobacter suboxydans contains membrane-bound D-glucose and alcohol dehydrogenases (GDH and ADH) as the primary dehydrogenases in the respiratory chain. These enzymes are known to be quinoproteins having pyrroloquinoline quinone as the prosthetic group. GDH reduces an artificial electron acceptor, ferricyanide, in the membrane, but not after solubilization with Triton X-100, while ADH can react with the electron acceptor even after solubilization and further purification. In this study, it has been shown that the ferricyanide reductase activity of GDH is restored by adding the supernatant solubilized with Triton X-100 to the residue, and also by incorporation of purified ADH into the membranes of an ADH-deficient strain, G. suboxydans var. a. In addition, the ferricyanide reductase activity of GDH was reconstituted in proteoliposomes from GDH, ADH, and ubiquinone-10. Thus, the results indicated that the electron transfer from GDH to ferricyanide was mediated by ubiquinone and ADH. The data also suggest that GDH and ADH transfer electrons mutually via ubiquinone in the respiratory chain.
It is well known that the oxidative bacteria such as acetic acid bacteria oxidize D-glucose to D-gluconic acid, or ethanol to acetic acid. The incomplete oxidation is carried out by quinoprotein D-glucose dehydrogenase or alcohol dehydrogenase (GDH or ADH) located on the outer surface of the cytoplasmic membrane and linked to the respiratory chain. GDH has been purified from the membranes of many bacteria {1-4) and shown to be a hydrophobic single polypeptide of 83 to 87 kDa, which contains pyrroloquinoline quinone as the prosthetic group (5, 6). Furthermore, GDH of enteric bacteria such as Escherichia coli has been shown to exist as the apo-form (7, 8). The purified enzymes are capable of reacting with several artificial dyes, although ubiquinone has now been shown to be a physiological electron acceptor for the enzyme (9, 10). During studies on GDH of Gluconobacter, a peculiar phenomenon was observed; although GDH exhibits an activity for ferricyanide reduction in the membranes, the purified enzyme loses the ferricyanide reductase activity at around neutral pH, the optimum pH observed in the membranes (2), while ADH retains the ferricyanide reductase activity after purification (11). Since the purified enzyme has normal GDH activity with other electron acceptors (2, 10), the membranes may contain a mediator transferring electrons from GDH to ferricyanide. There are some exceptional strains of Gluconobacter having no ferricyanide reductase activity of GDH in the membranes, 1 This work was supported by a Grant-in-Aid (No. 62440014) for Scientific Research from the Ministry of Education, Science and Culture of Japan. Abbreviations: GDH, D-glucose dehydrogenase [EC 1.1.99.17]; ADH, alcohol dehydrogenase; Qi0, ubiquinone-10; HQNO, 2-heptyl4-hydroxyquinoline iV-oxide; octylglucoside, /3-octyl-D-glucopyranoside; PMS, phenazine methosulfate; DCIP, dichlorophenol indophenol.
Vol. 107, No. 6, 1990
which are 5-keto-D-gluconate-producing G. suboxydans var. a strains. Since the strains contain a normal level of GDH which seems to oxidize D-glucose to produce Dgluconate, the precursor for 5-keto-D-gluconate, G. suboxydans var. a seems to lack the mediator described above. In this study, thus, the mediator between GDH and ferricyanide was investigated, and the results obtained here demonstrate that both ADH and ubiquinone are required for the electron transfer. Concomitantly, the data also suggested that ubiquinone mediates the direct electron transfer between the two quinoproteins, GDH and ADH. MATERIALS AND METHODS Materials—Ubiquinone-10 (Qio), Amphitol, and yeast extract were kindly supplied by Eisai, Kao, and Oriental Yeast Industry, respectively. /?-Octyl-D-glucopyranoside (octylglucoside) and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) were obtained from Calbiochem and Sigma, respectively. All other materials used in this study were commercial products of analytical grade. Phospholipids were extracted from G. suboxydans cells with isopropanol/ hexane, washed with acetone, dissolved in diethyl ether, and stored as described (12). GDH was purified from the membranes of G. suboxydans in the presence of 0.1% Triton X-100 and the detergent was replaced by 1% octylglucoside, as described (20). ADH was also purified from the membranes of the organisms essentially by the same methods as described previously (11). Triton X-100 in the enzyme solution was depleted by precipitating with 20% (w/v) polyethylene glycol 6000 and resuspending in 10 mM potassium phosphate buffer, pH 6.0, or was exchanged with octylglucoside by using hydroxyapatite as in the case of GDH.
Bacterial Strains, Growth Conditions, and Membrane 863
864 Preparation—G. suboxydans EFO 12528 and G. suboxydans var. a IFO 3254 were used throughout this work. The organisms were grown in a sugar-rich medium with rotary shaking or in a 50-liter jar fermentor at 30"C (23). Cells were harvested at the late exponential phase by centrifugation and then washed twice with distilled water. The cell paste was suspended in 10 mM potassium phosphate buffer, pH 6.0 at a concentration of 1 g wet weight of cells per 5 ml, and then passed through a French pressure cell press at 16,000 psi. After centrifugation to remove intact cells, the supernatant was centrifuged at 80,000 x g for 60 min. The resultant precipitates were homogenized in 10 mM potassium phosphate buffer, pH 6.0, at a protein concentration of 10 mg/ml to use as membrane fraction. Reconstitution of the Ferricyanide Reductase Activity of GDH from the Solubilized Supernatant and Membrane Residue—The membrane fraction prepared from G. suboxydans as described above was treated with 0.3 or 1.0% Triton X-100. After adding Triton X-100 to the membrane fraction, the suspension was incubated for 30 min on ice and then centrifuged at 80,000 Xg for 60 min to obtain the supernatant and the membrane residue. To the supernatant obtained by treatment with 0.3% Triton X-100, polyethylene glycol 6,000 was added to a final concentration of 25% . (w/v). The mixture was stirred for 30 min on ice and centrifuged at 12,000 X g for 10 min. The resultant precipitate was dissolved in 10 mM potassium phosphate buffer, pH 6.0, or in the same buffer containing 1% octylglucoside or 1% Amphitol. The suspension was mixed with a half volume of the membrane residues prepared by treatment with 1% Triton X-100 and suspended in 10 mM potassium phosphate buffer, pH 6.0, at a protein concentration of 10 mg/ml. The mixture was incubated for 60 min at room temperature, or dialyzed overnight against 10 mM potassium phosphate buffer, pH 6.0, below 10'C for reconstitution. Reconstitution of ADH into Membranes Prepared from G. suboxydans var. a—To the membrane fraction prepared from G. suboxydans var. a as described above, octylglycoside was added to a final concentration of 1%. The suspension (1 ml, 10 mg of protein) was allowed to stand for 30 min on ice, and then mixed with 0.5 ml (1 mg of protein) of purified ADH from which Triton X-100 had been depleted by polyethylene glycol-precipitation. After standing on ice for 60 min, the mixture was dialyzed overnight against 10 mM potassium phosphate buffer, pH 6.0, and used for enzyme assay. Reconstitution of Proteoliposomes with Ferricyanide Reductase Activity of GDH—Phospholipids containing Qi0 (10 nmol of Qio per mg of phospholipid) were prepared as described previously (20), and sonicated. The liposomes (1 mg) with or without Q[0 were mixed with GDH (10 ng of protein) in the presence of 1.25% octylglucoside, and the mixture was incubated on ice for 30 min. Thereafter, proteoliposomes were prepared by dialysis and centrifugation, as described {10). The proteoliposomes containing only GDH, or both Q lo and GDH, were mixed with 2 volumes of ADH solution containing 1% octylglucoside; the final octylglucoside concentration was 0.67%. The mixture was incubated on ice for 30 min and then dialyzed overnight against 50 mM potassium phosphate buffer, pH 6.5. Proteoliposomes were collected by centrifugation at 120,000 X g for 3 h and then suspended in the same buffer (0.1 ml per
E. Shinagawa et al. mg of phospholipid). Enzyme Assay—GDH and ADH activities were measured by using two different electron acceptor systems, ferricyanide and phenazine methosulfate (PMS)-dichlorophenol indophenol (DCIP). The ferricyanide reductase activity was determined colorimetrically under essentially the same conditions as described previously (2, 11), while the PMS-DCEP reductase activity was assayed spectrophotometrically (2). Other Analytical Procedures—Protein content was determined by the modified Lowry method, in which sodium dodecyl sulfate was included in the alkali solution {14). RESULTS Effect of Solubilization with Triton X-100 on Enzyme Activity of GDH and ADH in the Membranes of G. suboxydans—When the membranes of G. suboxydans were treated with Triton X-100, only ferricyanide reductase activity of GDH was selectively damaged, while other enzyme activities, i.e. ferricyanide reductase of ADH and PMS-DCIP reductase of GDH, were not affected (Table I). These enzyme activities were also examined after solubilization and subsequent separation into the solubilized supernatant and precipitate (Table I). Ferricyanide reductase activity of ADH was detected only in the supernatant, while PMS-DCIP reductase of GDH was only partially solubilized. At 1.0% Triton X-100, no ferricyanide reductase activity of GDH was detected in either the solubilized or the residual fraction. Although the ferricyanide reductase activity was observed over a rather wide range of pH in the native membrane, as shown in Fig. 1, solubilization of the membrane with Triton X-100 decreased the activity near neutral pH (pH 4-7) but not at acidic pH (below pH3), at which purified GDH exhibits ferricyanide reductase activity (2). Since glucose oxidase activity has been shown to be retained in the residual membrane after solubilizaiton with Triton {13), however, the respiratory chain itself may be not much damaged by the treatment. Reconstitution of Ferricyanide Reductase Activity of GDH from the Supernatant Solubilized with Triton X-100 TABLE I. Effect of solubilization with Triton X-100 on enzyme activities of GDH and ADH in the membranes of G. suboxydans. The membranes were incubated with 0.3 or 1.0% Triton X-100 for 30 min on ice (suspension), and then the supernatants and precipitates were obtained by centrifugation at 80,000 X g for 60 min. Ferricyanide reductase and PMS/DCIP reductase activity of GDH and ferricyanide reductase activity of ADH were measured as described in 'MATERIALS AND METHODS." Enzyme activities are expressed as the relative activity (%) to the native membranes. Specific activities (units/mg protein) of the enzymes in the native membranes are shown in parentheses. Samples Membranes 0.3% Triton X-100 Suspension Supernatant Precipitate 1.0% Triton X-100 Suspension Supernatant Precipitate
GDH
Ferricyanide 100 (0.68) 10 0 13 0 0.5 0
ADH
PMS/DCP 100 (3.68)
Ferricyanide 100 (1.70)
100 11 89
105 101 9
100 20 78
82 100 2 J. Biochem.
Ubiquinone-Dependent Electron Transfer in Quinoproteins and the Precipitate—The data presented above suggest that Triton X-100 solubilizes some mediators transferring electrons from GDH to ferricyanide. To examine whether the solubilized supernatant is able to restore ferricyanide reductase activity of GDH in the membrane residues, reconstitution of the activity was tried using the residual membranes solubilized with 1% Triton X-100 and the supernatant solubilized with 0.3% Triton X-100, which were used for the reasons that the residual membranes show almost no ferricyanide reductase activity of GDH and the supernatant has a relatively low level of solubilized GDH (about 10%). For the reconstitution, the solubilized Fig. 1. Effect of pH on ferricyanide reductase activity of GDH in the membranes of G. suboxydans before and after solnbilization with Triton X-100. The membranes were solubilized with 0.3 or 1.0% Triton X-100 as described in the footnote of Table I and the residual membranes were used for enzyme assay. Ferricyanide reductase activity (units/mg of protein) was measured in 50 mM citratephosphate buffer at various pHs. — • —, native membrane; — •—, residual membranes solubilized with 0.3% Triton X-100; —O—, residual membranes solubilized with 1.0% Triton X-100.
865 supernatant was precipitated with polyethylene glycol to deplete Triton X-100 and dissolved again in buffer with or without dialyzable detergent, Amphitol or octylglucoside. When the Triton-depleted supernatant was mixed with the residual membranes, some ferricyanide reductase activity (at around pH 5) of GDH was restored (Fig. 2A). Furthermore, dialysis of the mixture to remove detergent remarkably improved the restoration of ferricyanide reductase activity of GDH (Fig. 2B). As shown, the reconstitution was more efficient when performed in buffer containing Amphitol or octylglucoside. Although the data are not shown, the solubilized supernatant could also restore the ferricyanide reductase activity in the membranes of G. suboxydans var. a which have recently been shown to exhibit very low ferricyanide reductase activity of GDH in spite of having a normal level of GDH (unpublished). Reconstitution of Ferricyanide Reductase Activity of GDH from Purified ADH and the Membranes of G. suboxydans var. a—The results obtained above have shown that the supernatant from G. suboxydans membranes after treatment with Triton X-100 contains some components able to mediate electron transfer from GDH to ferricyanide, as well as almost all of the ADH activity (Table I). Furthermore, the membranes of G. suboxydans var. a, having a negligible level of ferricyanide reductase activity of GDH, have been shown to lack the second subunit of ADH complex and thus also ADH activity (unpublished). Thus, ADH is a candidate to mediate the electron transfer from GDH to ferricyanide. To examine the possibility of ADH mediating the electron transfer from GDH to ferricyanide, therefore, ADH purified from G. suboxydans
'as c 0.4 = 0.3 -
8
Fig. 3. Reconstitution of ferricyanide reductase activity of GDH from ADH and the membranes of G. suboxydans var. a. Reconstitution was performed as described in "MATERIALS AND METHODS." Ferricyanide reductase activity (units/mg of protein) was measured with the native ( o ) and reconstituted ( • ) membranes.
Fig. 2. Reconstitution of ferricyanide reductase activity of GDH from the supernatant and precipitate obtained by solubilizing the membranes of G. suboxydans with Triton X-100. Reconstitution was performed, as described in "MATERIALS AND METHODS," in buffer without detergent (— • —), with 1% octylglucoside (—O—), or with 1% Amphitol (—•—). Ferricyanide reductase activity (unita/mg of protein) was measured with the solubilized precipitate (— O —) and the reconstituted membranes before (A) and after (B) dialysis. Vol. 107, No. 6, 1990
TABLE II. Effect of HQNO on enzyme activities of GDH and ADH in the membranes of G. suboxydans. Each enzyme activity was measured after pre-incubation of the membranes (26 ^g of protein) with 3 //I of 1 or 10 mM HQNO dissolved in dimethylsulfoxide in the assay mixture (1 ml) at 25'C for 5 min. Remaining enzyme activities are expressed as the relative activity (%) with respect to the activity of the membranes treated with the same volume of dimethylsulfoxide. GDH
3//M HQNO 30 MM HQNO
Ferricyanide 83.5 65.9
ADH
PMS/DCIP 93.8 88.7
Ferricyanide 95.8 100.6
E. Shinagawa et al.
866
PH
Fig. 4. Reconstitution of ferricyanide reductase activity of GDH into proteoliposomes containing GDH, ADH, and ubiquinone. Reconstitution was performed, as described in "MATERIALS AND METHODS," with different combinations of GDH, ADH, andQ.o- Activity (ferricyanide reductase) of GDH is expressed as 100 times the [activity ratio of ferricyanide reductase versus PMS/DCIP reductase measured at pH 6.0]. (A) — o—, native membrane of G. suboxydans (ferricyanide reductase activity at pH 6.0 of GDH, 0.87 unit/mg protein; PMS/DCIP reductase activity at pH 6.0 of GDH, 3.68 units/mg; PMS/DCIP reductase activity at pH 6.0 of ADH, 0.99 unit/mg); — o—, proteoliposomes prepared only with GDH (PMS/ DCIP reductase activity, 147 units/mg; ferricyanide reductase activity at pH 3.0, 2.0 units/mg); — • —, proteoliposomes prepared with Q10 and GDH (PMS/DCIP reductasne activity, 83.7 units/mg; ferricyanide reductase activity at pH 3.0, 2.5 units/mg). (B) — o—, ADH dissolved in 0.1% Triton X-100 was reconstituted into proteoliposomes containing only GDH at 1 : 1 molar ratio of ADH to GDH (PMS/DCIP reductase activities of GDH and ADH, 131 and 16.9 units/mg; ferricyanide reductase activity at pH 3 of GDH, 3.4 units/ mg); — •—, ADH dissolved in 0.1% Triton X-100 was reconstituted into proteoliposomes containing both Q,o and GDH at 1 : 1 molar ratio of ADH to GDH (PMS/DCIP reductase activities of GDH and ADH, 73.3 and 14.3 units/mg; ferricyanide reductase activity at pH 5.0 of GDH, 3.4 units/mg); — O —, ADH dissolved in 1% octylglucoside was reconstituted into proteoliposomes containing both Q,o and GDH at 1 : 1 molar ratio of ADH to GDH (PMS/DCIP reductase activities of GDH and ADH, 106 and 22.8 units/mg; ferricyanide reductase at pH 5.0 of GDH, 9.2 units/mg); —•—, ADH dissolved in 1% octylglucoside was reconstituted into proteoliposomes containing both Q]o and GDH at 5 : 1 molar ratio of ADH to GDH (PMS/DCIP reductase activities of GDH and ADH, 35.0 and 24.6 units/mg; ferricyanide reductase activity at pH 4.0 of GDH, 5.2 units/mg).
was reconstituted into the membranes of G. suboxydans var. a. As shown in Fig. 3, reconstitution of ADH into the membranes generates ferricyanide reductase activity (at around neutral pH) of GDH which is not detected in the native membranes of G. suboxydans var. a. Reconstitution of Ferricyanide Reductase Activity of GDH from Purified GDH and ADH, and Ubiquinone—The data described above show that ADH can mediate the electron transfer reaction from GDH to ferricyanide. Since
no such activity was detected by simple mixing of GDH and ADH (data not shown), it is conceivable that another factor such as lipid environment or another respiratory components) such as ubiquinone may be required for the electron transfer. As shown in Table II, an ubiquinone analogue, HQNO, inhibits ferricyanide reductase of GDH but not of ADH, and only partially inhibits PMS-DCIP reductase of GDH. Although the inhibition is not so marked as in the case of terminal cytochrome o oxidase (15), the results suggest that ubiquinone may be involved in the electron transfer from GDH to ferricyanide. Therefore, we attempted to reconstitute such an electron transfer reaction into an artificial phospholipid bilayer by incorporating ADH into proteoliposomes containing GDH and/or ubiquinone (Fig. 4). When GDH alone was reconstituted into proteoliposomes, GDH exhibited low ferricyanide reductase activity only at pH 3.0 (Fig. 4A), like the membrane treated with Triton X-100 (Fig. 1). Although this situation was not much changed by double reconstitution with GDH and ADH, as shown in Fig. 4B, the ferricyanide reductase activity at neutral pH appeared when both GDH and ADH were reconstituted with phospholipids containing ubiquinone (Qio). In this experiment, Q,o was used at a concentration of 10 nmol per mg of phospholipid because the glucose oxidase respiratory chain consisting of GDH, Q10 and cytochrome o has been reconstituted most efficiently at the concentration of 5-10 nmol per mg of phospholipid (10). Thus, ubiquinone was shown to be indispensable for the reconstitution, but the quinone itself could not be a mediator for ferricyanide because the ferricyanide reductase activity was not reconstituted only by GDH and Qi0 (Fig. 4A). The efficiency of the reconstitution was dependent on the enzymes used; when ADH dissolved in 1% octylglucoside was used instead of the enzyme dissolved in Triton X-100, the ferricyanide reductase activity was increased (Fig. 4B). Furthermore, when ADH was added in five times greater amount, the activity was not much increased but the optimum pH shifted from pH 5 to pH 4 (Fig. 4B), which was different from that of the native membrane (Fig. 4A). Thus, the efficiency to transfer electrons from GDH to ferricyanide is about half in the reconstituted system as compared with the native membrane; the values of the ferricyanide reductase/PMS-DCIP reductase activity ratio were 0.1 and 0.23, respectively (Fig. 4). DISCUSSION Quinoproteins GDH and ADH are the primary dehydrogenases for the glucose and ethanol oxidase respiratory chains of acetic acid bacteria. Both dehydrogenases contain the same prosthetic group, pyrroloquinoline quinone, but exhibit different structural character from each other; GDH is a single polypeptide quinoprotein but ADH is composed of three distinct subunits, quinohemoprotein, cytochrome c, and a small unidentified peptide (for a review, see Ref. 16). Furthermore, although both enzymes can reduce ferricyanide in the membranes of acetic acid bacteria, only ADH can react with the artificial dye after isolation (16). In this study, the identity of the mediators for electron transfer between GDH and ferricyanide in the membranes was addressed. The results presented here provide a clear indication that electron transfer from GDH to ferricyanide is mediated by J. Biochem.
Ubiquinone-Dependent Electron Transfer in Quinoproteins
867
ubiquinone and ADH in the membranes of G. suboxydans. This conclusion is supported by the following observations, (i) Solubilization of the membranes with Triton X-100 diminishes ferricyanide reductase activity of GDH concomitantly with depletion of ADH from the membranes (Table I and Fig. 1). (ii) The solubilized supernatant, including ADH, has a capability of restoring the ferricyanide reductase activity of GDH in the residual membranes (Fig. 2). (iii) ADH can confer ferricyanide reductase activity of GDH upon the membranes of G. suboxydans var. a in which both ferricyanide reductase activities of GDH and ADH are deficient (Fig. 3). (iv) HQNO selectively inhibits ferricyanide reductase activity of GDH (Table II). (v) Finally, the ferricyanide reductase activity can be reconstituted into proteoliposomes from GDH, ADH, and Qlo (Fig. 4). Although the electron transfer reaction to ferricyanide is not physiological and the significance of the conclusions described above seems unsubstantial, the observations presented here give some important clues for understanding the electron transfer reaction in the respiratory chain of G. suboxydans. Reconstitution of ferricyanide reductase activity of GDH was not accomplished by only GDH and Q,o or by only GDH and ADH, and all three components were indispensable, for the reconstitution. Since GDH has been shown to be able to react directly with ubiquinone in a phospholipid bilayer (10), it is suggested that GDH donates electrons to ubiquinone first and then reduced ubiquinone reacts directly with ADH, which subsequently reduces ferricyanide. Recently, we have shown that the ethanol oxidase respiratory chain can be reconstituted into proteoliposomes from ADH, Q lo , and cytochrome o terminal oxidase (unpublished). In conjunction with the other observations described above, the results obtained in this study suggest that both quinoproteins, GDH and ADH, may transfer electrons mutually via ubiquinone in the respiratory membranes of G. suboxydans.
We wish to thank Ryozi Takagaki, Masanori Uchida, Naoko Hirayama, and Shinjirou Sakurai for their technical assistance.
Vol. 107, No. 6, 1990
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