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Originally published In Press as doi:10.1074/jbc.M310163200 on November 11, 2003

J. Biol. Chem., Vol. 279, Issue 4, 3078-3083, January 23, 2004
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Occurrence of a Bound Ubiquinone and Its Function in Escherichia coli Membrane-bound Quinoprotein Glucose Dehydrogenase*

MD. Elias{ddagger}, Satsuki Nakamura{ddagger}, Catharina T. Migita{ddagger}, Hideto Miyoshi§, Hirohide Toyama{ddagger}, Kazunobu Matsushita{ddagger}, Osao Adachi{ddagger}, and Mamoru Yamada{ddagger}

From the {ddagger}Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan and the §Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received for publication, September 12, 2003 , and in revised form, October 31, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The membrane-bound pyrroloquinoline quinone (PQQ)-containing quinoprotein glucose dehydrogenase (mGDH) in Escherichia coli functions by catalyzing glucose oxidation in the periplasm and by transferring electrons directly to ubiquinone (UQ) in the respiratory chain. To clarify the intramolecular electron transfer of mGDH, quantitation and identification of UQ were performed, indicating that purified mGDH contains a tightly bound UQ8 in its molecule. A significant increase in the EPR signal was observed following glucose addition in mGDH reconstituted with PQQ and Mg2+, suggesting that bound UQ8 accepts a single electron from PQQH2 to generate semiquinone radicals. No such increase in the EPR signal was observed in UQ8-free mGDH under the same conditions. Moreover, a UQ2 reductase assay with a UQ-related inhibitor (C49) revealed different inhibition kinetics between the wild-type mGDH and UQ8-free mGDH. From these findings, we propose that the native mGDH bears two ubiquinone-binding sites, one (QI) for bound UQ8 in its molecule and the other (QII) for UQ8 in the ubiquinone pool, and that the bound UQ8 in the QI site acts as a single electron mediator in the intramolecular electron transfer in mGDH.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Escherichia coli mGDH,1 which contains PQQ as a prosthetic group (1, 2), catalyzes a direct oxidation of D-glucose to D-gluconate in the periplasm and concomitantly transfers electrons to UQH2 oxidase via UQ in the respiratory chain (3-6). mGDH is an 88-kDa monomeric protein with an N-terminal hydrophobic domain and a large C-terminal periplasmic domain (6). The former consists of five transmembrane segments, and the latter has a {beta}-sheet propeller fold superbarrel structure that is a catalytic domain bearing the PQQ-binding (7) and Ca2+- or Mg2+-binding (8, 9) sites. A substantial amount of information on the domains, equivalent to the latter in PQQ-containing quinoproteins, has been accumulated from the modeled structures of mGDH (7) and membrane-bound ADH III (10) and from x-ray structures of MDH (11), ADH I (12), ADH IIB (13), and soluble glucose dehydrogenase (14), which have been further confirmed by mutagenic analysis on several of the amino acid residues surrounding PQQ (15-19).

Our understanding of the interaction with UQ or its involvement in catalytic reactions in membrane-bound PQQ-containing dehydrogenases, however, is limited. The UQ reduction site (interacting with bulk UQ) in mGDH has been shown to be located near the membrane surface (20), which idea was strengthened from the findings that its C-terminal periplasmic domain, interacting peripherally with the membrane, possesses the UQ reduction site (21). ADH III in Gluconobacter suboxydans has been postulated to have two discrete sites for UQH2 oxidation and UQ reduction in its subunit II (22). Among other primary dehydrogenases, both the FAD-containing succinate dehydrogenase and the subunit NuoM of NADH-UQ oxidoreductase in E. coli include at least one UQ-binding site (23, 24).

Most of the information on UQ-binding sites and their redox properties has come from the photosynthetic reaction center of blue-purple bacteria (25, 26) and several protein complexes involved in mitochondrial and bacterial respiratory chains (24, 27-31). These sites are categorized as reduction (acceptor), oxidation (donor), and electron pair-splitting sites (32). QA and QB of the reaction center (26), Qp of the E. coli-succinate dehydrogenase (31), and the UQ-binding site of E. coli NADH:UQ oxidoreductase (24) are reduction sites, whereas QL of the E. coli cytochrome bo3 is an oxidation site (30). The QO site in bovine heart and yeast cytochrome bc1 complexes functions as a splitting site by bifurcation in the flow of a pair of electrons from UQH2 (32, 33). In the E. coli cytochrome bo3 complex, bound UQ8 at the QH site acts as a transient electron reservoir between the two-electron oxidation of the UQH2 pool and the one-electron (at a time) reduction of oxygen at the heme-copper center (30). The reduction site (Qn) of the yeast cytochrome bc1 complex, in addition to the QA and QH sites, stabilize ubisemiquinone (UQ.) (26, 33, 34). However, there are no reports available on a bound UQ such as this in PQQ-containing dehydrogenases or on its physical role in the intramolecular electron transfer in primary dehydrogenases.

Here we have discovered a bound UQ8 in the E. coli mGDH molecule and have postulated its functional role in the enzyme turnover. By comparing EPR spectra and the effects of inhibitors on UQ2 reductase activity between native and UQ8-free mGDHs, we propose that mGDH possesses two UQ-binding sites, QI for bound UQ8 and QII for bulk UQ pool, and that UQ8 at the QI site functions as a single electron transfer gate in mGDH. Because mGDH has a simpler structure compared with other dehydrogenases, it can be a model for elucidating the mechanism of intramolecular electron flow to bulk UQ.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—UQn were kindly provided by Eizai Co., Ltd. (Tokyo, Japan). UQ analogues were synthesized as described previously (35). All other chemicals were of analytical grade and obtained from commercial sources.

Bacterial Strains—The E. coli K-12 strains used in this study were W3110 (IN (rrnD-rrnE) rph-1) (36), MU1227 (cyo+ cyd+ ubiA::cml) (37), and YU654 (W3110, ubiA::cml). YU654 was constructed by transferring ubiA::cml from MU1227, which was performed by P1 transduction (38). W3110 and YU654 were used as host strains for purification of the wild-type mGDH and {Delta}UbiA mGDH, respectively.

Purification of the Wild-type mGDH and {Delta}UbiA mGDH—W3110 cells harboring pUCGCD1 (39) and YU654 (ubiA::cml) cells harboring pUCGCD1 were grown in LB medium (1% Bacto-tryptone, 0.5% yeast extract, and 0.5% NaCl) containing ampicillin (50 µg/ml) for 8 h at 30 °C under aeration conditions (150 rpm). Harvesting of cells and the preparation of membrane fractions were carried out as described by Yamada et al. (6). Purification of mGDH from membrane fractions was performed at 4 °C by the following procedure modified from the previous one (6) with two column chromatographies of DEAE-Toyopearl (Toyo Soda) and ceramic hydroxyapatite (Bio-Rad).

In the case of purification with Triton X-100, membrane fractions (~10 mg/ml of protein) were washed with 10 mM potassium phosphate buffer (KPB) (pH 7.0) containing 0.1% Triton X-100, and subsequently mGDH was solubilized from the washed membrane with 10 mM KPB (pH 7.0) containing 1.0% Triton X-100 and 100 mM KCl. The solubilized sample was dialyzed and then applied onto a DEAE-Toyopearl column as described previously (6). Active fractions were pooled and dialyzed against 1 mM KPB (pH 6.8) containing 0.1% Triton X-100. The dialysate was applied onto a ceramic hydroxyapatite column (1-ml bed volume/~5 mg of protein) equilibrated with 1 mM KPB (pH 6.8) containing 0.1% Triton X-100. The column was washed with 10 bed volumes of 2 mM KPB (pH 6.8) containing 0.1% Triton X-100. The enzyme was eluted by a linear gradient composed of 10 bed volumes of 3 mM KPB (pH 7.0) containing 0.1% Triton X-100 and 10 bed volumes of 12 mM KPB (pH 7.0) containing 0.1% Triton X-100. The active fractions that resulted at ~6 mM KPB (pH 7.0) were pooled.

In the case of purification with DM, membrane fractions (~10 mg/ml of protein) were washed with 10 mM KPB (pH 7.0) containing 0.04% DM, and the washed membrane fractions were subjected to solubilization for 60 min in the presence of 10 mM KPB (pH 7.0) containing 0.2% DM and 100 mM KCl. The suspension was centrifuged at 86,000 x g for 90 min, and the supernatant obtained was dialyzed against the same buffer without DM. The dialysate was applied onto a DEAE-Toyopearl column (1-ml bed volume/~10 mg of protein) equilibrated with 10 mM KPB (pH 7.0) containing 0.1% DM. The column was washed with 10 bed volumes of the same buffer and successively with 10 bed volumes of 10 mM KPB (pH 7.0) containing 0.02% DM. The enzyme was eluted by a linear gradient composed of 10 bed volumes of the same buffer and 10 bed volumes of 10 mM KPB (pH 7.0) containing 0.02% DM and 120 mM KCl. The active fractions eluted at ~70 mM KCl were pooled and dialyzed against the same buffer containing 0.02% DM. The dialysate was applied onto a ceramic hydroxyapatite column (1-ml bed volume/~5 mg of protein) equilibrated with 10 mM KPB (pH 7.0) containing 0.02% DM. The column was washed with 10 bed volumes of 200 mM KPB (pH 7.0) containing 0.02% DM. mGDH was eluted by a linear gradient composed of 10 bed volumes of 200 mM KPB (pH 7.0) containing 0.02% DM and 10 bed volumes of 1 M KPB (pH 7.0) containing 0.02% DM. The active fractions (at ~700 mM KPB, pH 7.0) were pooled and dialyzed against 10 mM KPB (pH 7.0) containing 0.02% DM.

In all cases, the active fractions (pooled after the ceramic hydroxyapatite column) were concentrated by a DEAE-Toyopearl column (1-ml bed volume/~10 mg of protein) in which the enzyme adsorbed was eluted with a small volume of 10 mM KPB (pH 7.0) containing the respective detergents (0.2% Triton X-100 or 0.1% DM) and 150 mM KCl and were then finally dialyzed against 10 mM KPB (pH 7.0) to reduce salt concentration in the enzyme solution. These concentrated materials were found to have a homogeneity of ~95% (judging from SDS-7% polyacrylamide gel electrophoresis) and were used as the purified wild-type mGDH with Triton X-100 or wild-type mGDH and {Delta}UbiA mGDH with DM.

Quantitation of Bound Ubiquinone in Purified Wild-type and {Delta}UbiA mGDHs—Approximately 30 nmol of enzyme were treated with 10 bed volumes of 100% ethanol in the presence of 30 nmol of UQ10 as an internal standard and incubated for 1 h with gentle shaking at 30 °C. The solution was centrifuged at 3000 rpm for 10 min to remove denatured protein molecules. The supernatant was mixed with 2.5 bed volumes of n-hexane for 1 min. The upper phase was collected and dried, and the residue was then resolved in 0.1 ml of HPLC solvent (ethanol/methanol/acetonitrile, 4:3:3 v/v). The resolved materials were subjected to reverse-phase HPLC using a Zorbax ODS column (Mitsuitoatsu) at the flow rate of 0.8 ml/min. The elution was monitored at 278 nm by using an SPD-M6A photodiode array detector (Shimadzu). UQ was identified by comparison of its migration with those of standard UQs (UQ8 and UQ10), and the content was estimated from the ratio of the peak area to that of the internal standard UQ10.

Treatment of Purified Wild-type mGDH with an Excess Amount of C49 Inhibitor—Approximately 10 nmol of purified wild-type mGDH were incubated with 10 µmol of a synthetic capsaicin analogue, C49 (35), for 1 h at 25 °C. The following procedures were then carried out at 4 °C. The enzyme sample was charged on a DEAE-Toyopearl column that had been equilibrated with 10 mM KPB (pH 7.0) containing 0.1% DM. The column was washed with 10 bed volumes of the same buffer. The enzyme was eluted with the same buffer containing 150 mM KCl, and the eluted enzyme was dialyzed against 10 mM KPB (pH 7.0). Bound UQ8, in this eluted mGDH, was extracted and quantitated by reverse-phase HPLC as described above.

Matrix-assisted Laser Desorption and Ionization Time-of-flight Mass Spectroscopy (MALDI-TOF/MS)—Compounds eluted from the reverse-phase HPLC column were collected, dried, and resolved in 0.1 ml of 100% ethanol. The resolved materials were placed on the analyzing plate. The molecular mass of these compounds was determined on a Voyager MALDI-TOF/MS device (Perseptive Biosystems). The spectra obtained by this process were analyzed using Micromass software.

EPR Spectroscopy—X-band (9.36 GHz) continuous wave spectra were recorded on an Elexsys E-500 spectrophotometer (Bruker) at 20 K, which was equipped with an Oxford helium cryostat (ESR 900). The concentrated enzyme solution (30 µM) in extra high quality quartz tubes (5-mm outer diameter) was frozen in liquid nitrogen. The applied microwave power was 0.1 mW; field modulation frequency, 100 kHz; field modulation amplitude, 1 G; and temperature, 20 K. In the power saturation experiments, the microwave power was varied between 200 µW and 10 mW, and the temperature was varied between 6 and 77 K. Spectral intensity was estimated by the double integration of spectra and the compared obtained values to that from the signal of Methylobacterium extorquens MDH (30 µM) on the assumption that the PQQ in the MDH is in the 100% semiquinone radical form.

Measurement of Protein and Enzyme Activities—Protein content was determined according to the Dulley and Grieve method (40) using bovine serum albumin as a standard. Holoenzyme formation was performed by incubating the purified enzyme in 10 mM 4-morpholinepropanesulfonic acid (pH 7.0) containing 30 µM PQQ and 1 mM MgCl2 for 30 min at 25 °C. Using the holoenzyme thus prepared, the following enzyme activities were measured. Phenazine-methosulfate reductase activity was measured spectrophotometrically (U-2000A, Hitachi) with phenazine methosulfate and dichloroindophenol as an electron mediator and acceptor, respectively, as described previously (17). UQ2 reductase activity was spectrophotometrically measured as described by Elias et al. (17). Inhibition of UQ2 reductase activity was examined by incubating the reaction mixture with different concentrations of inhibitor for 4 min before starting the reaction. One unit of phenazine-methosulfate reductase or UQ2 reductase activity is defined as 1 µmol of dichloroindophenol or UQ2, respectively, reduced/min, both of which correspond to 1 µmol of glucose oxidized/min. In UQ2 reductase assay, the Km and Vmax values and the Ki value for C49 were estimated on the basis of the Lineweaver-Burk plots. Absolute absorption spectra of apoform and holoform of the wild-type mGDH and {Delta}UbiA mGDH were taken before and after the addition of glucose as described (21).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Determination of Bound UQ in mGDH—To examine whether bound UQ occurs in mGDH, the extract from the wild-type mGDH purified with DM was analyzed by a reverse-phase HPLC column. As shown in Fig. 1a, a compound corresponding to the position of UQ8 was detected at 17 min of retention time, which showed an absorption spectrum with a peak at 278 nm. Based on comparison of the retention time and absorption spectrum with those of standard UQs, it was suggested that the compound extracted is UQ8. The fraction at 17 min of retention time was then collected and subjected to MALDI-TOF/MS analysis. The obtained mass (727.44) of the compound in the fraction was found to be identical with that of UQ8. We further examined the UQ8-free mGDH ({Delta}UbiA mGDH) purified from the ubiA::cml strain, being defective of UQ biosynthesis. No such peak corresponding to that of UQ8 was detected in the {Delta}UbiA mGDH (Fig. 1b).



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  		FIG. 1.
Elution profiles of UQs extracted from the wild-type (a) and {Delta}UbiA (b) mGDHs. UQs were extracted from 30 nmol of purified enzyme mixed with an internal standard UQ10 (30 nmol) and then analyzed on a reverse-phase HPLC as described under "Experimental Procedures." Standard UQs (UQ8 and UQ10) of 30 nmol each were also analyzed (c). Inserts show the absorption spectra observed on a photodiode array detector.

 
Quantitative determination of UQ was performed by calculation of the peak area and by comparison with that of UQ10 as an internal standard (Fig. 1a). As a result, 0.9 ± 0.02 mol of UQ8/1 mol of mGDH was recovered from the wild-type mGDH purified with DM. In contrast, only a trace amount of UQ8 was detected from the wild-type mGDH purified with Triton X-100. This observation is consistent with the fact that bound UQ8 was lost in purified E. coli cytochrome bo complex when Triton X-100 was used as a detergent (30).

To test whether this UQ8 in the purified wild-type mGDH can be removed by the addition of UQ analogue, we incubated the purified wild-type mGDH with an excess amount of C49, a capsaicin analogue shown to be a competitive inhibitor against UQ2 in mGDH of E. coli (35). The recovery of UQ8 after treatment with a 1000-fold excess of C49 was identical to that without the inhibitor. Taken together, our results suggest that one molecule of UQ8 tightly binds to a specific site in the native mGDH molecule and the site seems to be different from the site for bulk UQ8.

EPR Spectra of the Wild-type mGDH and {Delta}UbiA mGDH— mGDH occurs as an apoenzyme in E. coli (9), and the active holoenzyme is reconstituted with authentic PQQ and a divalent cation. The incorporated PQQ is reduced to PQQH2 by the addition of glucose. To elucidate the role of bound UQ8 in intramolecular electron transfer during the catalytic reaction of mGDH, EPR spectra were recorded on the holoenzymes of wild-type and {Delta}UbiA mGDHs purified with DM, and the obtained spectra were compared.

No significant EPR signals were detected from both types of apo-mGDHs (Fig. 2). A small signal appeared at g = 2.0034 in the wild-type holo-mGDH (Fig. 2a). The signal was largely increased by the addition of glucose and then decreased by the subsequent addition of ferricyanide, an artificial single electron acceptor (data not shown). These results indicate the existence of a single electron acceptor inside the wild-type mGDH molecule, which may receive one electron from PQQH2 to form a PQQH semiquinone radical. We assume that the bound UQ8 in the enzyme, which was demonstrated above, acts as the single electron acceptor from PQQH2. On the other hand, completely different patterns of EPR spectra were observed when the {Delta}UbiA mGDH was examined. A large signal at g = 2.0034 was observed in the holo-{Delta}UbiA mGDH without the addition of glucose, suggesting generation of a semiquinone radical of PQQ. The signal was decreased by the addition of glucose (Fig. 2b). This reduction may be due to the formation of PQQH2 from the semiquinone radical. In the control experiments, an EPR signal was detected neither from PQQ only nor from PQQ plus Mg2+ in the same buffer as was used for the experiments with mGDH protein.



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  		FIG. 2.
EPR spectra of the wild-type (a) and {Delta}UbiA (b) mGDHs before and after addition of glucose. EPR spectra were recorded for 30 µM purified mGDHs in 10 mM KPB (pH 7.0) containing 0.1% DM (Apo-mGDH). Holoenzyme formation was performed by the addition of 30 µM PQQ and 30 µM MgCl2 at 25 °C for 30 min. Spectra were then recorded for the holomerized enzyme (Holo-mGDH) and subsequently after addition of 5 mM glucose (Holo-mGDH + Glu).

 
Spectra obtained from both glucose-PQQ wild-type mGDH and PQQ-{Delta}UbiA mGDH showed almost the same features with a small shoulder at the lower field side and a broadened half-signal at the higher field side of the first derivative spectrum, suggesting rhombic anisotropy. This feature did not change when the microwave power was varied between 200 µW and 10 mW at 20 K, and only saturation broadening of the signals was observed at the higher powers. Reported EPR spectra of the PQQ radicals in MDH (42), soluble glucose dehydrogenase (43), and ADH II (44) have features similar to that shown in Fig. 2, whereas EPR spectra of ubisemiquinone radicals in cytochrome-bo3 oxidase (34, 45) are apparently different, exhibiting hyperfine splitting because of the 5-methyl hydrogen nuclei. On the other hand, estimation of the signal intensity, using the spectra from PQQ-{Delta}UbiA mGDH as a reference, has revealed that 60-80% of PQQ is converted to the radical form. The magnitude of the spectrum from glucose-PQQ wild-type mGDH is ~40% of that of the PQQ-{Delta}UbiA mGDH spectrum. The spin-spin coupling between the closely positioned radical centers is, therefore, less probable. Accordingly, we conclude that the signals from glucose-PQQ wild-type mGDH and PQQ-{Delta}UbiA mGDH come from a single species, i.e. the PQQ radical.

Absolute Absorption Spectra of PQQ in mGDH—We found significant numbers of semiquinone radicals in purified wild-type and {Delta}UbiA mGDHs after and before the addition of glucose, respectively. The radical intensity of the latter, however, decreased after glucose addition. Such unexpected differences between both mGDHs prompted us to distinguish the state of PQQ, a spectrophotometrically oxidized or reduced form. As shown in Fig. 3, the spectra of both holo-mGDHs appeared to be almost the same under the conditions with or without glucose even though they exhibited largely different intensities of semiquinone signals under the same conditions (Fig. 2). The absorption spectra indicate that PQQ molecules were in the oxidized state in both holo-mGDHs and became a completely reduced form immediately after the addition of glucose to the holoenzyme solution as observed previously (17).



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  		FIG. 3.
Absolute absorption spectra of the wild-type (a) and {Delta}UbiA (b) mGDHs. Absorption spectra were taken for 12 µM of purified mGDHs in 10 mM KPB (pH 7.0) containing 0.1% DM (Apo-mGDH). Holoenzyme formation was performed by the addition of 12 µM PQQ and 12 µM MgCl2 at 25 °C for 30 min. Spectra were then taken for the holomerized enzyme (Holo-mGDH) and subsequently after the addition of 5 mM glucose (Holo-mGDH + Glu).

 
The reduction of PQQ and the radical formation following the addition of glucose in the wild-type mGDH suggest that at least PQQH· is responsible for the semiquinone radical signal. PQQH2 generated by glucose oxidation in the native mGDH may be converted to PQQH· following a single electron transfer from PQQH2 to bound UQ8, which in turn becomes ubisemiquinone (UQ.). Thus, the EPR signal at g = 2.0034 observed for the wild-type mGDH reduced by glucose may be a mixture of those of PQQH· and UQ.

In the presence of glucose, the semiquinone radical of PQQ in the wild-type holo-mGDH was assumed to be in a PQQH· state, but its absorption spectrum was similar to that of the {Delta}UbiA mGDH, presumably having PQQH2. Thus, two absorption spectra each of PQQH2 and PQQH· could not be differentiated as reported in MDH (46). On the other hand, the semiquinone radical of PQQ in bound UQ8-free {Delta}UbiA mGDH in the absence of glucose may be PQQ. but not PQQH·, as it is a spectrophotometrically oxidized state. The formation of PQQ. would be caused by donation of a single electron from the inside mGDH protein molecule. Interestingly, such a PQQ. formation seems to be limited by the presence of bound UQ8 in the wild-type mGDH. Furthermore, PQQ. might be in equilibrium with PQQ because the PQQH2 formation by substrate oxidation occurs in a PQQ state as proposed elsewhere (41, 47, 48).

UQ2 Reductase Activity and Effects of Inhibitors—To clarify further the role of bound UQ8 in intramolecular electron transfer of mGDH, UQ2 reductase activities of the wild-type and UQ8-free mGDHs were compared. No significant difference was found in the Vmax value between the wild-type and {Delta}UbiA mGDHs or in the Km values for UQ2, 13 and 11 µM for wild-type and {Delta}UbiA mGDHs, respectively (Fig. 4). This similarity in the Vmax values might be due to the incorporation of UQ2 into the bound UQ8 site in UQ8-free mGDH and its performance, which is similar to bound UQ8 in the wild-type mGDH. Moreover, both mGDHs showed nearly the same phenazine-methosulfate reductase activity (data not shown).



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  		FIG. 4.
Kinetic analyses of the UQ2 reductase activities of the wild-type mGDH and the {Delta}UbiA mGDH. Lines A, wild-type mGDH; B, wild-type mGDH with 2.5 µM C49; C, wild-type mGDH with 5 µM C49; D, {Delta}UbiA mGDH; E, {Delta}UbiA mGDH with 2.5 µM C49; F, {Delta}UbiA mGDH with 5 µM C49.

 
We next examined the effects of UQ-related inhibitors on the UQ2 reductase activity of the wild-type and UQ8-free mGDHs. Some synthetic capsaicins such as compound C14, C45, and C49 were found to be potent inhibitors for mGDH of E. coli (35). Consistent with this, C49 at 2.5 and 5 µM showed competitive inhibition in the wild-type mGDH as shown in Fig. 4. On the other hand, C49 at 2.5 and 5 µM showed mixed inhibition in {Delta}UbiA mGDH with a reduced apparent Vmax value and an increased apparent Km value for UQ2. The Ki values of the wild-type and {Delta}UbiA mGDHs were estimated to be 4 and 13 µM, respectively. From these findings, the inhibitor seems to bind to a common site, the UQ2-reactive site, in both of the mGDHs and to an additional site in {Delta}UbiA mGDH. The binding of the inhibitor to the site different from the UQ2-reactive site may change the local structure of the UQ2-reactive site to reduce UQ2 reductase activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study indicates that E. coli mGDH possesses a tightly bound UQ8 in its molecule, and the UQ8 is functionally important for the intramolecular electron transfer in this dehydrogenase. A significant increase in the EPR signal in native mGDH following glucose oxidation (Fig. 2a) implies that bound UQ8 works downstream of PQQ in the process of intramolecular electron transfer and accepts a single electron from PQQH2. Kinetic analysis on UQ2 reduction of the wild-type and bound UQ8-free mGDHs with C49 (Fig. 4) also suggests that mGDH has at least two UQ-binding sites. C49 seems to inhibit the interaction of UQ2 to the wild-type mGDH. Considering the molecular similarity, C49 may also occupy the bound UQ8 site, and the binding may cause a structural change in the bulk UQ site in {Delta}UbiA mGDH, which results in a reduction of the Vmax value of UQ2 reductase activity.

On the basis of the findings in this and previous studies (20, 21), we propose the following mechanism of intramolecular electron transfer in mGDH. There are two UQ-binding sites, one for bound UQ8 and another for the bulk UQ pool (designated as QI and QII, respectively). The QI site may be located close to the PQQ-binding site, and the QII site, which corresponds to the site indicated by using depth-dependent fluorescent inhibitors (20), may reside peripherally on mGDH and near to the membrane surface in the inner membrane. When glucose is oxidized to glucono-{delta}-lactone, which spontaneously becomes gluconate, PQQ is reduced to PQQH2. After that, two possible mechanisms of intramolecular electron flow in mGDH can be considered on the basis of the ubiquinone redox mechanism of several UQ-reactive proteins (24-28, 30-32). First, two electrons split from PQQH2 are separately transferred to bound UQ8 at the QI site and to UQ8 at the QII site. The one electron from the QI site then moves to the UQ8 at the QII site, which becomes UQ8H2. Second, the two electrons from PQQH2 are transferred in a one-by-one fashion to the UQ8 at the QII site through the UQ8 at the QI site. Further studies are required to decipher the mechanism of the electron flow in the mGDH molecule.

Many UQ-reactive proteins have two UQ/UQH2-binding sites (24-28, 30-32), although their ubiquinone redox mechanisms are different. One site often stabilizes a ubisemiquinone (Q.), and the other site is under a dynamic equilibrium with the UQ pool (26, 30, 34). Our study suggests that a similar mechanism operates in mGDH, where the QI site stabilizes a ubisemiquinone of bound UQ8 at the QI site after the transfer of a single electron from PQQH2, and the QII site faces to the lipid bilayer to interact with bulk UQ. The UQ8 at the QI site may thus operate as a single electron transfer gate as postulated for UQ8 at the QH site of cytochrome bo3 complex (30) and for UQ at the QA site of the reaction center (26).

The high and low level formations of PQQ. in the UQ8-free mGDH and native mGDH, respectively, in the absence of glucose suggest the preventive function of bound UQ8 in PQQ. formation. This may indicate the close location of the UQ8 to PQQ in the mGDH molecule. Such prevention, however, was not observed in UQ8-free holo-mGDH preincubated with UQ2 (data not shown). These results imply that UQ8 may be incorporated to be a bound form during folding of the mGDH molecule in protein synthesis and offers the structural influence to prevent formation of PQQ. The prevention of the radical formation would have some physiological importance. Sato et al. (43) have demonstrated that Ca2+ can stabilize electrochemically produced free PQQ. or the semiquinone radical of PQQ incorporated into soluble glucose dehydrogenase molecule under the condition without glucose, but such stabilization does not occur with Mg2+. Consistent with this, no EPR signal was observed in the mixture of PQQ and Mg2+ (data not shown). The stabilization of PQQ. in mGDH may thus occur only in an incorporated state with Mg2+ added to the UQ8-free molecule.

Recently, a bound form of UQ (UQ10) was also found in G. suboxydans ADHIII.2 It is thus possible that most membrane-bound quinoprotein dehydrogenases possess a bound UQ, which in each case will be highlighted by its function according to its intramolecular electron transfer and structural importance.


   FOOTNOTES
 
* This work was supported by a grant-in-aid for basic research from the Ministry of Education, Science, and Culture of Japan and by Grant-in-aid P02216 [GenBank] from the Japan Society for the Promotion of Science Fellows (to MD. E.) for Scientific Research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan. Tel.: 81-83-933-5869; Fax: 81-83-933-5869; E-mail: m-yamada{at}yamaguchi-u.ac.jp.

1 The abbreviations used are: mGDH, membrane-bound glucose dehydrogenase; PQQ, pyrroloquinoline quinone; UQn, ubiquinone-n; ADH I, soluble alcohol dehydrogenase I; ADH IIB, soluble alcohol dehydrogenase IIB; ADH III, membrane-bound alcohol dehydrogenase III; MDH, methanol dehydrogenase; KPB, potassium phosphate buffer; DM, N-dodecyl-{beta}-D-maltoside; HPLC, high pressure liquid chromatography; MALDI-TOF/MS, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry; W, watt. Back

2 K. Matsushita, M. Mizuguchi, Y. Kobayashi, C. T. Migita, H. Toyama, O. Adachi, K. Sakamoto, and H. Miyoshi, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank M. Kawamukai for providing a strain.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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