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 UQ 8 in its molecule. A significant increase in the EPR signal was observed following glucose addition in mGDH reconstituted with PQQ and Mg 2؉ , suggesting that bound UQ 8 accepts a single electron from PQQH 2 to generate semiquinone radicals. No such increase in the EPR signal was observed in UQ 8 -free mGDH under the same conditions. Moreover, a UQ 2 reductase assay with a UQ-related inhibitor (C49) revealed different inhibition kinetics between the wildtype mGDH and UQ 8 -free mGDH. From these findings, we propose that the native mGDH bears two ubiquinone-binding sites, one (Q I ) for bound UQ 8 in its molecule and the other (Q II ) for UQ 8 in the ubiquinone pool, and that the bound UQ 8 in the Q I site acts as a single electron mediator in the intramolecular electron transfer in mGDH. Escherichia coli mGDH,1 which contains PQQ as a prosthetic group (1, 2), catalyzes a direct oxidation of D-glucose to Dgluconate in the periplasm and concomitantly transfers electrons to UQH 2 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 ␤-sheet propeller fold superbarrel structure that is a catalytic domain bearing the PQQ-binding (7) and Ca 2ϩ -or Mg 2ϩ -binding (8, 9) sites. A substantial amount of information on the domains, equivalent to the latter in PQQcontaining 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)(16)(17)(18)(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 UQH 2 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 ...

The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (e aq ؊ ) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of e aq ؊ , although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both with the bound UQ and PQQ in mGDH.The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) 2 belongs to the quinoprotein family with PQQ as a coenzyme (1, 2), and it catalyzes D-glucose oxidation to D-gluconate at the periplasmic side to transfer electrons to ubiquinol oxidase via UQ in the respiratory chain (3-5). Topological analysis revealed that mGDH consists of an N-terminal hydrophobic domain with five membrane-spanning segments and a large C-terminal domain residing in the periplasm, which contains PQQ and Ca 2ϩ -or Mg 2ϩ -binding sites in a superbarrel structure, conserved in quinoproteins (6 -8). Although its tertiary structure has not been resolved, the arrangement of amino acid residues around PQQ has been modeled on the basis of the crystal structure of the quinoprotein methanol dehydrogenase (6) as depicted in Fig. 1. The arrangement has been confirmed by results of several experiments with site-directed amino acid substitutions (9 -13). The orthoquinone portion of PQQ is a vital part for the catalytic reaction, to which Lys-493 hydr...