Menaquinone as Well as Ubiquinone as a Bound Quinone Crucial for Catalytic Activity and Intramolecular Electron Transfer in Escherichia coli Membrane-bound Glucose Dehydrogenase

Abstract

Escherichia coli membrane-bound glucose dehydrogenase (mGDH), which is one of quinoproteins containing pyrroloquinoline quinone (PQQ) as a coenzyme, is a good model for elucidating the function of bound quinone inside primary dehydrogenases in respiratory chains. Enzymatic analysis of purified mGDH from cells defective in synthesis of ubiquinone (UQ) and/or menaquinone (MQ) revealed that Q-free mGDH has very low levels of activity of glucose dehydrogenase and UQ 2 reductase compared with those of UQ-bearing mGDH, and both activities were significantly increased by reconstitution with UQ 1 . On the other hand, MQ-bearing mGDH retains both catalytic abilities at the same levels as those of UQ-bearing mGDH. A radiolytically generated hydrated electron reacted with the bound MQ to form a semiquinone anion radical with an absorption maximum at 400 nm. Subsequently, decay of the absorbance at 400 nm was accompanied by an increase in the absorbance at 380 nm with a first order rate constant of 5.7 ؋ 10 3 s ؊1 . This indicated that an intramolecular electron transfer from the bound MQ to the PQQ occurred. EPR analysis revealed that characteristics of the semiquinone radical of bound MQ are similar to those of the semiquinone radical of bound UQ and indicated an electron flow from PQQ to MQ as in the case of UQ. Taken together, the results suggest that MQ is incorporated into the same pocket as that for UQ to perform a function almost equivalent to that of UQ and that bound quinone is involved at least partially in the catalytic reaction and primarily in the intramolecular electron transfer of mGDH.Facultative anaerobic bacteria and lower eukaryotes are known to have capabilities to adapt to environmental changes for survival. One such capability is acquired by synthesis of both UQ 2 and MQ(1). A fine control of biosynthesis and the relative concentrations of the two Qs is crucial for growth, in respect to oxygen supply under aerobic and anaerobic conditions (2-5). UQ n as an electron carrier is primarily involved in aerobic respiration, whereas MQ n is involved in anaerobic respiration. Bekker et al.(6) reported significant changes in both size and redox state of the Q pool when the environment changes from a well aerated environment to an environment with low oxygen availability. Some respiratory components that intrinsically interact with Q are capable of catalyzing redox reactions with both UQ and MQ (7-11). Such interactions may be important in physiological and evolutionary aspects in addition to enzymatic function. DsbB, which is involved in the formation of disulfide bonds in Escherichia coli extracytoplasmic space, and fumarate reductase differentially employ Q under aerobic and anaerobic conditions (7-9). The respiratory oxidation in E. coli of ␣-glycerophosphate and D-lactate is promoted by both UQ and MQ, whereas NADH oxidase and succinate oxidase require only UQ for activity (12).E. coli membrane-bound mGDH, which is known as a quinoprotein, catalyzes the oxidation of D-glucose to D-gluconate at the periplas...

Menaquinone as Well as Ubiquinone as a Crucial Component in the Escherichia coli Respiratory Chain

“…External addition of UQ-1 showed UQ-1 dosedependent increase in PMS reductase activity in Q-free mGDH. A similar level of increase in UQ-2 reductase activity was observed in the presence of UQ-1 in Q-free mGDH (Mustafa et al, 2008b). A radiolytically generated hydrated electron reacted with the bound MK to form a semiquinone anion radical with an absorption maximum at 400 nm.…”

“…Recent mGDH analysis provided the first evidence that the primary dehydrogenase in respiratory chains utilizes both MK and UQ as a bound Q and suggest that bound MK occurs in a fashion similar to that of bound UQ in the mGDH molecule and functions as an electron acceptor from PQQ (Mustafa et al, 2008b). We also presented the data for the first time that suggest the requirement of bound Q for catalytic reaction in quinoprotein dehydrogenases (Mustafa et al, 2008b).…”

“…Pulse radiolysis analysis revealed that the two redox centers are closely located at a distance of 11-13 Å and that Asp466 and Lys493 are involved in proton donation to the semiquinone anion radical of bound Q and in electron transfer from bound UQ to PQQ, respectively (Elias et al, 2000;Mustafa et al, 2008a). Recent mGDH analysis provided the first evidence that the primary dehydrogenase in respiratory chains utilizes both MK and UQ as a bound Q and suggest that bound MK occurs in a fashion similar to that of bound UQ in the mGDH molecule and functions as an electron acceptor from PQQ (Mustafa et al, 2008b). We also presented the data for the first time that suggest the requirement of bound Q for catalytic reaction in quinoprotein dehydrogenases (Mustafa et al, 2008b).…”

“…The latter activity reflects the total ability of catalytic reaction and the successive intramolecular electron transfer from PQQ to UQ-2 at the Q II site. The Q-free enzyme, however, exhibited only 18% of the dehydrogenase activity and 6% of the UQ-2 reductase activity of UQbearing mGDHs (Mustafa et al, 2008b).…”

“…mGDH expressed in the ubiA mutant defective in UQ biosynthesis was found to contain MK-8, and its MK content in purified mGDH protein was estimated to be 0.9 ± 0.03 mol/mol of mGDH (Mustafa et al, 2008b). Functional activities of purified bound MK-containing mGDH (MK-mGDH) and non-bound quinone-containing mGDH (Q-free mGDH) purified from ubiA menA cells were compared with those of bound UQ-containing mGDH (UQ-mGDH) from menA mutant cells.…”