Comparison of the Structural Features of Ubiquinone Reduction Sites Between Glucose Dehydrogenase in Escherichia coli and Bovine Heart Mitochondrial Complex I

Abstract

To characterize the structural features of the ubiquinone reduction site of glucose dehydrogenase (GlcDH) in Escherichia coli, we performed structure/activity studies of a systematic set of synthetic ubiquinone analogues and specific inhibitors (synthetic capsaicins) of this site. Considering the proposed similarity of the quinone binding domain motif between GlcDH and one subunit of mitochondrial complex I [Friedrich, T., Strohdeicher, M., Hofhaus, G., Preis, D., Sahm, H. & Weiss, H. (1990) FEBS Lett. 265, 37-40], we compared the structure/activity profiles of the substrates and inhibitors for GlcDH with those for bovine heart mitochondrial complex I. With respect to GlcDH, replacement of one or both methoxy groups in the 2 and 3 positions of benzoquinone ring by ethoxy group(s) resulted in a drastic decrease in the electron accepting activity. The presence of a 5-methyl group and the conformational property of the 6-alkyl side chain did not significantly contribute to the activity. These results suggested that only half of the benzoquinone ring (the moiety corresponding to the 2 and 3 positions) is recognized by the quinone reduction site in a strict sense. In contrast, quinone analogues with structural modifications at all positions in the benzoquinone ring retained the activity with mitochondrial complex I. This finding indicated that the catalytic site of complex I is spacious enough to accommodate a variety of structurally different quinone derivatives. The correlation of the inhibitory potencies of a series of synthetic capsaicins between the two enzymes was very poor. These findings indicated that the binding environment of ubiquinone in GlcDH is very specific and differs from that in mitochondrial complex I.Keywords: glucose dehydrogenase; NADH-ubiquinone oxidoreductase; ubiquinone; respiratory inhibitor; structure/activity relationship.Glucose dehydrogenase (GlcDH), which resides in the inner membrane of Escherichia coli, functions in the direct oxidation of D-glucose to D-gluconate and concomitantly transfers electrons to cytochrome oxidase through ubiquinone in the respiratory chain [I, 21. This enzyme is assumed to possess the binding sites for glucose, ubiquinone (Q), and metal ion as well as pyrroloquinoline quinone (PQQ) [3, 41. Friedrich et al. [5] have indicated that the membrane spanning domain of GlcDH in Acinetobacter calcoaceticus was similar to that of one subunit of mitochondrial NADH-ubiquinone oxidoreductase (complex I), and predicted that its interaction site with Q resides at the cytoplasmic side of the inner membrane. They also speculated that this enzyme transfers an electron to Q located at the cytoplasmic side thus forming a proton electrochemical gradient across the inner membrane. However, Yamada et al. [6] have demonstrated that GlcDH in E. coli, which is highly homologous to that in A. calcoaceticus (70%), has a Q reduction site close to the periplasmic side of the membrane and that its electron transfer to Q is incapable of generating a proton electrochemical gradient. To ...

Occurrence of a Bound Ubiquinone and Its Function in Escherichia coli Membrane-bound Quinoprotein Glucose Dehydrogenase

“…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 M showed competitive inhibition in the wild-type mGDH as shown in Fig.…”

“…To test whether this UQ 8 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 UQ 2 in mGDH of E. coli (35). The recovery of UQ 8 after treatment with a 1000-fold excess of C49 was identical to that without the inhibitor.…”

“…UQ analogues were synthesized as described previously (35). All other chemicals were of analytical grade and obtained from commercial sources.…”

“…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 mol of a synthetic capsaicin analogue, C49 (35), for 1 h at 25°C. The following procedures were then carried out at 4°C.…”

Probing the Ubiquinone Reduction Site of Mitochondrial Complex I Using Novel Cationic Inhibitors

“…This specificity in complex I is conspicuous because diethoxy-Q 2 and its reduced form proved to be very poor substrates in studies with other electron transfer enzymes such as bovine heart mitochondrial complexes II and III (31), 4 glucose dehydrogenase (25), and terminal ubiquinol oxidases (18) in Escherichia coli. This fact supports our previous conclusion that the ubiquinone reduction site in complex I is spacious enough to accommodate bulky exogenous ubiquinones and a variety of structurally different inhibitors in a dissimilar manner (25).…”