Topological analysis of quinoprotein glucose dehydrogenase in Escherichia coli and its ubiquinone-binding site.

Topological structure of quinoprotein glucose dehydrogenase in the inner membrane of Escherichia coli was determined by constructing protein fusions with alkaline phosphatase or beta-galactosidase. Analysis of the fusions revealed that the dehydrogenase possesses five membrane-spanning segments, and the N-terminal and C-terminal portions resided at the cytoplasmic and periplasmic side of the membrane, respectively. These results agreed with the hydropathy profile based on its primary structure. The topological structure suggests that the predicted binding site of the prosthetic group pyrroloquinoline quinone is located at the periplasmic side and that the amino acid residues corresponding to those that were presumed to interact with ubiquinone in one subunit of mitochondrial NADH dehydrogenase also occur at the periplasmic side. When the purified glucose dehydrogenase and cytochrome o ubiquinol oxidase were reconstituted together with ubiquinone into liposomes, a membrane potential could be generated by the electron transfer at the site of the ubiquinol oxidase but not of the dehydrogenase. These results suggest that glucose dehydrogenase has a ubiquinone reacting site close to the periplasmic side of the membrane, and thus its electron transfer to ubiquinone appears to be incapable of forming a proton electrochemical gradient across the inner membrane of E. coli.