Molecular Mechanism and Structural Basis of Ubiquinone-Redox Reaction in Bacterial Respiratory Chains

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In this research project, search for ubiquinone-binding site and molecular mechanism of redox reaction of ubiquinone was carried out in several ubiquinone-reacting enzymes in the respiratory chains of Escherichia coli and Gluconobacter suboxydans. The objective ubiquinone-reacting enzymes were cytochrome bo oxidase (Cyo), glucose dehydrogenase (GDH), and succinate dehydrogenases (SDH) of E. coli, and alcohol dehydrogenases (ADH) of G. suboxydans. In order to do this research, detection of the bound quinone in the enzymes and also photo-affinity labeling with azidoquinone of the enzymes followed by analysis of the labeled subunit and further peptides by MALDI-TOF-MS were mainly performed.1) 2-azido-Q2 and also 3-azido-Q2 were successfully synthesized via the precursors, 2- and 3- metylated quinone compounds, and also the azido-moiety was found to be decomposed by UV irradiation. 2) Labeling conditions for Cyo of E. coli with both 2-azido- and 3-azido-Q2 were established. 3) Ubiquinone-reacting site of E. coli GDH was shown to be present in the C- terminal hydrophilic PQQ domain, which could be done by preparing the C-terminal domain by site-directed mutagenesis and the reactivity toward ubiquinone. 4) In E. coil SDH, ubiquinone-reacting site was shown to be present in the membrane-anchoring subunit by photo-affinity labeling with azidoquinone. 5) When purified G. suboxydans ADH with dodecyl maltoside, the enzyme retained tightly ubiquinone-10 inside the molecule, and also shown to be as the ubisemiquinone form. 6) In G. suboxydans ADH, the subunit II was shown to be labeled with azidoquinone, especially the ubiquinone reduction site, but not the ubiquinol oxidation site. And also the labeled quinone was found to work as electron mediator to pooled ubiquinone.