title: Reaction of Escherichia coli cytochrome bo(3) and mitochondrial cytochrome bc(1) with a photoreleasable decylubiquinol.
abstract: In order to probe the reaction chemistry of respiratory quinol-oxidizing enzymes on a rapid time scale, a photoreleasable quinol substrate was synthesized by coupling decylubiquinol with the water-soluble protecting group 3',5'-bis(carboxymethoxy)benzoin (BCMB) through a carbonate linkage. The resulting compound, DQ-BCMB, was highly soluble in aqueous detergent solution, and showed no reactivity with quinol-oxidizing enzymes prior to photolysis. Upon photolysis in acetonitrile, 5, 7-bis(carboxymethoxy)-2-phenylbenzofuran, carbon dioxide, and decylubiquinol were formed. In aqueous media, free 3', 5'-bis(carboxymethoxy)benzoin was also produced. Photolysis of DQ-BCMB with a 308 nm excimer laser led to the release of the BCMB group in less than 10(-6) s. Decylubiquinol was released in the form of a carbonate monoester, which decarboxylated with an observed first-order rate constant of 195-990 s(-1), depending on the reaction medium. Yields of decylubiquinol as high as 35 microM per laser pulse were attained readily. In the presence of Escherichia coli cytochrome bo(3), photolysis of DQ-BCMB led to the oxidation of quinol by the enzyme with a rate that was limited by the rate of the decylubiquinol release. Mitochondrial cytochrome bc(1) reacted with photoreleased decylubiquinol with distinct kinetic phases corresponding to rapid b heme reduction and somewhat slower c heme reduction. Oxidation of photoreleased ubiquinol by this enzyme showed saturation kinetics with a K(m) of 3.6 microM and a k(cat) of 210 s(-1). The saturation behavior was a result of decylubiquinol being released as a carbonate monoester during the photolysis of DQ-BCMB and interacting with cytochrome bc(1) before decarboxylation of this intermediate yielded free decylubiquinol. The reaction of cytochrome bc(1) and photoreleased decylubiquinol in the presence of antimycin A led to monophasic b heme reduction, but also yielded slower quinol oxidation kinetics. The discrimination of kinetic phases in the reaction of cytochrome bc(1) with ubiquinol substrates has provided a means of exploring the bifurcation of electron transfer that is central to the operation of the Q-cycle in this enzyme.
authors.affiliation: Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: K C
authors.initials: KC
authors.lastname: Hansen
authors.forename: B E
authors.initials: BE
authors.lastname: Schultz
authors.forename: G
authors.initials: G
authors.lastname: Wang
authors.forename: S I
authors.initials: SI
authors.lastname: Chan
mesh_terms: D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism*; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010777:Photochemistry; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C083850:cytochrome bo3, E coli; D014451:Ubiquinone; D014450:Electron Transport Complex III; C003741:ubiquinol