title: Tightly-bound ubiquinone in the Escherichia coli respiratory complex I.
abstract: NADH:ubiquinone oxidoreductase (Complex I), the electron input enzyme in the respiratory chain of mitochondria and many bacteria, couples electron transport to proton translocation across the membrane. Complex I is a primary proton pump; although its proton translocation mechanism is yet to be known, it is considered radically different from any other mechanism known for redox-driven proton pumps: no redox centers have been found in its membrane domain where the proton translocation takes place. Here we studied the properties and the catalytic role of the enzyme-bound ubiquinone in the solubilized, purified Complex I from Escherichia coli. The ubiquinone content in the enzyme preparations was 1.3±0.1 per bound FMN residue. Rapid mixing of Complex I with NADH, traced optically, demonstrated that both reduction and re-oxidation kinetics of ubiquinone coincide with the respective kinetics of the majority of Fe-S clusters, indicating kinetic competence of the detected ubiquinone. Optical spectroelectrochemical redox titration of Complex I followed at 270-280nm, where the redox changes of ubiquinone contribute, did not reveal any transition within the redox potential range typical for the membrane pool, or loosely bound ubiquinone (ca. +50-+100mV vs. NHE, pH 6.8). The transition is likely to take place at much lower potentials (E(m) ≤-200mV). Such perturbed redox properties of ubiquinone indicate that it is tightly bound to the enzyme's hydrophobic core. The possibility of two ubiquinone-binding sites in Complex I is discussed.
authors.affiliation: University of Helsinki, Finland.
authors.forename: Michael
authors.initials: M
authors.lastname: Verkhovsky
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Bloch
authors.forename: Marina
authors.initials: M
authors.lastname: Verkhovskaya
mesh_terms: D001665:Binding Sites; D004563:Electrochemistry; D042967:Electron Transport Complex I / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D014451:Ubiquinone / Q000378:metabolism*
chemical_list: D014451:Ubiquinone; D042967:Electron Transport Complex I