title: Activation of respiratory Complex I fromEscherichia colistudied by fluorescent probes.
abstract: Respiratory Complex I fromE. colimay exist in two interconverting forms: resting (R) and active (A). The R/A transition of purified, solubilized Complex I occurring upon turnover was studied employing two different fluorescent probes, Annine 6+, and NDB-acetogenin. NADH-induced fluorescent changes of both dyes bound to solubilized Complex I fromE. coliwere characterized as a function of the protein:dye ratio, temperature, ubiquinone redox state and the enzyme activity. Analysis of this data combined with time-resolved optical measurements of Complex I activity and spectral changes indicated two ubiquinone-binding sites; a possibility of reduction of the tightly-bound quinone in the resting state and reduction of the loosely-bound quinone in the active state is discussed. The results also indicate that upon the activation Complex I undergoes conformational changes which can be mapped to the junction of the hydrophilic and membrane domains in the region of the assumed acetogenin-binding site.
authors.affiliation: Institute of Biotechnology, University of Helsinki, PO Box 65 (Viikinkaari 1), FIN-00014, Finland.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Belevich
authors.affiliation: Institute of Biotechnology, University of Helsinki, PO Box 65 (Viikinkaari 1), FIN-00014, Finland.
authors.forename: Galina
authors.initials: G
authors.lastname: Belevich
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Subhash C
authors.initials: SC
authors.lastname: Sinha
authors.affiliation: Institute of Biotechnology, University of Helsinki, PO Box 65 (Viikinkaari 1), FIN-00014, Finland.
authors.forename: Marina
authors.initials: M
authors.lastname: Verkhovskaya
keywords: Biochemistry; Biological Sciences; Biophysics