title: A novel chloride-binding site modulates the heme-copper binuclear center of the Escherichia coli bo-type ubiquinol oxidase.
abstract: Cytochrome bo-type ubiquinol oxidase in Escherichia coli belongs to a superfamily of the heme-copper respiratory oxidases and catalyzes the redox-coupled proton pumping. Previous studies [Y. Orii, T. Mogi, M. Sato-Watanabe, T. Hirano, and Y. Anraku (1995) Biochemistry 34, 1127-1132] suggest that it requires chloride ions for the facilitated heme b-to-heme o intramolecular electron transfer. To extend our previous studies on chloride binding by bo-type ubiquinol oxidase, we prepared two kinds of chloride-bound enzymes, UQO-412 and UQO-409, and a chloride-depleted enzyme, UQO-407, and examined their spectroscopic and enzymatic properties. UQO-412, which exhibits the Soret peak at 412 nm in the air-oxidized state, was obtained by purification with anion-exchange liquid chromatography, and UQO-409 was derived from UQO-412 by extensive washing and showed a 3-nm blue shift. UQO-407 was obtained from UQO-409 by omitting chloride ions from buffers throughout purification and showed a further blue shift in the Soret peak and the pronounced chloride-sensitive EPR signals at g=6 and g=3.15, which are attributable to spin-spin exchange interaction at the binuclear center. Kinetic studies on chloride binding by UQO-407 revealed the presence of a chloride-binding site with a K(d) value of 3.5 mM. Flow-flash experiments demonstrated that the heme b-to-heme o electron transfer was perturbed in both UQO-409 and UQO-407, although steady state enzyme activities of three UQOs were indistinguishable. The present studies demonstrated that the E. coli bo-type ubiquinol oxidase is endowed with a novel chloride-binding site which controls the electromagnetic state of the heme-copper binuclear center. Further, we suggest that the intramolecular electron transfer in the enzyme requires diffusible molecules other than the bound chloride ion.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo.
authors.forename: T
authors.initials: T
authors.lastname: Hirano
authors.forename: T
authors.initials: T
authors.lastname: Mogi
authors.forename: M
authors.initials: M
authors.lastname: Tsubaki
authors.forename: H
authors.initials: H
authors.lastname: Hori
authors.forename: Y
authors.initials: Y
authors.lastname: Orii
authors.forename: Y
authors.initials: Y
authors.lastname: Anraku
mesh_terms: D001665:Binding Sites; D002712:Chlorides / Q000737:chemistry*; D003300:Copper / Q000032:analysis*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry*; D055590:Electromagnetic Phenomena; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006418:Heme / Q000032:analysis*; D007700:Kinetics; D010100:Oxygen; D017670:Sodium Compounds / Q000494:pharmacology
chemical_list: D002712:Chlorides; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D017670:Sodium Compounds; D006418:Heme; D003300:Copper; C017168:cytochrome bo, E coli; D010100:Oxygen