title: Effects of proton motive force on the structure and dynamics of bovine cytochrome C oxidase in phospholipid vesicles.
abstract: A conventional method for reconstituting cytochrome c oxidase (CcO) into phospholipid vesicles (COV) has been modified to permit resonance Raman (RR) analysis in the presence and absence of proton motive force (ΔμH(+)). The COV has an average diameter of 20 nm and contains one CcO molecule within a unified orientation with CuA located outside the COV. The process of generation of ΔμH(+) across the membrane was monitored spectrophotometrically with rhodamine123 dye. The COV exhibits a respiratory control ratio (RCR) value of >30 and is tolerant to RR measurements with 10 mW laser illumination for 60 min at 441.6 nm. Structural perturbations at the heme sites caused by incorporation into vesicles were clarified by spectral comparisons between solubilized CcO and COV. Absorption spectroscopy revealed that the rate of electron transfer from cytochrome c to O2 is reduced significantly more in the presence of ΔμH(+) than in its absence. RR spectroscopic measurements indicate that CcO in COV in the "respiratory-controlled" state adopts a mixed-valence state (heme a(2+) and heme a3(3+)). This study establishes a supramolecular model system for experimentally examining the energy conversion protein machinery in the presence of ΔμH(+).
authors.affiliation: Laboratory of Spectroscopy, Graduate School of Life Science, University of Hyogo , RSC-UH Leading Program Center, Koto 1-1-1, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nomura
authors.forename: Sachiko
authors.initials: S
authors.lastname: Yanagisawa
authors.forename: Kyoko
authors.initials: K
authors.lastname: Shinzawa-Itoh
authors.forename: Shinya
authors.initials: S
authors.lastname: Yoshikawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Ogura
mesh_terms: D000818:Animals; D002417:Cattle; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000502:physiology*; D006418:Heme / Q000031:analogs & derivatives; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D053078:Membrane Potential, Mitochondrial; D008929:Mitochondria, Heart / Q000187:drug effects / Q000502:physiology; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D061209:Proton Ionophores / Q000494:pharmacology; D018892:Proton-Motive Force; D013059:Spectrum Analysis, Raman
chemical_list: D008051:Lipid Bilayers; D010743:Phospholipids; D061209:Proton Ionophores; C027728:heme a; D006418:Heme; D003576:Electron Transport Complex IV