title: Proton pumping by an inactive structural variant of cytochrome c oxidase.
abstract: The aa3-type cytochrome c oxidases (CytcOs) from e.g. Rhodobacter sphaeroides and Paracoccus denitrificans harbor two proton-transfer pathways. The K pathway is used for proton uptake upon reduction of the CytcO, while the D pathway is used after binding of O2 to the catalytic site. The aim of the present study was to determine whether or not CytcO in which the K pathway is blocked (by e.g. the Lys362Met replacement) is capable of pumping protons. The process can not be studied using conventional assays because the O2-reduction activity is too low when the K pathway is blocked. Consequently, proton pumping with a blocked K pathway has not been demonstrated directly. Here, the Lys362Met and Ser299Glu structural variants were reconstituted in liposomes and allowed to (slowly) become completely reduced. Then, the reaction with O2 was studied with μs time resolution after flash photolysis of a blocking CO ligand bound to heme a3. The data show that with both the inactive Lys362Met and partly active Ser299Glu variants proton release occurred with the same time constants as with the wild-type oxidase, i.e. ~200μs and ~3ms, corresponding in time to formation of the ferryl and oxidized states, respectively. Thus, the data show that the K pathway is not required for proton pumping, suggesting that D and K pathways operate independently of each other after binding of O2 to the catalytic site.
authors.affiliation: Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Emelie
authors.initials: E
authors.lastname: Svahn
authors.affiliation: Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Kristina
authors.initials: K
authors.lastname: Faxén
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana Champaign, Urbana, IL 61801, United States.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.affiliation: Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address: peterb@dbb.su.se.
authors.forename: Peter
authors.initials: P
authors.lastname: Brzezinski
mesh_terms: D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017494:Proton Pumps
chemical_list: D017494:Proton Pumps; D003576:Electron Transport Complex IV
keywords: Cytochrome aa(3); Electrochemical potential; Electron transfer; Membrane protein; Redox reaction; Respiration