Internal charge transfer in cytochrome c oxidase at a limited proton supply: Proton pumping ceases at high pH |
| |
Authors: | Hå kan Lepp,Peter Brzezinski |
| |
Affiliation: | Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden |
| |
Abstract: | BackgroundIn the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O2 is linked to proton uptake from solution to form H2O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.MethodsIn this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O2. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.ResultsThe results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O2 reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.ConclusionsThe data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the “wrong” side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme.General significanceThese results provide new insights into the function of cytochrome c oxidase. |
| |
Keywords: | CytcO, cytochrome c oxidase boldFont" >R boldFont" >2, CytcO with a two-electron reduced catalytic site boldFont" >P boldFont" >3 (also boldFont" >P boldFont" >R), the &ldquo peroxy&rdquo intermediate formed at the catalytic site upon reaction of the four-electron reduced CytcO with O2 boldFont" >F boldFont" >3, oxo-ferryl intermediate boldFont" >O boldFont" >0, fully-oxidized CytcO N-side, negative side of the membrane P-side, positive side of the membrane, Time constants are given as (rate constant)&minus 1 |
本文献已被 ScienceDirect 等数据库收录! |
|