Internal charge transfer in cytochrome c oxidase at a limited proton supply: proton pumping ceases at high pH.
BACKGROUND
In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O(2) is linked to proton uptake from solution to form H(2)O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.


METHODS
In 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 O(2). These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.


RESULTS
The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O(2) reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.


CONCLUSIONS
The 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 approximately 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.


CONCLUSIONS
These results provide new insights into the function of cytochrome c oxidase.
Lepp
Håkan
H
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Brzezinski
Peter
P
D004579:Electron Transport / Q000502:physiology; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000502:physiology; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017494:Proton Pumps / Q000737:chemistry / Q000378:metabolism*; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
D017494:Proton Pumps; D011522:Protons; D003576:Electron Transport Complex IV