Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway.
Cytochrome c oxidase is a membrane-bound enzyme, which catalyses the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of O(2) to water. Electron transfer through the enzyme is coupled to proton pumping across the membrane. Protons that are pumped as well as those that are used for O(2) reduction are transferred though a specific intraprotein (D) pathway. Results from earlier studies have shown that replacement of residue Asn139 by an Asp, at the beginning of the D pathway, results in blocking proton pumping without slowing uptake of substrate protons used for O(2) reduction. Furthermore, introduction of the acidic residue results in an increase of the apparent pK(a) of E286, an internal proton donor to the catalytic site, from 9.4 to ~11. In this study we have investigated intramolecular electron and proton transfer in a mutant cytochrome c oxidase in which a neutral residue, Thr, was introduced at the 139 site. The mutation results in uncoupling of proton pumping from O(2) reduction, but a decrease in the apparent pK(a) of E286 from 9.4 to 7.6. The data provide insights into the mechanism by which cytochrome c oxidase pumps protons and the structural elements involved in this process.
Lepp
Håkan
H
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Salomonsson
Lina
L
Zhu
Jia-Peng
JP
Gennis
Robert B
RB
Brzezinski
Peter
P
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism; D004563:Electrochemistry; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D054328:Proton Pump Inhibitors / Q000494:pharmacology*; D017494:Proton Pumps / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology
D001426:Bacterial Proteins; D054328:Proton Pump Inhibitors; D017494:Proton Pumps; D011994:Recombinant Proteins; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV