The proton collecting function of the inner surface of cytochrome c oxidase from Rhodobacter sphaeroides.
The experiments presented in this study address the problem of how the cytoplasmic surface (proton-input side) of cytochrome c oxidase interacts with protons in the bulk. For this purpose, the cytoplasmic surface of the enzyme was labeled with a fluorescein (Flu) molecule covalently bound to Cys223 of subunit III. Using the Flu as a proton-sensitive marker on the surface and phiOH as a soluble excited-state proton emitter, the dynamics of the acid-base equilibration between the surface and the bulk was measured in the time-resolved domain. The results were analyzed by using a rigorous kinetic analysis that is based on numeric integration of coupled nonliner differential rate equations in which the rate constants are used as adjustable parameters. The analysis of 11 independent measurements, carried out under various initial conditions, indicated that the protonation of the Flu proceeds through multiple pathways involving diffusion-controlled reactions and proton exchange among surface groups. The surface of the protein carries an efficient system made of carboxylate and histidine moieties that are sufficiently close to each other as to form a proton-collecting antenna. It is the passage of protons among these sites that endows cytochrome c oxidase with the capacity to pick up protons from the buffered cytoplasmic matrix within a time frame compatible with the physiological turnover of the enzyme.
Laser Laboratory for Fast Reactions in Biology, Department of Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69978 Israel.
Y
Y
Marantz
E
E
Nachliel
A
A
Aagaard
P
P
Brzezinski
M
M
Gutman
D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D019793:Fluorescein / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
D011522:Protons; D003576:Electron Transport Complex IV; D019793:Fluorescein