Lateral pH gradient between OXPHOS complex IV and F(0)F(1) ATP-synthase in folded mitochondrial membranes.
Ion-driven ATP synthesis by rotary F0F1 ATP-synthase powers aerobic life. Since Mitchell's seminal hypothesis, this synthesis has been discussed in terms of the proton-motive force between two bulk phases, each in equilibrium. In active mitochondria, a steady proton flow cycles between pumps and the distant ATP synthase. Here we determine the lateral pH profile along the p-side of cristae in situ by attaching a ratiometric fluorescent pH-sensitive GFP variant to OXPHOS complex IV, a proton pump, and the dimeric F0F1 ATP-synthase, a proton consumer. In respiring HeLa cells, we observe that the local pH at F0F1 dimers is 0.3 units less acidic than that at complex IV. This finding is consistent with the calculated pH profile for steady proton diffusion from CIV to F0F1. The observed lateral variation in the proton-motive force necessitates a modification to Peter Mitchell's chemiosmotic proposal. The experimental technique can be extended to other pH-dependent reactions in membrane microcompartments.
Division of Mitochondrial Dynamics, Fachbereich Biologie/Chemie, Universität Osnabrück, D-49076 Osnabrück, Germany.
Bettina
B
Rieger
Niedersachsen-Professur für Biophysik, Fachbereich Biologie/Chemie, Universität Osnabrück, D-49076 Osnabrück, Germany.
Wolfgang
W
Junge
Division of Mitochondrial Dynamics, Fachbereich Biologie/Chemie, Universität Osnabrück, D-49076 Osnabrück, Germany.
Karin B
KB
Busch
D002138:Calibration; D004058:Diffusion; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051336:Mitochondrial Membranes / Q000378:metabolism*; D008958:Models, Molecular; D010085:Oxidative Phosphorylation; D021122:Protein Subunits / Q000378:metabolism; D017494:Proton Pumps / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*
C485187:PHluorin; D021122:Protein Subunits; D017494:Proton Pumps; D049452:Green Fluorescent Proteins; D003576:Electron Transport Complex IV; D006180:Proton-Translocating ATPases