Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization.
Visible spectroscopy was used to measure real-time changes in the oxidation state of cytochrome c (cyt c) and the a-cytochromes (cyt aa(3)) of cytochrome oxidase during mitochondrial outer membrane permeabilization (MOMP) initiated by anisomycin in HL-60 cells. The oxidation state of mitochondrial cyt c was found to be approximately 62% oxidized before MOMP and became approximately 70% oxidized after MOMP. In contrast, the cytosolic pool of cyt c was found to be almost fully reduced. This oxidation change allows cyt c release to be continuously and quantitatively monitored in real time. Anoxia and antimycin were used to fully reduce and fully oxidize, respectively, the mitochondrial pool of cyt c and it was found that the release of cyt c was independent of it oxidation state consistent with a simple model of cyt c passively diffusing down a concentration gradient through a pore or tear in the outer membrane. After MOMP was complete, the flux of cyt c diffusing back into the mitochondria was measured from the residual mitochondrial oxygen consumption after complete inhibition of the bc(1) with antimycin and myxothiazol. The outer membrane was found to be highly permeable after MOMP implying that the reduction of cyt c in the cytosol must be very rapid. The permeability of the outer membrane measured in this study would result in the release of cyt c with a time constant of less than 1 s.
Ripple
Maureen O
MO
Department of Radiology, Dartmouth Medical School, Hanover, NH 03755, USA.
Abajian
Michelle
M
Springett
Roger
R
D000841:Anisomycin / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004579:Electron Transport / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation; D018922:HL-60 Cells; D006801:Humans; D008689:Methacrylates / Q000494:pharmacology; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010101:Oxygen Consumption; D010539:Permeability; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012402:Rotenone / Q000494:pharmacology; D013057:Spectrum Analysis / Q000379:methods*; D013844:Thiazoles / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D000935:Antifungal Agents; D008689:Methacrylates; D011500:Protein Synthesis Inhibitors; D013844:Thiazoles; D014475:Uncoupling Agents; D012402:Rotenone; C032456:antimycin; D000968:Antimycin A; D000841:Anisomycin; C030517:myxothiazol; D045304:Cytochromes c; D053148:Caspase 3; D053453:Caspase 9; D010100:Oxygen