Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

• Published in: Apoptosis (London) Vol. 15; no. 5; pp. 563 - 573
• Main Authors: Ripple, Maureen O., Abajian, Michelle, Springett, Roger
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.05.2010; Springer Nature B.V
• Subjects: Anisomycin - pharmacology; Anoxia; Antifungal Agents - pharmacology; Antimycin A - analogs & derivatives; Antimycin A - pharmacology; Biochemistry; Biomedical and Life Sciences; Biomedicine; Cancer Research; Caspase 3 - metabolism; Caspase 9 - metabolism; Cell Biology; Cytochrome; Cytochromes c - metabolism; Cytosol - metabolism; Electron Transport - drug effects; Electron Transport - physiology; Enzyme Activation; HL-60 Cells; Humans; Membranes; Methacrylates - pharmacology; Mitochondrial Membranes - drug effects; Mitochondrial Membranes - metabolism; Oncology; Original Paper; Oxidation; Oxidation-Reduction; Oxygen - metabolism; Oxygen Consumption; Permeability; Protein Synthesis Inhibitors - pharmacology; Rotenone - pharmacology; Spectrum Analysis - methods; Thiazoles - pharmacology; Uncoupling Agents - pharmacology; Virology; Cytochrome; Caspase; Cytochrome oxidase; Mitochondrial outer membrane permeabilization (MOMP); Oxidation state; Electron transport chain (ETC)
• ISSN: 1360-8185; 1573-675X
• DOI: 10.1007/s10495-010-0455-2

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 ≈62% oxidized before MOMP and became ≈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.