Selective Cytochrome c Displacement by Phosphate and Ca2+ in Brain Mitochondria

In brain mitochondria, phosphate- and Ca2+-dependent cytocrome c (cyt c) release reveals pools that interact differently with the inner membrane. Detachment of the phosphate-dependent pool did not influence the pool released by Ca2+. Cyt c pools were also detected in a system of cyt c reconstituted in cardiolipin (CL) liposomes. Gradual binding of cyt c (1 nmol) to CL/2–[12-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]dodecanoyl-1-hexadecan oyl-sn-glycero-3-phosphocholine (NBDC12-HPC) liposomes (10 nmol) produced NBD fluorescence quenching up to 0.4 nmol of added protein. Additional bound cyt c did not produce quenching, suggesting that cyt c-CL interactions originate distinct cyt c pools. Cyt c was removed from CL/NBDC12-HPC liposomes by either phosphate or Ca2+, but only Ca2+ produced fluorescence dequenching and leakage of encapsulated 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide. In mitochondria, complex IV activity and mitochondrial membrane potential (Δψm) were not affected by the release of the phosphate-dependent cyt c pool. Conversely, removal of cyt c by Ca2+ caused inhibition of complex IV activity and impairment of Δψm. In a reconstituted system of mitochondria, nuclei and supernatant, cyt c detached from the inner membrane was released outside mitochondria and triggered events leading to DNA fragmentation. These events were prevented by enriching mitochondria with exogenous CL or by sequestering released cyt c with anti-cyt c antibody.