Cytochrome c release from mitochondria occurs through a two-step process. This study demonstrates that cytochrome c is bound to the inner mitochondrial membrane via cardiolipin, existing in two conformations: loosely bound through electrostatic interactions and tightly bound via hydrophobic interactions. To release cytochrome c, the electrostatic interaction with cardiolipin must first be disrupted, generating a soluble pool. Permeabilization of the outer mitochondrial membrane by Bax then allows cytochrome c to be released into the extramitochondrial environment. Neither step alone is sufficient to trigger release. The study also shows that cytochrome c release is significantly reduced when the electrostatic interaction with cardiolipin remains intact, indicating the importance of both steps. The release of cytochrome c is a key event in apoptosis, as it leads to the activation of caspases and subsequent cell death. The study used isolated liver mitochondria to show that cytochrome c release can be either Ca²⁺-dependent or Ca²⁺-independent. In the Ca²⁺-dependent pathway, mitochondrial Ca²⁺ overload leads to the opening of the permeability transition pore, causing membrane swelling and cytochrome c release. In the Ca²⁺-independent pathway, Bax oligomers stimulate cytochrome c release, though the exact mechanism of membrane permeabilization is unclear. The study further demonstrates that cytochrome c can be mobilized into a soluble form by either electrostatic changes or oxidative stress. Electrostatic changes, such as altering ionic strength, can disrupt the electrostatic interaction between cytochrome c and cardiolipin, leading to release. Oxidative stress, such as cardiolipin peroxidation, can also mobilize cytochrome c by altering membrane structure. These findings support the two-step model of cytochrome c release, where the protein must first detach from cardiolipin and then be released through outer membrane permeabilization. The study provides evidence that cytochrome c release is a critical step in apoptosis, requiring both steps of membrane detachment and permeabilization.Cytochrome c release from mitochondria occurs through a two-step process. This study demonstrates that cytochrome c is bound to the inner mitochondrial membrane via cardiolipin, existing in two conformations: loosely bound through electrostatic interactions and tightly bound via hydrophobic interactions. To release cytochrome c, the electrostatic interaction with cardiolipin must first be disrupted, generating a soluble pool. Permeabilization of the outer mitochondrial membrane by Bax then allows cytochrome c to be released into the extramitochondrial environment. Neither step alone is sufficient to trigger release. The study also shows that cytochrome c release is significantly reduced when the electrostatic interaction with cardiolipin remains intact, indicating the importance of both steps. The release of cytochrome c is a key event in apoptosis, as it leads to the activation of caspases and subsequent cell death. The study used isolated liver mitochondria to show that cytochrome c release can be either Ca²⁺-dependent or Ca²⁺-independent. In the Ca²⁺-dependent pathway, mitochondrial Ca²⁺ overload leads to the opening of the permeability transition pore, causing membrane swelling and cytochrome c release. In the Ca²⁺-independent pathway, Bax oligomers stimulate cytochrome c release, though the exact mechanism of membrane permeabilization is unclear. The study further demonstrates that cytochrome c can be mobilized into a soluble form by either electrostatic changes or oxidative stress. Electrostatic changes, such as altering ionic strength, can disrupt the electrostatic interaction between cytochrome c and cardiolipin, leading to release. Oxidative stress, such as cardiolipin peroxidation, can also mobilize cytochrome c by altering membrane structure. These findings support the two-step model of cytochrome c release, where the protein must first detach from cardiolipin and then be released through outer membrane permeabilization. The study provides evidence that cytochrome c release is a critical step in apoptosis, requiring both steps of membrane detachment and permeabilization.