In the mitochondrion, pyruvate is oxidized by the pyruvate dehydrogenase complex to the acetyl group, which is fully oxidized to carbon dioxide by the citric acid cycle (also known as the Krebs cycle). Every "turn" of the citric acid cycle produces two molecules of carbon dioxide, one equivalent of ATP guanosine triphosphate (GTP) through substrate-level phosphorylation catalyzed by succinyl-CoA synthetase, as succinyl-CoA is converted to succinate, three equivalents of NADH, and one equivalent of FADH. NADH and FADH are recycled (to NAD and FAD, respectively) by oxidative phosphorylation, generating additional ATP. The oxidation of NADH results in the synthesis of 2–3 equivalents of ATP, and the oxidation of one FADH yields between 1–2 equivalents of ATP. The majority of cellular ATP is generated by this process. Although the citric acid cycle itself does not involve molecular oxygen, it is an obligately aerobic process because O is used to recycle the NADH and FADH. In the absence of oxygen, the citric acid cycle ceases.
The generation of ATP by the mitochondrion from cytosolic NADH relies on the malate-aspartate shuttle (and to a lesser extent, the glycerol-phosphate shuttle) because the inner mitochondrial membrane is impermeable to NADH and NAD. Instead of transferring the generated NADH, a malate dehydrogenase enzyme converts oxaloacetate to malate, which is translocated to the mitochondrial matrix. Another malate dehydrogenase-catalyzed reaction occurs in the opposite direction, producing oxaloacetate and NADH from the newly transported malate and the mitochondrion's interior store of NAD. A transaminase converts the oxaloacetate to aspartate for transport back across the membrane and into the intermembrane space.
In oxidative phosphorylation, the passage of electrons from NADH and FADH through the electron transport chain releases the energy to pump protons out of the mitochondrial matrix and into the intermembrane space. This pumping generates a proton motive force that is the net effect of a pH gradient and an electric potential gradient across the inner mitochondrial membrane. Flow of protons down this potential gradient – that is, from the intermembrane space to the matrix – yields ATP by ATP synthase. Three ATP are produced per turn.
Although oxygen consumption appears fundamental for the maintenance of the proton motive force, in the event of oxygen shortage (hypoxia), intracellular acidosis (mediated by enhanced glycolytic rates and ATP hydrolysis), contributes to mitochondrial membrane potential and directly drives ATP synthesis.
Most of the ATP synthesized in the mitochondria will be used for cellular processes in the cytosol; thus it must be exported from its site of synthesis in the mitochondrial matrix. ATP outward movement is favored by the membrane's electrochemical potential because the cytosol has a relatively positive charge compared to the relatively negative matrix. For every ATP transported out, it costs 1 H. Producing one ATP costs about 3  H. Therefore, making and exporting one ATP requires  4H The inner membrane contains an antiporter, the ADP/ATP translocase, which is an integral membrane protein used to exchange newly synthesized ATP in the matrix for ADP in the intermembrane space.