Once the fatty acid is inside the mitochondrial matrix, beta-oxidation occurs by cleaving two carbons every cycle to form acetyl-CoA. The process consists of 4 steps.
# A long-chain fatty acid is dehydrogenated to create a trans double bond between C2 and C3. This is catalyzed by acyl CoA dehydrogenase to produce trans-delta 2-enoyl CoA. It uses FAD as an electron acceptor and it is reduced to FADH.
# Trans-delta 2-enoyl CoA is hydrated at the double bond to produce L-3-hydroxyacyl CoA by enoyl-CoA hydratase.
# L-3-hydroxyacyl CoA is dehydrogenated again to create 3-ketoacyl CoA by 3-hydroxyacyl CoA dehydrogenase. This enzyme uses NAD as an electron acceptor.
# Thiolysis occurs between C2 and C3 (alpha and beta carbons) of 3-ketoacyl CoA. Thiolase enzyme catalyzes the reaction when a new molecule of coenzyme A breaks the bond by nucleophilic attack on C3. This releases the first two carbon units, as acetyl CoA, and a fatty acyl CoA minus two carbons. The process continues until all of the carbons in the fatty acid are turned into acetyl CoA.
This acetyl-CoA then enters the mitochondrial tricarboxylic acid cycle (TCA cycle). Both the fatty acid beta-oxidation and the TCA cycle produce NADH and FADH, which are used by the electron transport chain to generate ATP.
Fatty acids are oxidized by most of the tissues in the body. However, some tissues such as the red blood cells of mammals (which do not contain mitochondria) and cells of the central nervous system do not use fatty acids for their energy requirements, but instead use carbohydrates (red blood cells and neurons) or ketone bodies (neurons only).
Because many fatty acids are not fully saturated or do not have an even number of carbons, several different mechanisms have evolved, described below.