Mitochondrial trifunctional enzyme catalyzes the last three of the four reactions of the mitochondrial beta-oxidation pathway. The mitochondrial beta-oxidation pathway is the major energy-producing process in tissues and is performed through four consecutive reactions breaking down fatty acids into acetyl-CoA. Among the enzymes involved in this pathway, the trifunctional enzyme exhibits specificity for long-chain fatty acids. Mitochondrial trifunctional enzyme is a heterotetrameric complex composed of two proteins, the trifunctional enzyme subunit alpha/HADHA carries the 2,3-enoyl-CoA hydratase and the 3-hydroxyacyl-CoA dehydrogenase activities, while the trifunctional enzyme subunit beta/HADHB described here bears the 3-ketoacyl-CoA thiolase activity. acetyl-CoA + an acyl-CoA = a 3-oxoacyl-CoA + CoA acetyl-CoA + butanoyl-CoA = 3-oxohexanoyl-CoA + CoA acetyl-CoA + hexanoyl-CoA = 3-oxooctanoyl-CoA + CoA acetyl-CoA + octanoyl-CoA = 3-oxodecanoyl-CoA + CoA acetyl-CoA + decanoyl-CoA = 3-oxododecanoyl-CoA + CoA acetyl-CoA + dodecanoyl-CoA = 3-oxotetradecanoyl-CoA + CoA acetyl-CoA + tetradecanoyl-CoA = 3-oxohexadecanoyl-CoA + CoA Lipid metabolism; fatty acid beta-oxidation. Heterotetramer of 2 alpha/HADHA and 2 beta/HADHB subunits; forms the mitochondrial trifunctional enzyme (By similarity). Also purified as higher order heterooligomers including a 4 alpha/HADHA and 4 beta/HADHB heterooligomer which physiological significance remains unclear (By similarity). The mitochondrial trifunctional enzyme interacts with MTLN (PubMed:29949755). Interacts with RSAD2/viperin (By similarity). Protein stability and association with membranes require HADHA. Acetylation of Lys-202 is observed in liver mitochondria from fasted mice but not from fed mice. Belongs to the thiolase-like superfamily. Thiolase family.