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 described here carries the 2,3-enoyl-CoA hydratase and the 3-hydroxyacyl-CoA dehydrogenase activities while the trifunctional enzyme subunit beta/HADHB bears the 3-ketoacyl-CoA thiolase activity. Independently of the subunit beta, the trifunctional enzyme subunit alpha/HADHA also has a monolysocardiolipin acyltransferase activity. It acylates monolysocardiolipin into cardiolipin, a major mitochondrial membrane phospholipid which plays a key role in apoptosis and supports mitochondrial respiratory chain complexes in the generation of ATP. Allows the acylation of monolysocardiolipin with different acyl-CoA substrates including oleoyl-CoA for which it displays the highest activity. a (3S)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O a 4-saturated-(3S)-3-hydroxyacyl-CoA = a (3E)-enoyl-CoA + H2O (3S)-hydroxyoctanoyl-CoA = (2E)-octenoyl-CoA + H2O (3S)-3-hydroxydodecanoyl-CoA = (2E)-dodecenoyl-CoA + H2O (3S)-hydroxyhexadecanoyl-CoA = (2E)-hexadecenoyl-CoA + H2O a long-chain (3S)-3-hydroxy fatty acyl-CoA + NAD(+) = a long-chain 3-oxo-fatty acyl-CoA + H(+) + NADH (3S)-hydroxyoctanoyl-CoA + NAD(+) = 3-oxooctanoyl-CoA + H(+) + NADH (3S)-hydroxydecanoyl-CoA + NAD(+) = 3-oxodecanoyl-CoA + H(+) + NADH (3S)-3-hydroxydodecanoyl-CoA + NAD(+) = 3-oxododecanoyl-CoA + H(+) + NADH (3S)-hydroxytetradecanoyl-CoA + NAD(+) = 3-oxotetradecanoyl-CoA + H(+) + NADH (3S)-hydroxyhexadecanoyl-CoA + NAD(+) = 3-oxohexadecanoyl-CoA + H(+) + NADH 1'-[1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phospho]-3'-[1-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phospho]-glycerol + hexadecanoyl-CoA = 1'-[1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phospho]-3'-[1-(9Z,12Z-octadecadienoyl)-2-hexadecanoyl-sn-glycero-3-phospho]-glycerol + CoA (9Z)-octadecenoyl-CoA + 1'-[1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phospho]-3'-[1-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phospho]-glycerol = 1'-[1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phospho]-3'-[1-(9Z,12Z-octadecadienoyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phospho]-glycerol + CoA (9Z,12Z)-octadecadienoyl-CoA + 1'-[1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phospho]-3'-[1-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phospho]-glycerol = 1',3'-bis-[1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phospho]-glycerol + 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 (PubMed:1730633). The mitochondrial trifunctional enzyme interacts with MTLN (By similarity). Protein stability and association with mitochondrion inner membrane do not require HADHB. In the N-terminal section; belongs to the enoyl-CoA hydratase/isomerase family. In the central section; belongs to the 3-hydroxyacyl-CoA dehydrogenase family.