Involved in the initial and rate-limiting step of peroxisomal beta-oxidation of straight-chain saturated and unsaturated very-long-chain fatty acids (PubMed:12758125). Catalyzes the desaturation of fatty acyl-CoAs such as palmitoyl-CoA (hexadecanoyl-CoA) to 2-trans-enoyl-CoAs ((2E)-enoyl-CoAs) such as (2E)-hexadecenoyl-CoA, and donates electrons directly to molecular oxygen (O(2)), thereby producing hydrogen peroxide (H(2)O(2)) (PubMed:12758125). Isoform 2 shows higher activity with hexadecanoyl-CoA as substrate than isoform 1 (PubMed:12758125). a 2,3-saturated acyl-CoA + O2 = a (2E)-enoyl-CoA + H2O2 a 2,3-saturated acyl-CoA + O2 = a (2E)-enoyl-CoA + H2O2 hexadecanoyl-CoA + O2 = (2E)-hexadecenoyl-CoA + H2O2 hexadecanoyl-CoA + O2 = (2E)-hexadecenoyl-CoA + H2O2 dodecanoyl-CoA + O2 = (2E)-dodecenoyl-CoA + H2O2 O2 + octanoyl-CoA = (2E)-octenoyl-CoA + H2O2 decanoyl-CoA + O2 = (2E)-decenoyl-CoA + H2O2 O2 + tetradecanoyl-CoA = (2E)-tetradecenoyl-CoA + H2O2 hexadecanedioyl-CoA + O2 = (2E)-hexadecenedioyl-CoA + H2O2 O2 + tetracosanoyl-CoA = (2E)-tetracosenoyl-CoA + H2O2 glutaryl-CoA + O2 = (2E)-glutaconyl-CoA + H2O2 hexanoyl-CoA + O2 = (2E)-hexenoyl-CoA + H2O2 O2 + octadecanoyl-CoA = (2E)-octadecenoyl-CoA + H2O2 (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoyl-CoA + O2 = (2E,5Z,8Z,11Z,14Z,17Z)-icosahexaenoyl-CoA + H2O2 (6Z,9Z,12Z,15Z,18Z,21Z)-tetracosahexaenoyl-CoA + O2 = (2E,6Z,9Z,12Z,15Z,18Z,21Z)-tetracosaheptaenoyl-CoA + H2O2 Lipid metabolism; peroxisomal fatty acid beta-oxidation. Homodimer (By similarity). Interacts with LONP2 (By similarity). Belongs to the acyl-CoA oxidase family.