Delta(3,5),Delta(2,4)-Dienoyl-CoA isomerase (DI), an auxiliary enzyme of unsaturated fatty acid beta-oxidation, was purified from rat mitochondria and peroxisomes and subjected to N-terminal sequencing to facilitate a mechanistic study of this enzyme. The mature mitochondrial DI from rat heart was lacking its 34 N-terminal amino acid residues that have the properties of a mitochondrial targeting sequence. The peroxisomal isomerase was identified as a product of the same gene with a truncated and ragged N terminus. Expression of the cDNA coding for the mature mitochondrial DI in Escherichia coli yielded an enzyme preparation that was as active as the native DI. Because the recombinant DI also exhibited Delta(3,5,7),Delta(2,4,6)-trienoyl-CoA isomerase (TI) activity, both isomerases reside on the same protein. Mutations of any of the 3 acidic amino acid residues located at the active site (Modis, Y., Filppula, S. A., Novikov, D. K., Norledge, B., Hiltunen, J. K., and Wierenga, R. K. (1998) Structure 6, 957-970) caused activity losses. In contrast to only a 10-fold decrease in activity upon replacement of Asp(176) by Ala, substitutions of Asp(204) by Asn and of Glu(196) by Gln resulted in 10(5)-fold lower activities. Such activity losses are consistent with the direct involvement of these latter two residues in the proposed proton transfers at carbons 2 and 6 or 8 of the substrates. Probing of the wild-type and mutants forms of the enzyme with 2,5-octadienoyl-CoA as substrate revealed low Delta(2),Delta(3)-enoyl-CoA isomerase and Delta(5),Delta(4)-enoyl-CoA isomerase activities catalyzed by Glu(196) and Asp(204), respectively. Altogether, these data reveal that positional isomerizations of the diene and triene are facilitated by simultaneous proton transfers involving Glu(196) and Asp(204), whereas each residue alone can catalyze, albeit less efficiently, a monoene isomerization.