Spectroscopic studies of rat liver acyl-CoA oxidase with reference to recognition and activation of substrate.

Abstract

Two forms of rat peroxisomal acyl-CoA oxidase (ACO-I and -II) interact with the substrate analogs, 3-ketoacyl-CoAs, forming a complex characterized by the so-called charge-transfer (CT) band around 575 nm in the absorption spectra. The CT band of ACO-I exhibited a broad dependency on the acyl chain-length from C4 to C16, whereas that of ACO-II showed increased intensity with a longer acyl chain to reach a maximum with a chain-length of C12. These chain-length dependencies of the CT bands were compared with those of the enzymatic activities reported previously [Setoyama et al. (1995) Biochem. Biophys. Res. Commun. 217, 482-487]. The differences in spectroscopic and enzymatic properties between ACO-I and -II suggest that the amino acid stretch corresponding to the third exon in the ACO sequence affects the binding of the ligand and substrate, since the difference in the primary structure between ACO-I and -II lies in the short amino acid stretch corresponding to the third of the total of 14 exons. On the other hand, resonance Raman spectra of the complexes of ACO-I and -II with 3-ketoacyl-CoAs excited in the CT band showed similar features. The two prominent FAD bands II and III, associated with the C(4a)=N(5) moiety of FAD, were observed at 1,577 and 1,545 cm(-1), respectively. In contrast, the bands at 1,615 and 1,493 cm(-1) in the ACO-I x 3-keto-C8-CoA complex were assigned to the stretching modes of C=O at positions 3 and 1 of the ligand, respectively, by using the isotopically labeled ligands. Both C=O stretching bands were shifted to lower wave numbers upon complex formation with ACO-I, implying that the C=O bond involves the single bond (C-O-) character in the active site cavity. The downshift of the C(1)=O stretching band was larger than that of the C(3)=O stretching band. Therefore, the ligand lies in the active site as the anionic form with a major contribution from C(1)-O-. These observations demonstrate that the CT band around 575 nm arises from the charge-transfer interaction between the oxidized FAD and the enolate transformed after the elimination of the a-proton. The band II of FAD in the complexes reveals a significant decrease in the frequency in comparison with the complexes of medium-chain acyl-CoA dehydrogenase (MCAD) with 3-ketoacyl-CoA. This observation suggests a difference between ACO and MCAD in the hydrogen-bonding network associated with enzyme-bound FAD.