Molecular Properties of L-Kynurenine 3-Hydroxylase from Rat Liver Mitochondria

Abstract

L-Kynurenine 3-hydroxylase [EC 1. 14. 1. 2] has been solubilized with Triton X-100 and purified to homogeneity from rat liver mitochondria. The purified enzyme exists in aqueous media as an oligomeric aggregate and in 0.2% Triton X-100 as a dimer with a molecular weight of about 345, 000. Its monomeric molecular weight is approximately 160, 000, and 4 mol of FAD are associated with 1 mol of the monomeric unit of the enzyme. The addition of FAD to the apoenzyme resulted in remarkable increase in both L-kynurenine 3-hydroxylase activity and FAD fluorescence. The enzyme molecule migrated as a single band in polyacrylamide gel electrophoresis and in chromatography on a Sephadex G-200 column, and was resolved into two bands only after complete denaturation by high concentrations of SDS or urea. SDS-polyacrylamide gel electrophoresis in the presence of SDS at a concentration of more than 1 and Sephadex G-100 gel chromatography in the presence of 5M urea caused cleavage of the monooxygenase molecule into a protein having a molecular weight of about 137, 500 and a smaller fragment. Upon dissociation into these fragments, the enzyme activity was completely lost. The visible absorption spectrum of the oxidized holo-L-kynurenine 3-hydroxylase showed an absorption maximum at 408nm with a prominent shoulder at around 445 run. In contrast to the oxidized form, the dithionite-reduced form had peaks at about 420 and 554nm. In spite of its electrophoretic homogeneity, appreciable amounts of cytochrome b₅-like heme-protein were detected in our highly purified preparation of L-kynurenine 3-hydroxylase. The purified L-kynurenine 3-hydroxylase catalyzed the reductions by NADH or NADPH of DCPI and ferricyanide.