EPR Study of Homogentisate Oxygenase from Aspergillus Sp.

Abstract

Homogentisate oxygenase from the fungus, Aspergillus, was isolated and partially purified. We used this enzyme for the EPR study of homogentisate oxygenase reaction. The strain of Aspergillus was isolated from soil in Saitama, Japan, and was cultivated in a medium containing ferrous iron and l-tyrosine as the sole carbon source. As well as the mammalian and bacterial homogentisate oxygenase, the fungal enzyme required ferrous iron as a cofactor, but not Fe³⁺, Cu²⁺, Co²⁺, or Mn²⁺. This fungal oxygenase was found to be very labile in the presence of O₂. We performed EPR experiments on this homogentisate oxygenase reaction, and examined how the iron component was involved in oxidation of the substrate. When the homogentisate was added in an oxygen environment, an EPR signal of high spin iron (ferric) appeared at g = 4.28, which disappeared after incubation for 5 min at 25°C. Based on this result, we considered the oxygenation of homogentisate and proposed a reaction mechanism for homogentisate oxygenase from Aspergillus.