Oxygenated Form of Protocatechuate 3,4-Dioxygenase, a Non-heme Iron-containing Dioxygenase, as Reaction Intermediate

Abstract

Abstract A short-lived new spectral species of protocatechuate 3,4-dioxygenase, a trivalent non-heme iron-containing enzyme, was observed in the early stage of the reaction. This new spectral species was characterized by a broad absorption band with a maximum between 500 and 520 mµ, distinct from those of the enzyme or the enzyme-protocatechuic acid complex. It could be demonstrated only in the presence of both protocatechuic acid and molecular oxygen. The rate constant for the decomposition of this species determined by the stopped flow analysis agreed quite well with the turnover number of the enzyme as determined by the over-all reaction. During the steady state of the reaction, a similar spectral species could be shown with substrate analogues which were metabolized more slowly than protocatechuic acid. Based on these findings, the new spectral species was interpreted to represent an oxygenated form of the enzyme, namely a ternary complex of oxygen, substrate, and enzyme, and to be an obligatory intermediate in the reaction.