Substrate and Cofactor Requirements of Indoleamine 2,3-Dioxygenase in Interferon-Gamma-Treated Cells: Utilization of Oxygen Rather Than Superoxide

Ernst   R.   Werner; G.      Werner-Felmayer

Abstract

Much attention has been paid in initial biochemical studies on the ability of indoleamine 2,3-dioxygenase to use superoxide as substrate to cleave tryptophan to N-formyl kynurenine. This ability, however, is limited to the ferric form of the enzyme only, whereas the ferrous form requires oxygen rather than superoxide as substrate. As long as the enzyme is held in the ferrous form, high yield formation of product proceeds from the ferrous oxygen tryptophan ternary complex without the participation of superoxide. Enzyme assays in homogenates are carried out in presence of Methylene Blue, ascorbate and catalase. Ascorbate can be replaced by other reductants like e.g. tetrahydrobiopterin. Experiments with alteration of intracellular tetrahydrobiopterin concentrations in intact interferon-gamma treated cells clearly showed that tetrahydrobiopterin is not required for the indoleamine 2,3-dioxygenase reaction. In homogenates of interferongamma treated T-24 cells, substrates of xanthine oxidase did not stimulate the indoleamine 2,3-dioxygenase reaction, nor did allopurinol inhibit the reaction, nor did superoxide dismutase alter indoleamine 2,3-dioxygenase activity irrespective of the reductant used. From these experiments we concluded that molecular oxygen rather than superoxide is used in cell homogenates by indoleamine 2,3- dioxygenase to cleave L-tryptophan. A detailed analysis of available reports on oxygen and superoxide utilization by indoleamine 2,3- dioxygenase gives a comprehensive picture that the enzyme uses oxygen bound to the ferrous enzyme for cleavage of tryptophan, that the enzyme needs to be held by reductants in the ferrous state in enzyme incubations, and that superoxide is one of the reductants capable performing this reduction.

Keywords: Indoleamine-pyrrole 2,3-dioxygenase, tetrahydrobiopterin, oxygen, superoxide, lavinmononucleotide, superoxide dismutase, tryptophan, interferon-gamma