On the role of copper in activation of and catalysis by tryptophan-2,3-dioxygenase.

L-Tryptophan-2,3-dioxygenases (EC 1.13.1.12), purified to homogeneity from Pseudomonas acidovorans and from rat liver, have been shown to contain as bound cofactors, 2 g atoms copper and 2 moles of heme per mole of tetrameric enzyme. Kinetic and spectral studies, utilizing the metal complexing agents diethyldithiocarbamate (Cu2+ specific), bathocuproinesulfonate (Cu+ specific), and bathophenanthrolinesulfonate (Cu+ and Fe2+ chelator), have established that copper, as well as heme, is essential for the catalytic activity of both hepatic and bacterial tryptophanoxygenases. Diethyldithiocarbamate does not inhibit the ongoing catalytic reaction but does prevent reductive activation by sodium ascorbate of aerobically aged, oxidatively inactivated tryptophan oxygenase, indicating the presence of chelatable Cu2f only in inactive enzyme molecules. Bathocuproinesulfonate and bathophenanthrolinesulfonate inhibit the ongoing catalytic reaction, indicating the presence of chelatable Cu+ in functioning molecules of tryptophan oxygenase. Oxidatively inactivated tryptophan oxygenase contains (CU~+)~ (ferriheme)2, and fully active tryptophan oxygenase contains (Cu+), (ferriheme)*. Bathocuproinesulfonate inhibits pseudomonad tryptophan oxygenase competitively with respect to L-tryptophan (Ki = 31 PM) and noncompetitively with respect to 02. Difference spectra of the bathocuproinesulfonate-pseudomonad enzyme complex versus the pseudomonad enzyme alone are indistinguishable from a bathocuproinesulfonate-Cu+ complex in solution. The bathocuproinesulfonate difference spectrum disappears in parallel with reductant independent catalytic activity as pseudomonad tryptophan oxygenase is aerobically aged in the absence of L-tryptophan. This supports the view that the catalytically active form of tryptophan oxygenase contains Cuf and that as the enzyme develops a requirement for an exogenous reductant, such as ascorbate,