Tryptophan Oxygenase of Pseudomonas acidovorans

Tryptophan oxygenase (L-tryptophan: oxygen oxidoreductase, EC 1.13.1.12) was purified to a state of homogeneity from substrate-induced Pseudomonas acidovorans. The sedimentation and diffusion coefficients of the homogeneous protein are SU),~, 6.26 x lo-l3 set, and DzO w, 4.78 X 10d7 cm2 set-l, and from these values a molecular weight, M,,D, of 121,000 is obtained. High and low speed equilibrium sedimentation experiments give molecular weights, Mesuii, of 118,000 and 123,000, respectively. Ammo acid analyses reveal no sulfur-containing amino acid residues other than methionine. Quantitative analytical data show 1 mole of ferriprotoporphyrin IX and only trace amounts of copper and nonheme iron per mole of holoenzyme. The major absorption bands in the optical spectrum of the native holoenzyme occur at 280 and 405 mp, and the specific absorbance values (EF~Z) are 1.20 and 1.88, respectively. Optical and electron paramagnetic resonance spectral data indicate that the single protoheme iron moiety of both the native (ferric) and the chemically reduced (ferrous) form of the holoenzyme exists predominantly in the high spin configuration. Any of three kinds of treatment (alkaline pH, guanidinium chloride, or sodium dodecyl sulfate) results in dissociation of the native protein into subunits which are devoid of enzymatic activity and which have profoundly altered optical spectra. The subunit species produced in each case appear monodisperse by velocity and equilibrium sedimentation criteria and are characterized by the following sedimentation coefficients (s~~,,J and molecular weights (iWequil): alkaline pH, 2.46 S and 31,300; guanidinium chloride, 1.73 S and 33,200; sodium dodecyl sulfate, 2.67 S and 35,800. These data indicate that native tryptophan oxygenase is composed of a single heme moiety affixed to a protein molecule containing four polypeptide chains, of essentially equivalent mass, which are stabilized in a specific tetrameric structure exclusively by noncovalent interactions.