Acid-alkaline transition and thermal spin equilibrium of the heme in ferric L-tryptophan 2,3-dioxygenases.

Ltryptophan, to the enzyme greatly facilitated the acidalkaline transition. By titrating the spectral change as a function of pH, the pK values for the acid-alkaline transition were determined to be 8.4 and 9.7 for the rat liver and Pseudomonas enzyme, respectively, in the absence of L-tryptophan. Upon addition of L-tryptophan, however, these pK values shifted to 6.9 and 8.0 for the rat liver and Pseudomonas enzyme, respectively. Thus, the binding of substrate to the enzyme caused a shift in pK values for the acid-alkaline transition by about 1.6 pH units from an alkaline to a neutral region. These pK values as well as the shifts in pK values upon binding of L-tryptophan were confirmed further by measuring the pK from pH dependence curve of midpoint potentials of the heme-iron in the enzymes. By the aid of low temperature spectroscopic techniques, the intensities of low spin bands in the spectra of the alkaline forms were found to increase with concomitant decreases in the high spin bands when the temperature was lowered from a room to liquid nitrogen temperature. Contrariwise, elevation of the temperature increased the high spin components with parallel decreases in the low spin components. These temperature-dependent changes in the spin state were confirmed by an electron paramagnetic and a proton nuclear magnetic resonance spectroscopy. Thus, the spin state of the alkaline form of ferric L-tryptophan 2,3dioxygenase is in a thermal spin equilibrium between high and low spin state in either enzymes. The thermodynamic parameters for the spin state change were also described.