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

R Makino,K Sakaguchi,T Iizuka,Y Ishimura

doi:10.1016/s0021-9258(19)70217-8

Abstract

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.

Highlights

THEJOURNAL OF BIOLOOICACHLEMISTRYUpon addition of L-trypto- in “a low or a high affinity form” for the binding of the phan, these pK values shifted to 6.9 and 8.0 exogenous ligands depending upon the presence and absence for the rat liver and Pseudomonasenzyme,respectively
An acid-alkaline transition-and temperature-de- L-Tryptophan 2,3-dioxygenase is a unique hemoprotein pendent changes in thespin state of ferric L-tryptophan which catalyzes the insertion of molecular oxygen into the
Previous studies have demonstrated that the reactivities of the enzyme-heme for the binding of ligands such as oxygen, carbon monoxide, and cyanide were greatly augmented by the binding of L-tryptophan to the enzyme [7, 8, 10, 11].Upon binding of L-tryptophan, the dissociation constant of cyanide complex of the ferric Pseudomonas enzyme was altered from 280 to 1.5 PM and thatfor carbon monoxide complex of the ferrous enzyme changed from 280 to 5 p~ ( 7 )

Summary

THEJOURNAL OF BIOLOOICACHLEMISTRY

Upon addition of L-trypto- in “a low or a high affinity form” for the binding of the phan, these pK values shifted to 6.9 and 8.0 exogenous ligands depending upon the presence and absence for the rat liver and Pseudomonasenzyme,respectively. The binding of substrate to theenzyme caused a phan toferric rat liver and Pseudomonas enzymes was shown shift in pK values for the acid-alkaline transition by to cause significant spectral changes both in the visible and about 1.6 pHunits from an alkaline to a neutral region. The alkaline form in the high state of the alkaline form of ferric L-tryptophan 2,3- spin state was suggested to be the “high affinity form” of the dioxygenase is in a thermal spin equilibrium between enzyme for the binding of exogenousligands toward the hemehigh and low spin state in either enzymes. Modynamic parameters for the spin state change were described

EXPERIMENTAL PROCEDURES

The anaerobic vessel and procedure for the potential measurements

Optical spectrum

Findings

DISCUSSION