THE ROOM-TEMPERATURE POTENTIOMETRY OF XANTHINE-OXIDASE - PH-DEPENDENT REDOX BEHAVIOR OF THE FLAVIN, MOLYBDENUM, AND IRON-SULFUR CENTERS

  • 1 January 1982
    • journal article
    • research article
    • Vol. 257  (19) , 1617-1626
Abstract
A series of potentiometric titrations of xanthine oxidase were performed at room temperature in the pH range 61.-9.9. Reduction of the 2 Fe/S centers was monitored by CD [circular dichroism], and that of the FAD and Mo center by EPR. The Fe/S centers behave as centers having a protonable group whose pKa changes with reduction state (E [midpoint potential] = -344 mV, pKo = 6.4 and pKr = 8.0 for Fe/S II). The flavin and the 2 types of Mo centers show varying behavior, but, in all cases, electron addition is accompanied by protonation. The sequence for FAD is reduction, protonation, reduction, protonation with E1 = -398 mV, E2 = -240 mV, pK1 = 9.5, pK2 = 7.4. For rapid Mo, the sequence is protonation, reduction, protonation, reduction with E1 = -369 mV, E2 = -301 mV, pK1 = 7.9, pK2 = 8.4; and for slow Mo, protonation, reduction, protonation with E1 = 320 mV, E2 = -477 mV, pK1 = 7.5, pK2 = 9.5. Comparison to data obtained previously at cryogenic temperatures showed the centers to have significant temperatures dependence, which calls for a re-evaluation of conclusions reached using cryogenic techniques (e.g., rapid-freeze). The optical absorbance characteristics of the enzyme were also investigated and a possible absorbance for Mo was suggested.