The room temperature potentiometry of xanthine oxidase. pH-dependent redox behavior of the flavin, molybdenum, and iron-sulfur centers.
A series of potentiometric titrations of xanthine oxidase have been performed at room temperature in the pH range 6.1-9.9. Reduction of the two Fe/S centers was monitored by CD, 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 = -344 mV, pKo = 6.4, and pKr = 8.1 for Fe/S I; E = -249 mV, pKo = 6.4, and pKr = 8.0 for Fe/S II). The flavin and the two types of molybdenum 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" molybdenum, the sequence is protonation, reduction, protonation, reduction with E1 = -369 mV, E2 = -301 mV, pK1 = 7.9, pK2 = 8.4; and for slow molybdenum, protonation, reduction, protonation with E1 = 320 mV, E2 = -477 mV, pK1 = 7.5, pK2 = 9.5. Comparison to data obtained previously at cryogenic temperatures (Cammack, R., Barber, M. J., and Bray, R. C. (1976) Biochem. J. 157, 469-468 and Barber, M. J., and Seigel, L. M. (1982) in Flavins and Flavoproteins (Massey, V., and Williams, C. H., eds) pp. 796-804, Elsevier/North-Holland, New York) showed the centers to have significant temperature 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 molybdenum was suggested.
D000818:Animals; D002417:Cattle; D005260:Female; D005182:Flavin-Adenine Dinucleotide; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism*; D008892:Milk / Q000201:enzymology; D008982:Molybdenum; D010084:Oxidation-Reduction; D011199:Potentiometry; D013696:Temperature; D014969:Xanthine Oxidase / Q000378:metabolism*
D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D005182:Flavin-Adenine Dinucleotide; D008982:Molybdenum; D014969:Xanthine Oxidase