STOICHIOMETRY OF ELECTRON UPTAKE AND THE EFFECT OF ANIONS AND PH ON THE MOLYBDENUM AND HEME REDUCTION POTENTIALS OF SULFITE OXIDASE

Microcoulometric titrations of chicken liver sulfite oxidase under varied conditions at 25-degrees-C have shown that the enzyme requires three electrons for complete subunit reduction. Reduction potentials (vs NHE) for the molybdenum site at pH 9.00 in the absence or coordinating anions (Mo(VI/V) = 0.057 V, Mo(V/IV) = -0.233 V) are considerably more negative than at pH 6.00 in 0.10 M KCl (Mo(VI/V) = 0.131 V, Mo(V/IV) = -0.086 V), consistent with the formation of chloride complexes of the Mo(V) and Mo(IV) states. Phosphate (H2PO4-) competes effectively with chloride for the Mo(V) site of the enzyme at lower concentrations (0.020 M phosphate vs 0.10 M KCl, pH 7.00) and complexes the Mo(VI) state, as seen in the significant negative shifts of the reduction potentials (Mo(VI/V) = 0.038 V, Mo(V/IV) = -0.239 V). Dissociation constants for proton and phosphate complexes or the Mo(VI) state and the chloride complex of the Mo(IV) state have been estimated. The heme reduction potentials decrease with increase in pH and are relatively unaffected by anions. The results are consistent with the results of recent EPR and EXAFS studies of the enzyme regarding active-site structure.