The pH Dependence of the Spectral and Anion Binding Properties of Iron Containing Superoxide Dismutase from E. Coli B: An Explanation for the Azide Inhibition of Dismutase Activity

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Abstract
Examination of the optical and EPR properties of the ferric form of the iron containing superoxide dismutase from E. coli B, at pH values ranging from 4.5 to 10.9, has revealed two reversible structural transitions affecting the Fe3+ ion. The apparent pKa. values of these transitions are 5.1±0.3 and 9.0±0.3. The binding of azide has been studied over the pH range 4.5 to 10.7; the affinity of the Fe3+ for N3− is independent of pH from 4.5 to ∼ 7.5, after which the dissociation constant decreased by a factor of 10 per unit increase in pH. The apparent pKa. which affects N3− binding to the iron is 8.6±0.2. The association of N3− with the iron has been examined using the temperature‐jump method at pH 7.4 and 9.3. The kinetics of ligand association were shown to conform to the minimal mechanism: magnified image K1 was found to be essentially unaffected by pH whereas K2 was much lower at pH 9.3 than at 7.4. The value of K1 at pH 7.4 (100 M−1) corresponds very closely to that obtained for the inhibition constant of azide, 10 mM.12 A scheme is presented in which N3− inhibits the iron containing dismutase by competing with O2− for an anion binding site near, but not on the Fe3+.