The Catalytic Mechanism of the Manganese-Containing Superoxide Dismutase of Escherichia coli Studied by Pulse Radiolysis

The dismutation of O₂ ^-, catalyzed by Escherichia coli Mn dismutase, has been investgated. O₂ ^- was generated in formate aqueous solutions by pulse radiolysis. When the initial concentration of O₂ ^-, [O₂ ^-]₀, is less than 10 times the total concentration of the dismutase, [E]₀, a reaction first order in both [O₂ ^-] and [E]₀ is observed, the apparent reaction rate constant of which is 1.5 ±0.15×10⁹ M^-1 sec^-1. When [O₂ ^-]0/[E]₀×15, a biphasic process is observed. Under these conditions only about 15 O₂ ^- radical ions per each dismutase molecule react with a relatively fast rate. Excess O₂ ^- is removed by a less efficient reaction, also first order in [E]₀ and nearly first order in [O₂ ^-], which has an apparent rate constant 1.6 ± 0.25×10⁸ M^-1 sec^-1. The results are interpreted in terms of four oxidation and reduction reactions, such as: (i) E + O₂ ^- → E^- + O₂, k = 1.3 ± 0.15×10⁹ M^-1 sec^-1; or (i)′ E + O₂ ^- + 2H⁺ → E+ + H₂O₂; (ii) E^- + O₂ ^- + 2H⁺→E + H₂O₂, k=1.6 ± 0.6×10⁹M^-1 sec^-1; or (ii)′ E+ + O₂ ^- → E + O₂; (iii) E^- + O₂→ E^2- + O₂, k≃2×10⁸ M^-1 sec^-1; or (iii)′ E + O₂ ^- → E^- + O₂; (iv) E^2- + O₂ ^- + 2H⁺ → E^- + H₂O₂, k≃1×10¹ M^-1 sec^-1; or (iv)′ E^- + O₂ ^- + 2H⁺→E + H₂O₂. E⁺, E, E^-, and E^2- may represent forms of enzymes in which Mn^IV, Mn^III, Mn^II, and Mn^I are respectively present. Alternative mechanisms involving additional oxidation states of the Mn are discussed. After the end of the catalytic process the high enzyme activity is fully regenerated within less than 30 sec.