Azide protection of bacteroides superoxide dismutases from inactivation by hydrogen peroxide

The anaerobes Bacteroides fragilis, B. distasonis and B. thetaiotaomicron produce an iron-containing superoxide dismutase (FeSOD). These FeSODs are reversibly inhibited by 1 mM azide (NaN3) and are irreversibly inactivated upon incubation with hydrogen peroxide (H2O2). H2O2 inactivation of the enzyme likely depends on a Fenton type reaction with the production of hydroxyl radical (OH). Addition of NaN3 to the enzyme solution decreased the rate of inactivation by H2O2. After 20 minutes incubation of purified B. distasonis FeSOD with 2.5 mM H2O2, 61% of the initial enzymatic activity remained when 1 mM NaN3 was also present compared with 29% activity without NaN3. Similar results were seen with FeSOD from B. fragilis and B. thetaiotaomicron. Metal analyses of the native, peroxidized, and NaN3 protected samples are consistent with loss of Fe from the enzyme upon peroxidation, but retention of Fe and enzymatic activity in the NaN3 protected sample. Protection of FeSOD activity from H2O2 inactivation was dependent on NaN3 concentration. Anionic hydroxyl radical scavengers, such as urate and xanthine did not significantly protect the enzyme. The results are consistent with binding of azide to the active site either preventing entry of H2O2 or altering Fe redox potential, preventing OH production