THE CONSERVED RESIDUE TYROSINE-34 IS ESSENTIAL FOR MAXIMAL ACTIVITY OF IRON-SUPEROXIDE DISMUTASE FROM ESCHERICHIA-COLI

We have expressed, purified, and analyzed the iron-containing superoxi de dismutase (FeSOD) of Escherichia coli with mutations directed at ty rosine position 34 to introduce phenylalanine (SODY34F), serine (SODY3 4S), or cysteine (SODY34C). FeSOD and mutant enzymes were purified fro m SOD-deficient cells using a GST-FeSOD fusion protein intermediate wh ich was subsequently cleaved with thrombin and repurified. Specific ac tivities were measured using the xanthine-xanthine oxidase method and gave 3148 u/mg for wild-type FeSOD. The SODY34S mutation virtually ina ctivates the enzyme (42 u/mg); mutation to cysteine greatly reduces ac tivity (563 u/mg), but the SODY34F mutant retains nearly 40% of the ac tivity of wild type (1205 u/mg). Fusion protein intermediates were als o shown to be active and were demonstrated to protect SOD-deficient E. coli cells from the induced effects of oxidative stress, with growth rates directly proportional to the specific activities of the expresse d mutant enzymes. SODY34F exhibited decreased thermal stability, reduc ed activity at high pH, and a pronounced increase in sensitivity to th e inhibitor sodium azide compared with wild-type FeSOD. These results suggest that tyrosine at position 34 is multifunctional and plays a st ructural role (probably through hydrogen bonding to glutamine at posit ion 69) in maintaining the integrity of the active site, a stabilizing role at high pH, and a steric role in obstructing access to the activ e site of both substrate and inhibitor molecules.