Superoxide reductase from Desulfoarculus baarsii: Reaction mechanism and role of glutamate 47 and lysine 48 in catalysis

Superoxide reductase (SOR) is a small metalloenzyme that catalyzes reductio n of O-2(.-) to H2O2 and thus provides an antioxidant mechanism against sup eroxide radicals. Its active site contains an unusual mononuclear ferrous c enter, which is very efficient during electron transfer to O-2(.-) [Lombard , M., Fontecave, M., Touati, D., and Niviere, V. (2000) J. Biol. Chem. 275, 115-121]. The reaction of the enzyme from Desulfoarculus baarsii with supe roxide was studied by pulse radiolysis methods. The first step is an extrem ely fast bimolecular reaction of superoxide reductase with superoxide, with a rate constant of (1.1 +/- 0.3) x 10(9) M-1 s(-1). A first intermediate i s formed which is converted to a second one at a much slower rate constant of 500 +/- 50 s(-1). Decay of the second intermediate occurs with a rate co nstant of 25 +/- 5 s(-1). These intermediates are suggested to be iron-supe roxide and iron-peroxide species. Furthermore, the role of glutamate 47 and lysine 48, which are the closest charged residues to the vacant sixth iron coordination site, has been investigated by site-directed mutagenesis. Mut ation of glutamate 47 into alanine has no effect on the rates of the reacti on. On the contrary, mutation of lysine 48 into an isoleucine led to a 20-3 0-fold decrease of the rate constant of the bimolecular reaction, suggestin g that lysine 48 plays an important role during guiding and binding of supe roxide to the iron center Il. In addition, we report that expression of the lysine 48 sor mutant gene hardly restored to a superoxide dismutase-defici ent Escherichia coli mutant the ability to grow under aerobic conditions.