Superoxide reductases (SORs) are non-heme iron-containing enzymes that remove superoxide by reducing it to hydrogen peroxide. The active center of SORs consists of a ferrous ion coordinated by four histidines and one cysteine in a square-pyramidal geometry. In the 2Fe-SOR, a distinct family of SORs, there is an additional desulforedoxin-like site that does not appear to be involved in SOR activity. Our previous studies on recombinant Archaeoglobus fulgidus neelaredoxin (1Fe-SOR) have shown that the reaction with superoxide involves the formation of a transient ferric form that, upon protonation, decays to yield an Fe3+-OH species, followed by binding of glutamate to the ferric ion via replacement of hydroxide (Rodrigues et al. in Biochemistry 45:9266-9278, 2006). Here, we report the characterization of recombinant desulfoferrodoxin from the same organism, which is a member of the 2Fe-SOR family, and show that the steps involved in the superoxide reduction are similar in both families of SOR. The electron donation to the SOR from its redox partner, rubredoxin, is also presented here.