An Fe-preferring cambialistic superoxide dismutase from Aneurinibacillus thermoaerophilus: biochemical...

Cambialistic superoxide dismutases (CamSODs) can utilize Fe²⁺ or Mn²⁺ as cofactors, but Fe-preferring enzymes are rare. Here, AtSODB exhibited a 23.69 kDa monomer band on SDS-PAGE and formed a homodimer natively, with a high specific activity of 4352.78 U·mg^-1, exhibiting an optimal temperature of 25 °C and an optimal pH of 8.0. The enzyme retained 81.6% activity after 60 min at 50 °C, remained >90% active from pH 6.00 to 11.00, and possessed a K_m of 0.15 mM and a V_max of 1.15 U·mg^-1. It showed strong chemical resilience, keeping 92.09% activity in Tween-20 and 101.90% activity with 5 mM EDTA. Metal supplementation increased activity to 5268.05 U·mg^-1 with Fe²⁺ versus 3309.43 U·mg^-1 with Mn²⁺, and in vitro reconstitution confirmed a clear iron preference (Fe²⁺: 111.69%; Mn²⁺: 98.57%). Phylogenetic and structural modeling placed AtSODB within the Fe/MnSOD family, with conserved catalytic architecture, and suggested that Val161 may contribute to Fe preference. Heterologous expression improved E. coli growth under paraquat-induced oxidative stress. Collectively, AtSODB represents a rare, Fe-preferring CamSOD with high stability, broad pH tolerance, and the potential to scavenge oxygen free radicals. This provides direct evidence of the non-exclusive selectivity for metal ions in thermophilic SODs. These findings advance our understanding of metal specificity in CamSODs and highlight AtSODB as a robust candidate for oxidative-stress applications.