Metal specificity in superoxide dismutases

Abstract

Superoxide dismutases (SODs) are essential metalloenzymes that protect cells from oxidative stress by catalysing the dismutation of superoxide radicals. While iron and manganese SODs are structurally similar, they exhibit strict metal specificity, and the precise determinants of this selectivity remain incompletely understood (Dudev, 2022). This study aimed to investigate the molecular basis of metal specificity in FeSOD isolated from Escherichia coli by sequentially introducing mutations within and near its active site. Building on the established FeSOD[dblDGF] mutant which included five substitutions (Q69G, A141Q, G142D, A68G, Y76F), an additional V70H mutation was introduced to create FeSOD[dblDGF_VH]. The mutant was expressed in E. coli under iron- and manganese-supplemented conditions, purified via immobilized metal affinity chromatography, and characterized through BCA assay, native PAGE with NBT staining, and Fe content analysis. The results demonstrated that FeSOD[dblDGF_VH] displayed higher relative activity when grown in manganese media, indicating a partial shift in metal preference, although overall activity remained lower than wild-type. Iron assays further suggested reduced iron incorporation in manganese-grown samples, supporting the proposed change in metal specificity. These findings highlight the critical role of active site residues in modulating metal selectivity and provide a possible foundation for further research in the V70H mutation.