The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.Keywords: superoxide dismutase; site-directed mutagenesis; metal specificity; thermostability.Iron superoxide dismutases (FeSOD, E.C.1.15.1.1) and manganese superoxide dismutases (MnSOD) constitute a class of structurally equivalent metalloenzymes prevalent in prokaryotes and in eukaryotic mitochondria, respectively. They exhibit a very high degree of homology in both sequence and structure (Fig. 1). The SODs are active only in dimeric association and all share structural homology in this respect [1]. The metal cofactors are required to catalyse the disproportionation of the superoxide radical into hydrogen peroxide and molecular oxygen [2] in a cyclic, two-stage oxidation-reduction mechanism:where M represents either iron or manganese. Selectivity of the proteins for their metal cofactor has been demonstrated in vivo [3] and although apoenzymes of each type of SOD may be reconstituted by the addition of metals, the resulting enzyme is active only with the authentic metal at its active centre [4][5][6][7]. A small number of cambialistic SODs have been shown to be active with either iron or manganese, though only those of Propionibacterium shermanii [8], Bacteroides gingivalis [9] and Bacteroides fragilis [10] demonstrate similar specific activities with either metal.In all structures solved for the mononuclear SODs, the metal ion is held in place by an absolutely conserved quartet of residues comprising three histidines and one aspartic acid which act as ligands to the metal (H26, H81, D167 and H171 for Escherichia coli MnSOD, Fig. 1B. This numbering will be used throughout except where indicated) [11][12][13][14][15][16][17][18][19][20][21]. This conservation is also reflected in all sequences elucidated for this large group of ubiquitous enzymes. A fifth metal ligand, either water or hydroxide, present in all structures produces a trigonal-pyramidal geometry at the active site. A distorted-octahedral geometry is assumed during catalytic turnover or inhibition,