A conserved active site residue favours metal ion binding by destabilizing apo‐protein.

Abstract

The active sites of Fe‐containing SOD and Mn‐containing SOD (FeSOD and MnSOD respectively) are organized around a single redox‐active ion, Fe or Mn, which alternates between its 3+ and 2+ states during turnover. The proteins are homologues, and some members of the family support activity with either Fe or Mn (Fe&MnSODs), while others bind either metal ion but display activity with only one. A second‐sphere Gln/His residue is the most commonly conserved difference between FeSODs and MnSODs, which supply the Gln from postion 69 or 146, respectively. Mutation of Gln69 of E. coli FeSOD to Glu or His produced large changes in the E° and catalytic activity indicating that the residue at position 69 has a large influence on the affinity for at least one of the Fe oxidation states (Yikilmaz et al 2006, Biochemistry 45:1151). Analogous studies of MnSOD have found that replacement of Gln146 results in changes in activity and redox tuning, but the Q146E‐MnSOD mutant has evaded study because it fails to bind Mn. We have compared the stability and metal ion binding of a series of mutants of the MnSOD protein wherein different amino acids occupy position 146. These studies shed light on the relationship between metal ion binding and protein stability in this series of related variants of MnSOD. In particular, Q146E‐apoMn‐SOD displays a melting temperature elevated over that of WT‐apoMn‐SOD by 30 °C, and other mutations produce intermediate behaviour. Thus the WT apoMn‐SOD is less thermally stable than many of the variants. We propose that the WT protein conserves a destabilizing amino acid at position 146 as part of a strategy for favouring metal ion binding, as well as redox tuning.