A closely related pair of superoxide dismutase isozymes from Staphylococcus aureus show distinct stabilities and proton-exchange dynamics.

Changes in biochemical properties, caused by iterative mutations in amino acid sequence, underlie the alterations in protein function over time that underpin the evolutionary process. An example is the switching of an enzyme's reliance from one essential metal to an alternative as their catalytic cofactor. We previously described such neofunctionalization in Staphylococcus aureus, which altered a superoxide dismutase (SOD) enzyme from being an ancestral manganese-dependent (MnSOD) into an extant isozyme that can equally utilize either manganese or iron, termed cambialism (camSOD). Yet it is unclear whether camSOD emergence involved selection solely for cofactor flexibility or whether other biochemical properties also diverged during neofunctionalization. Here, we have investigated an independent biochemical property of the S. aureus SODs, their structural stability. We demonstrate that the neofunctionalized camSOD exhibits increased stability relative to the ancestral MnSOD. S. aureus camSOD is more resistant to both chemical and thermal unfolding in vitro. Crucially, while both isozymes possess a stable "core" at the heart of their fold, consisting of regions of the protein localized around the metal cofactor that resist hydrogen-deuterium exchange when exposed to isotopically labelled solvent, this core is larger and more exchange-resistant in camSOD than MnSOD. Thus, during the recent divergence of this SOD pair, two distinct biochemical properties have undergone substantial and rapid evolutionary change. This study paves the way for investigations of the structural and functional relationship between these properties, a SODs metal-preference and stability, and of how these properties were concomitantly selected during neofunctionalization in the S. aureus lineage.