A metalloenzyme's metal preference evolves through redox modulation driven by the cofactor's secondary coordination sphere

Changes in protein properties and functions are central to the evolution of life. Metalloproteins can evolve by changing their preference from one metal cofactor to another. Recently, we demonstrated that the widely distributed iron or manganese dependent superoxide dismutase (SodFM) family have undergone numerous metal-preference changes, including during evolutionary adaptation of pathogenic bacteria to altered metal availability within the host. Yet the underlying properties of metal-binding sites that control metalloenzyme metal-preference are unclear, and thus we lack an understanding of how enzymatic metal-preference can be re-shaped by evolution. Here, we used spectral features of bound iron or manganese, whose intensities reflect their oxidation state, to assess how their redox properties are manipulated during SodFM evolution. We systematically analysed the metal oxidation state across diverse SodFMs from multiple phylogenetic groups with different catalytic metal-preferences, including those known to have undergone evolutionary metal-preference switching. We observed a striking relationship between resting oxidation state and catalytic metal-preferences. Mutagenesis of second-sphere residues previously identified as determining metal preference revealed that they modulate metal-dependent activity and cofactor oxidation state in tandem, demonstrating these properties are linked. Together, these data argue that the differing SodFM metal preferences observed across the tree of life evolved through tuning of their redox properties by the secondary coordination sphere. This study gives insight into the process by which a metalloenzyme originally optimised for one metal cofactor can evolve a new metal preference, under suitable selection pressure, through re-optimisation of its active site for catalytic reactivity of the new metal cofactor. Significance statementMetal cofactors are needed by almost half of all enzymes. Catalytic metal-preference of metalloenzymes can evolve, for example to adapt to altered environmental metal availability. Yet, it is unclear how this evolutionary process occurs, enabling an active site optimised for one metal to change to become optimised for the new metal. Here, we have investigated this evolutionary process in a family of superoxide dismutase (SodFM) enzymes. We found that spectral features, which reflect the enzymes redox properties, of a diverse array of SodFMs with differing metal-preferences, and of mutated forms with artificially altered preferences, correlate with the metal-dependence of their activity. The data show that metal-preference change in SodFMs involves evolutionary tuning of the redox properties of SodFMs.