An ancient metalloenzyme evolves through metal preference modulation

Abstract Evolutionary processes create functional diversity of natural proteins, the essential building blocks of all biological systems. Although descriptions of general protein evolution are extensive1, studies of natural proteins sampled across the tree of life and evaluated in a single experimental system are virtually non-existent. Almost half of all enzymes require metals2,3 and metalloproteins tend to optimally utilise the physicochemical properties of a specific metal cofactor. Life must constantly adapt to changes in metal-bioavailability, including exposure of pathogens to nutritional immunity4,5 and biogeological oxygenation of Earth’s atmosphere6,7. These changes can challenge the ability of metalloenzymes to maintain activity and presumptively drive their evolution. Here, we studied metal-preference evolution within the natural diversity of the iron/manganese superoxide dismutase (SodFM) enzyme family, important scavengers of toxic reactive oxygen species. We identified and experimentally verified residues with critically conserved roles in determining metal-preference that, when combined with an understanding of the protein’s evolutionary history, enabled improved prediction of metal-utilisation across the five SodFM subfamilies defined herein. By combining phylogenetics, bioinformatics, biochemistry, and structural biology we demonstrate that SodFM metal-utilisation can be evolutionarily fine-tuned by sliding on a continuous scale between perfect manganese- and iron-specificities. Over the history of life, SodFM metal-preference has been modulated multiple independent times within different evolutionary and ecological contexts and can be changed within short evolutionary timeframes. Our data shed light on the evolutionary mechanisms by which diverse metalloenzyme families can change their metal preference over time in nature or by design in man-made systems.