Determinants of metal cofactor specificity in iron/manganese superoxide dismutase metalloenzymes

Abstract

Metal ions are a crucial feature of biology, playing vital roles in the structure and/or function of over one-third of all proteins. The ubiquitous Fe/Mn superoxide dismutase (SOD) metalloenzyme superfamily exemplifies this; all members require a redox-active metal to enable the detoxification of harmful reactive oxygen species (ROS). All Fe/Mn SODs were originally thought to be either Fe-specific or Mn-specific, i.e. only able to use one metal cofactor for catalysis. The identification of cambialistic SODs, active with both Fe and Mn, challenged this binary categorisation. However, it remains unclear how differences in metal preferences of activity have evolved in Fe/Mn SODs, which are highly conserved from primary to tertiary structure. Investigation of the redox properties of select SODs has led to the hypothesis that tuning of the cofactor’s reduction potential by its secondary coordination sphere underlies metal-dependent activity. Utilising pairs of related SODs which have evolved distinct metal preferences, found in the human pathogens Staphylococcus aureus and Bacillus anthracis, it was found that metal preference correlated with redox-dependent absorption spectra. Reciprocal mutagenesis of secondary coordination sphere residues XD-2/XD-1 altered metal preference in both SOD pairs, also causing concomitant changes in these spectrally-monitored redox properties. Extending this analysis to a diverse set of bacterial Fe/Mn SODs and their mutated variants, spanning a range of metal preferences, supported the role of residues XD-2/XD-1 in determining metal preference and revealed a clear correlation between activity and the redox state of the metal cofactor. This work provides empirical evidence in support of the hypothesis that evolutionary changes in metal preference are conferred through changes to the metal cofactor’s redox properties, enacted through the mutation of key secondary sphere residues. Understanding mechanisms of metal specificity in such a widely-studied and prevalent metalloenzyme family has implications for understanding metal toxicity and bioengineering of artificial metalloenzymes.