An evolutionary path to altered cofactor specificity in a metalloenzyme

Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal’s redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity. Many metalloenzymes are highly specific for their cognate metal ion but the molecular principles underlying this specificity often remain unclear. Here, the authors characterize the structural and biochemical basis for the different metal specificity of two evolutionarily related superoxide dismutases.

M etalloproteins are critical to all aspects of life. They are ubiquitous, with as many as half of all enzymes requiring a metal cofactor for function 1,2 . Metalloenzymes are often highly specific for their cognate metal ion, exhibiting reduced activity with non-native metal cofactors in vitro and in vivo [3][4][5] . As a result, understanding the principles that govern the specificity of metal-protein interactions is relevant to nearly all aspects of biology, medicine and biotechnology. Yet the underlying biochemical principles that determine metal cofactor specificity, and the evolutionary processes that drive the optimization of enzyme active sites to utilize a specific metal cofactor remain unclear. This in turn limits our ability to predict the metal used by proteins from their sequence, to produce synthetic metalloenzymes to perform novel reactions for industrial applications, and to rationally design metalloenzyme inhibitors for medicine.
Our limited appreciation of how metalloprotein architecture dictates metal specificity is exemplified by the superoxide dismutases (SODs) 6 . SODs detoxify superoxide, a reactive oxygen radical produced by aerobic metabolism and by the mammalian immune system in response to infection 7 . The biological importance of SODs is illustrated by their presence in most organisms, including obligate anaerobes, and the independent evolution of three distinct dismutase protein families. These SOD families are defined by the cofactor they utilize as nickel SODs, copper and zinc SODs, and manganese (Mn) or iron (Fe) SODs 6 . Mn/Fedependent SODs are the most common, and historically were thought to be strictly either manganese-or iron-specific. We recently established that a Mn/Fe SOD from the bacterium Staphylococcus aureus exhibits equal activity with either manganese or iron. These versatile enzymes are termed cambialistic SODs (camSOD) 8 . In addition to the camSOD (SodM), S. aureus also possesses a second, manganese-dependent SOD (SodA) [8][9][10] . Although cambialistic SODs had previously been described 6,[11][12][13][14][15] , their biological importance was questioned. However, the S. aureus camSOD contributes to infection by enabling the bacterium to maintain a defense against superoxide when manganese starved by the host 8,16,17 . All members of the Mn/Fe SOD family are related in sequence, exhibit identical protein folds, and coordinate their metal ion using identical ligands 6 , making it unclear why some enzymes absolutely require manganese for catalysis (MnSOD), while others require iron (FeSOD), and still others show metal cofactor flexibility (camSOD).
The metal used by a protein is not permanently fixed, and can change in response to environmental pressures 2,18 . For example, iron was readily soluble in the anaerobic oceans during life's early evolution and early organisms are thus thought to have been iron-philic 18,19 . However, oxygenation by early photosynthetic organisms reduced the availability of iron 18,19 . The resulting biological iron deficiency would have imposed selective pressure to adapt iron-dependent enzymes to use non-iron cofactors [18][19][20] . While supported by bioinformatic analyses 18,21 , no experimental evidence has been presented demonstrating the evolutionary process by which a change in metal specificity has evolved through iterative mutation 20 .
Here, we exploit the close relationship between the staphylococcal SODs to understand how evolutionary changes in metal utilization occur. Genomic analysis shows the camSOD likely evolved from a manganese-specific predecessor that subsequently underwent neofunctionalization, a defined evolutionary process in which mutations rapidly accumulated in the duplicated gene during a period of functional redundancy, resulting in gain of a new beneficial function 22 . Integrated structural, biochemical, and electron paramagnetic resonance (EPR) studies reveal that two such mutations have altered amino acid residues in close spatial proximity to the SOD active site, driving the change in camSOD metal specificity. When these residues are reciprocally swapped, the metal specificities of the MnSOD and camSOD are largely interconverted. Remarkably, these residues possess non-polar sidechains located in the metal's secondary coordination sphere, and make no direct contacts to the metal-coordinating ligands. These subtle changes regulate the electronic structure and redox properties of the catalytic metal ion, dictating which metals the enzymes can use. Leveraging these findings reveals that small increases in iron-dependent catalysis by camSOD enhance the ability of S. aureus to overcome the immune response. Collectively, our data show how subtle changes to metalloenzyme architecture can dramatically alter the metal ion's reactivity and drive the evolution of isozymes with new cofactor specificity.

Results
The two S. aureus SODs exhibit extensive similarity. Initially, we comprehensively characterized which metals the MnSOD and camSOD could use. The MnSOD was active only with manganese, while the camSOD was active with manganese or iron (Supplementary Fig. 1 and Table 1). As with other characterized cambialistic SODs 12,14,23 , the activity of S. aureus camSOD is significantly lower than that of the MnSOD, suggesting that cambialism represents an evolutionary compromise, where catalytic activity is sacrificed at the expense of enabling cofactor flexibility. Alignment of the S. aureus SOD amino acid sequences demonstrated that the two proteins share 75% identity (Supplementary Fig. 2). Next, we examined the S. aureus SODs for structural differences using circular dichroism (CD) spectroscopy. The iron-and manganese-loaded forms of both proteins showed very similar CD spectra (Fig. 1a), consistent with both SODs containing comparable secondary structure composition in solution (Supplementary Table 1). We then determined three dimensional crystal structures of the iron-loaded forms of each SOD by X-ray diffraction and compared them with structures 24 of the manganese-loaded counterparts (Supplementary Table 2). All four forms adopted near-identical architectures ( Fig. 1b and Supplementary Fig. 3) whose polypeptide backbones could be overlaid with only minor deviations (Supplementary Table 3). Enzymatic activities of all variants of S. aureus MnSOD and camSOD, in each of their metalloaded forms, were assayed using a commercial SOD Activity Assay kit (Sigma). Each enzyme was assayed in triplicate using independent biological replicates, and error values quoted represent the standard deviation (SD) from the mean value given. All samples were assayed n = 3 except for three forms of the triple mutant variants that were assayed n = 4. Source data are provided as a source data file. a Values represent Units mg -1 protein ± SD. b The cambialism ratio (CR) is calculated by dividing the iron-dependent activity by the manganese-dependent activity, with a value of 1 representing a perfectly cambialistic enzyme. Enzymes that are cambialistic are defined herein as having a CR in the range 0.1-10.00. c Whereas all other samples were assayed simultaneously to circumvent noticeable day-to-day variability in the absolute activity detected using the commercial assay, the single-mutant variants with mutations at position 160 were assayed separately. To ensure comparability between the datasets, these variants were assayed simultaneously alongside samples of all wildtype forms, and small discrepancies in the absolute values were normalized by scaling using those wild-type activities as controls.
Crucially, the metal-binding ligands adopted near-identical spatial positions in all four structures (Fig. 1c), with insignificant changes to metal-ligand bond lengths or angles within the crystallographic resolution (Supplementary Tables 4 and 5). This is consistent with our previous EPR study that showed the positions of the protons on the metal-coordinated solvent and histidine ligands are identical 24 .
Identification of residues proximal to the active site. The structural similarity of the two SODs suggested that subtle differences must explain their different metal specificity. By overlaying the crystal structures of MnSOD and camSOD, all 50 amino acids that differed between the two proteins were spatially localized (Fig. 2a). Few of these were at the dimerization interface ( Supplementary Fig. 4), consistent with this region being highly conserved in SODs (Fig. 2b), and most were surface localized (Fig. 2c). However, we identified three residues (19, 159, and 160) internal to the protein that varied between MnSOD and camSOD that were spatially close (<10 Å) to the metal (Fig. 2d), where non-polar sidechains (Leu or Ile for Phe, or Gly for Leu) were swapped. Notably, these sidechains in the secondary coordination sphere made no direct contacts with the metal-coordinating sidechains in the primary coordination sphere.
Metal-specificity is determined by sequence positions 159/160. To study their role in metal specificity, we prepared variant forms  Fig. 1 Extensive structural similarity of S. aureus MnSOD and camSOD. a Overlaid CD spectra (n = 1) of the (upper panel) manganese-loaded (MnSOD in blue; camSOD in gray) and (lower panel) iron-loaded (MnSOD in gold; camSOD in teal) isoforms, demonstrating similar secondary structure content of the two isozymes in solution. b Superimposed crystal structure cartoons of (upper panel) manganese-loaded forms 24 and (lower panel) iron-loaded forms of each enzyme, with the proteins colored as per panel (a), and with manganese ions, iron ions, and waters shown as purple, orange, and red spheres, respectively. Analysis of all four structures show that the polypeptide backbones of all isoforms of the staphylococcal SODs are essentially identical to within the~2 Å resolution of the structural data (Supplementary Tables 3-5). c No significant differences within the structural resolution were detectable in the metal coordination environment that could explain the disparate metal specificity of their catalysis.  Fig. 2 Mutation of two residues located close to the active site inverts specificity. a Superimposed ribbon representation structures of the polypeptide backbones showing conserved (C) regions of the manganese-loaded forms of MnSOD (blue) and camSOD (gray), with all variable (V) residues highlighted (yellow and orange, respectively). b Analogous ribbon representation of the structure of S. aureus MnSOD, illustrating the localization of variable (cyan) and conserved (purple) regions of SOD sequence based on ConSurf analysis 59 of 500 SOD sequences (calculated conservation scores represented by the color spectrum key). Approximate dimerization interface is indicated (gray). c Superimposed space-filling models of MnSOD and camSOD, colored as in panel (a), illustrating that >85% of sidechains that vary between the S. aureus SODs are surface-exposed (2.5 Å 2 surface exposure cutoff used). d Magnified view of the superimposed cartoon representations of MnSOD and camSOD structures, colored as in panel (a), showing the spatial location of the three residues targeted for mutagenesis in proximity to the manganese ion (purple). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16478-0 ARTICLE NATURE COMMUNICATIONS | (2020) 11:2738 | https://doi.org/10.1038/s41467-020-16478-0 | www.nature.com/naturecommunications of MnSOD and camSOD in which the amino acids at the three candidate positions were reciprocally swapped. The recognition that SOD metal specificity in vitro and in vivo is better described as a spectrum than as binary possibilities highlights the need for a quantitative measure of cofactor plasticity to be used alongside absolute activity. Herein, we define the term cambialism ratio (CR) as such a measure, being the ratio of iron-dependent activity to manganese-dependent activity detectable using a spectrophotometric assay 8 ; this value is close to zero for MnSODs (CR = 0.002 for S. aureus MnSOD), close to unity for camSODs (CR = 0.996 for S. aureus camSOD), and has large values for FeSODs (CR > 800 for E. coli FeSOD 25 ).
Reciprocal mutations introduced at position 19 had negligible effects on the metal specificity of either enzyme (Table 1). Conversely, exchanging the residues at position 159 significantly impacted metal specificity. In the MnSOD, conversion of Gly159 to Leu (MnSOD Gly159Leu) increased its Fe-dependent activitỹ 10-fold relative to wild-type MnSOD while decreasing its manganese-dependent activity~2-fold (Table 1). While remaining a highly manganese-specific enzyme, this variant exhibited slightly increased cambialism (CR = 0.048). The reciprocal mutation in camSOD, converting Leu159 to Gly (camSOD Leu159Gly), diminished its iron-dependent activity >10-fold relative to wild-type camSOD while increasing its manganesedependent activity >3-fold, resulting in an enzyme variant with a MnSOD-like activity profile (CR = 0.030) ( Table 1). These results indicate that the residue at position 159 has a major effect on the ability of the S. aureus SODs to use iron and manganese, but the contribution of this residue alone was insufficient to fully explain the disparate metal specificities of the two S. aureus enzymes.
Interconversion of the residue at position 160 alone also influenced metal specificity ( Table 1). The MnSOD variant (Leu160Phe) was similar to the MnSOD Gly159Leu variant, exhibiting diminished manganese-dependent activity and increased iron-dependent activity (CR = 0.016) but to a lesser extent than in Gly159Leu, whereas the camSOD variant (Phe160Leu) showed slightly modified activity with both metals but remained largely cambialistic (CR = 0.325). Crucially, combining this mutation with that at position 159 further inverted the enzymes' metal specificity. The MnSOD double mutant (Gly159Leu-Leu160Phe) showed a substantial difference in metal utilization from that of the wild type or the singlemutant variants. The iron-dependent activity of the MnSOD double mutant was increased >20-fold relative to wild-type MnSOD, resulting in a highly cambialistic enzyme that displayed activity with manganese and with iron that differed only~2-fold (CR = 0.486). It's notable that, like in wild-type camSOD, this highly cambialistic variant exhibited diminished activity with manganese, illustrating that gaining cofactor flexibility requires a compromise with catalytic efficiency. It further demonstrates the importance of comparing both the absolute activity and the cambialism ratio when assessing metal specificity. Conversely, the camSOD double mutant (Leu159Gly-Phe160Leu) had greater activity with manganese and reduced activity with iron relative to the wild type or the single mutant (camSOD Leu159Gly). This shift in camSOD metal preference resulted in a highly manganese-specific enzyme (CR = 0.016) ( Table 1). Cumulatively, these results demonstrate that just two mutations in the secondary coordination sphere largely interconvert the metal specificity of camSOD and MnSOD.
Next, triple mutant variants, in which residues 19, 159 and 160 were all swapped, were evaluated. Unexpectedly, the MnSOD triple mutant (Phe19Ile-Gly159Leu-Leu160Phe) exhibited greater activity with manganese and less activity with iron (CR = 0.108) than the double mutant ( Table 1). The camSOD triple mutant (Ile19Phe-Leu159Gly-Phe160Leu) had increased activity with both metals relative to the double mutant (camSOD Leu159Gly-Phe160Leu). These studies demonstrate that positions 159 and 160 are important in regulating the reactivity of the metal cofactor. Strikingly, mutations at these positions produced profound effects on metal specificity, yet did so with essentially no changes to the protein backbone structure, at least within the resolution of the crystal structures ( Supplementary  Fig. 5). The physical structures of the metals' primary ligand spheres were the same, as were the substrate access channels and hydrogen-bonding networks that are important for protoncoupled electron transfer 6 .
Metal utilization correlates with the metal's electronic properties. Despite the similarity of the crystal structures, the mutations did have direct effects on the electronic structure and electrochemical properties of manganese centers. High-field electron paramagnetic resonance (HFEPR) spectra of the Mn (II)-loaded S. aureus SOD wild-type forms 24 and the six variants were distinct (Fig. 3a). These spectra are determined by the D and E values of the zero-field interactions (ZFI) of the Mn(II) centers ( Supplementary Fig. 6, Supplementary Eqs. 1-6, and Supplementary Note 1). Since the ZFI arises from the spin-orbit and magnetic interactions of the five unpaired Mn(II) electrons, this unambiguously demonstrated that the electronic structures of the metal centers were different. The sum, |D|+E, which measures the size of the Mn(II) ZFI, has been shown to correlate semiquantitatively with manganese-dependent activity, with cambialistic SODs exhibiting intermediate values between the low values of Mn(II)-loaded FeSODs and the high values of MnSODs 26 . As reported previously, the wild-type S. aureus proteins were consistent with this trend 24 , but the current measurements shed greater light. The values of |D| + E and the manganese-dependent enzymatic activities of seven of the enzyme forms (Supplementary Table 6) exhibited a significant linear correlation ( Supplementary  Fig. 7). The furthest departure from this correlation was camSOD Leu159Gly-Phe160Leu. This exception notwithstanding, the significance of this correlation can be interpreted from the results of studies on Mn(II/III)-and Fe(II/III)-containing, 4′-substituted 2,2′:6′,2′′-terpyridine complexes. These compounds are structurally homologous and exhibit metal-specific or cambialistic SODlike activity depending on the electron-donating/withdrawing strength of the 4' substituent, quantified by its Hammett σ values 27 . The reduction potentials of both the manganese and iron complexes exhibit a linear dependence on σ, as do the ZFI of the Mn(II) complexes 26 . Hence, the relationship between metalspecific activity, redox tuning through indirect secondary coordination sphere electronic effects, and Mn(II) ZFI in the enzymes is entirely consistent with the effects of subtle charge polarization in the simpler, better understood chemical systems.
Further evidence that the secondary coordination sphere residues influence the redox properties of the manganese cofactor comes from observations of their auto-oxidation properties. Mn (III) weakly absorbs in the 400-600 nm region 25,28,29 . The spectra of the two wild-type SODs in their resting state (equilibrated with ambient oxygen) had similar shapes, but the MnSOD peak intensity was greater (Fig. 3b). This demonstrated the MnSOD was more susceptible to auto-oxidation under these conditions than the camSOD, whereas the converse was true for the double mutants (Fig. 3b). These trends in auto-oxidation correlated with manganese-dependent enzyme activity (Table 1). One half reaction catalyzed by SODs is the oxidation of superoxide to molecular oxygen, and auto-oxidation is formally the reverse of this reaction (Supplementary Eq. 7). If auto-oxidation proceeds by the reverse mechanism (Supplementary Eqs. 8-12), then any changes in metal reduction potential will affect this reaction in both directions (Supplementary Note 2). There are other factors that could differentially affect the two reactions, namely substrate accessibility and proton availability. However, within the limits of the resolution of the crystal structures, we found no evidence of changes in structure that were likely to affect superoxide/ dioxygen motion to/from the metal ion or proton availability, with the positions of the residues around the active site remaining unchanged ( Supplementary Fig. 5). While our structural analysis suggested these alternative possibilities were unlikely, we also measured the susceptibility of the oxidized form of the manganese-loaded SODs to reduction by absorption spectroscopy. Each enzyme was oxidized with permanganate, and then titrated with increasing concentrations of dithionite while monitoring the disappearance of the oxidized Mn(III) species using UV/visible detection ( Supplementary Fig. 8). The camSOD was fully reduced at lower concentrations of dithionite than the MnSOD, consistent with the proposed difference in reduction potentials between the wild-type isozymes (Supplementary Eqs. [13][14][15][16][17]. Importantly, this trend was also reversed in the double mutant variants, with MnSOD G159L-L160F becoming reduced at lower concentrations of dithionite than camSOD L159G-F160L. The SOD metal site is only accessible via a narrow solvent channel, making small redox-active molecules inefficient electrochemical mediators for SODs, thus precluding direct reduction potential measurements 30 . Altogether, the spectroscopic data indicate that the two wildtype S. aureus SODs and the two double mutants differ in their manganese redox properties. The HFEPR spectra demonstrated that the electronic structure of their Mn(II) centers were significantly different. The auto-oxidation data and dithionite reduction data both suggest that the two wild-type SODs have different reduction potentials. These differences are inverted in the double mutants in both assays, which correlates with the inversion of manganese-dependent activity in these variants (Table 1). We conclude therefore that the altered metal specificity caused by the mutations is related to changes in reduction potential of the metal ions. The double mutation in MnSOD resulted in an altered electronic structure and re-tuning of the manganese reduction potential to more closely match that of camSOD and more catalytically active with iron, whereas the camSOD mutations altered reduction potential to make it more similar to MnSOD and more catalytically active with manganese.

S. aureus camSOD likely arose after a duplication of MnSOD.
A challenge in studying metalloenzyme neofunctionalization is a lack of knowledge regarding the properties of the predecessor from which it evolved. To evaluate the evolutionary relationship between the S. aureus SODs, we performed a maximum likelihood phylogenetic analysis using 2691 aligned Mn/Fe SOD sequences from bacteria, archaea and eukaryotes (Supplementary Data 1). The overall topology of the resulting phylogenetic tree was consistent with that derived from a previous analysis of a more limited sequence library 19 (Fig. 4a). Combining amino acid correlation analysis (Supplementary Data 2) with the phylogenetics ( Supplementary Fig. 9) showed that the analyzed SOD sequences could be grouped into two distinct subtypes based on the presence of highly conserved GGH or AAQ motifs, as well as a third, diverse group lacking either motif (Fig. 4a, b and Supplementary Fig. 10). We annotated known SOD metal specificities onto the tree, including only examples where activity with both manganese and iron cofactors were definitively tested, which demonstrated that the GGH subgroup contains predominantly manganese-dependent SODs 14,31 and the AAQ subgroup contains primarily iron-dependent SODs 23,25 .   MnSOD exhibits greater auto-oxidation at rest than camSOD, illustrated by increased absorbance of Mn(III) at~480 nm, a trend which is reversed in the double mutant variants that also exhibited reciprocal changes in metal specificity (Table 1). All samples (800 µM) were equilibrated with ambient aerobic conditions in 100 mM phosphate buffer, pH 7.5, 100 mM KCl, 1 mM EDTA before spectra were acquired. Spectra were collected on independent biological replicates (n = 2), mathematically converted to extinction coefficient (ε) using the Beer-Lambert law, and a representative spectrum is shown for each variant.
Nonetheless, confirmed cambialistic SODs were present in both subgroups 15,32 and a GGH-containing enzyme has been shown to be an FeSOD 33 (Fig. 4a, b), implying that the three metal specificities can and do interchange evolutionarily. Inspection of the staphylococcal SOD protein similarity network and the tree ( Supplementary Fig. 11) demonstrated that MnSOD is present in all staphylococcal genomes, whereas camSOD was identified only in the genomes of S. aureus, and of S. argenteus and S. schweitzeri, both recognized as earlybranching lineages of the S. aureus tree 34,35 . As MnSOD is the closest homolog to camSOD, the most parsimonious explanations are that the sodM gene that encodes camSOD arose from a duplication of sodA, encoding MnSOD, or that sodM was acquired by horizontal gene transfer from a close relative. Regardless, either model predicts that the newly acquired sodM would have originally encoded a manganese-specific enzyme. Increased branch length indicates an increased rate of evolutionary change for the ancestral SodM, while SodA remained under purifying selection 22 (Fig. 4c). The camSOD sequence is highly conserved across all S. aureus (99% identity), and this homology extends to the second SOD found in S. argenteus (97.5%) and S. schweitzeri (99%) (Supplementary Fig. 2). Notably Leu159 and Phe160 in camSOD are strictly conserved between S. aureus, S. schweitzeri and S. argenteus. Thus, we conclude that the acquired sodM gene originally encoded a MnSOD, which subsequently evolved into the modern camSOD during the emergence of S. aureus.
Acquisition of camSOD coincided with virulence factor expansion. To understand the wider context of camSOD emergence within the S. aureus lineage, we performed whole genome comparisons and a protein similarity network analysis (Supplementary Data 3) of genomes sampled across the staphylococcal phylogenetic tree (Supplementary Data 4). The sodA gene encoding MnSOD was located within a genomic region rich in essential genes [36][37][38] (Supplementary Data 5) that are highly conserved across the staphylococci (Fig. 5a, b). Conversely, the sodM gene encoding camSOD is located within a highly variable genomic region close to the chromosomal origin that is enriched in virulence factors 39 Fig. 4 S. aureus camSOD evolved from a manganese-specific predecessor. a Amino acid correlation (defined in the text as the AAQ group, blue, and the GGH group, magenta) and unrooted maximum likelihood phylogenetic tree based on alignment of amino acid (a.a.) sequences of 2691 Mn/Fe SOD homologs from bacteria (green circles), eukaryotes (sky blue circles), and archaea (red circles). Known metal-specificities of characterized enzymes (MnSOD = yellow, FeSOD = brown; camSOD = orange) are annotated with triangles. b Fragment of the alignment of selected SOD sequences of known metal specificity, with the metal-binding residues (red) and three residues in S. aureus camSOD (yellow) that were targeted for mutagenesis shown. Groups 1 and 2 of coevolving residues, identified using amino acid correlation analysis, were mapped onto the alignment, illustrating the GGH or AAQ motifs and associated correlated amino acids. The correlated residues, biological origin and metal specificity are indicated using the color scheme described in a, and sequence numbering is based on the S. aureus SODs. c Sub-tree extracted from the SOD tree in panel a containing all identified staphylococcal SOD sequences. Support values correspond to maximum likelihood bootstrap values from 100 rapid bootstrap replicates, with values >90 shown in green. The scale bar indicates number of substitutions per site. Macrococcus and the oxidase-positive staphylococci form an out-group to a strongly supported grouping of oxidase-negative staphylococci, including S. aureus, consistent with published Staphylococcaceae phylogenies 19,40,41 . Within the oxidasenegative staphylococci, all MnSOD (yellow) grouped together while camSOD homologs (orange) formed an out-group to the MnSODs.
these factors having been primarily acquired via horizontal transfer of mobile elements 40,41 . We screened the protein similarity networks to identify proteins with distributions similar to that of camSOD (Supplementary Data 3); proteins present in all S. aureus/S. argenteus but absent in other staphylococci, likely to have been present in their last common ancestor. We observed that the genomes that encoded camSOD possessed an expanded set of proteins involved in microbial competition, host interaction, immune evasion and virulence 40,42 (Fig. 5c and Supplementary Data 3 and 5). Some of these were unique to S. aureus/S. argenteus, whereas others were present in other staphylococci but were enriched within this clade, and included proteins involved in capsule biosynthesis (Cap), immunomodulation (Flr, Efb, Emp), secreted effectors (Esx) and toxins (Luk). A component of the Isd heme-uptake system (IsdD) was also unique to this clade (Fig. 5c). Indeed, we observed that S. aureus genomes are enriched in components that function in iron uptake via heme or siderophores 43 (Fig. 5c, Supplementary Fig. 12, and Supplementary Data 6). We conclude that the evolution of camSOD in the last common ancestor of S. aureus/S. argenteus coincided with the expansion of their repertoire of virulence genes 40,41 , and that its genomic context are consistent with its established role in pathogenicity 8 .
A single mutation alters resistance to host-derived stresses. The camSOD is a virulence factor that enables S. aureus to resist oxidative stress during infection when starved of manganese by the host 8 . Our analysis suggested that camSOD likely evolved from a manganese-specific predecessor, implying that evolution subsequently adapted the manganese-specific precursor, resulting in the extant cambialistic enzyme. We thus aimed to leverage our ability to produce camSOD enzymes with altered metal specificity to test whether the camSOD's iron-dependent catalysis directly contributes to S. aureus overcoming the host immune response, and if small changes in iron-dependent activity are sufficient to provide a functional advantage in the presence of natural stressors. A single mutation was introduced into the S. aureus chromosome, converting the wild-type camSOD to its Leu159Gly variant, which shows reduced ability to utilize iron (Table 1). This strain was evaluated, alongside wild-type S. aureus and a ΔsodM mutant that lacks camSOD entirely, for its ability to resist conditions representative of dual host-imposed stresses, manganese starvation and oxidative stress. Manganese starvation was imposed using the manganese-binding immune effector calprotectin, which can accumulate at sites of infection 8,16,17 at concentrations in excess of 1 mg ml -1 , while superoxide was generated by addition of paraquat 8,17 . Consistent with previous studies 8, 17 , the strain lacking camSOD was more sensitive to these stresses than wild type (Fig. 6a). The strain carrying the Leu159Gly variant camSOD enzyme was also more sensitive to these stresses (Fig. 6a), and its SOD activity showed altered susceptibility to inhibition by peroxide (Fig. 6b, c)    ability of camSOD to perform iron-dependent catalysis enables S. aureus to resist oxidative stress during physiological manganese deprivation. Collectively, our data are consistent with a model in which the evolution of camSOD was driven by interaction of the ancestor of S. aureus with the host immune system (Fig. 7). Acquisition of a redundant gene encoding a second manganese-specific SOD was followed by a period of rapid evolution of the protein sequence, which conferred the ability to utilize iron for catalysis. This period coincided with the expansion of the organism's repertoire of virulence-associated proteins, enabling S. aureus to survive immune assault and establish infection better than less pathogenic relatives 40 . We thus propose that the selection pressure that drove the neofunctionalization of camSOD from a manganesespecific precursor after acquisition involved the combined immune-mediated stresses of the oxidative burst and manganese starvation 7,8,16,17 .

Discussion
Metalloproteins are ubiquitous in biology, thus understanding the principles that govern metal cofactor utilization has implications for diverse aspects of biology, medicine, and biotechnology. However, the molecular factors that dictate the metal cofactor utilized by metalloenzymes, and how iterative changes to the protein sequence over time result in a change in metal specificity, remain poorly understood. Our inability to accurately predict the metal utilized by the Mn/Fe SODs, a widely distributed family of metalloenzymes, exemplifies these deficiencies. In this study, we developed the pair of S. aureus SODs as a powerful model system with which to interrogate how metal specificity can change over evolutionary time, enabling future studies to determine how the architecture of a metalloenzyme imposes cofactor specificity. Exploiting this platform demonstrated that evolution can effect substantial changes in metal specificity by making modest changes in the chemical nature of secondary coordination sphere residues that regulate metal reactivity.
Evolutionary distance between SODs with differing metal specificity has limited our ability to elucidate which residues dictate metal utilization 6,12,26,29 . Leveraging the extensive structural and sequence homology between the staphylococcal SODs identified two key residues that dictate metal-specificity. Remarkably, the chemical differences between the sidechains of these residues are minor, with Gly (MnSOD) and Leu (camSOD) at position 159, and Leu (MnSOD) and Phe (camSOD) at position 160. While both residues are close to the active site, neither sidechain makes direct contact with the metal or its coordinating ligands, nor do they affect the overall architecture of the active site. Despite this, HFEPR and UV/visible spectroscopy revealed that interconversion of these residues profoundly alters the electronic structure and redox properties of the manganese relative to the wild-type enzymes 24 . These data confirmed that MnSOD is more susceptible to auto-oxidation than camSOD, and crucially, this difference was inverted in the variants that also exhibited inverted metal specificities. These results support a model in which the SOD architecture selectively tunes the cofactor's reduction potential 6,28,44 , and identify positions 159 and 160 as playing a critical role in this electronic modulation. Broadly, these results highlight the important contribution of secondary coordination sphere residues in determining metal specificity. Further, they illustrate that even minor secondary sphere changes can have profound impacts on metal-specificity, which is relevant to the study of natural metalloproteins, and the de novo design of synthetic metalloenzymes [45][46][47][48][49] .
The residue equivalent to Gly159 in S. aureus MnSOD was reported as being generally Gly in both manganese-dependent   (Table 1) renders S. aureus less capable of overcoming the host immune response. b, c In-gel SOD activity assays using extracts (5.4 μg) prepared from b wild type S. aureus and c the strain expressing the Leu159Gly camSOD variant, cultured under identical conditions to those used in a, demonstrate that both forms of the SodM enzyme are detectable at similar levels (upper panels). Treatment of the extracts (left) or recombinant enzymes (right) with H 2 O 2 (lower panels), a specific inhibitor of the iron-loaded form 8 , demonstrated that the camSOD was loaded with iron in wild-type cells. The MnSOD enzyme is labeled as SodA, camSOD as SodM. This is a representative gel from a triplicate analysis of independent biological replicates (n = 3). Note that molecular weight markers were not used in these native gels, with SOD bands identified in the cell extracts by comparison of their mobility with the purified recombinant proteins. Source data for all panels are provided as a source data file. and cambialistic SODs, but Thr in iron-dependent SODs 12,29,50 . Introduction of Thr in this position in manganese-specific or cambialistic SODs increased the cambialism ratio 12,29 . This was previously explained by modification of a hydrogen-bonding network involving the mechanistically important metalcoordinated solvent 29 . Our data confirm that a Gly residue in this position strongly favors manganese-specificity, but show that a small aliphatic sidechain at this position confers cambialism when combined with an aromatic sidechain on the neighboring residue at position 160. Importantly, our structural analyses of the staphylococcal SODs and their variants did not identify changes to the hydrogen-bonding network or substrate access channel, and 1 H electron nuclear double resonance (ENDOR) spectroscopy demonstrated the positions of the solvent protons are unchanged between MnSOD and camSOD 24 , and likely also in the mutants. These observations cast doubt on the hydrogenbonding network explanation in our system, and highlight the potential of the staphylococcal SODs as a model system with which to uncover the mechanisms by which redox tuning is controlled.
Our data suggest that camSOD evolved from a MnSOD predecessor in the last common ancestor of S. aureus/S. argenteus/S. schweitzeri. The acquisition and subsequent altered metal utilization of camSOD coincided with the acquisition of numerous other factors that contribute to infection 8,39,41,42,51 . We exploited our ability to produce manganese-specific camSOD variants to determine whether its iron-dependent catalytic activity provides a direct benefit to S. aureus during exposure to stresses encountered within the host. A S. aureus strain expressing the Leu159Gly variant camSOD showed reduced growth under manganese-restricted and oxidative stress-exposed conditions relative to wild type. As this variant possesses reduced irondependent activity but increased catalysis with manganese, this result clearly demonstrates that S. aureus growth under these conditions requires iron-dependent camSOD activity. We previously showed that camSOD is more important than MnSOD to the establishment of infection of wild-type mice 8 . Collectively, these data support a model in which the manganese-specific camSOD antecedent, acquired by the last common ancestor of the extant S. aureus clade 41 , underwent neofunctionalization through selection for increased iron-dependent activity, a pressure likely imposed by the manganese starvation it experiences during infection.
This study presents experimental evidence of how a change in metal specificity has naturally evolved in a metalloenzyme. We provide evidence that the specificity switch was selected for by  Fig. 7 Evolution of camSOD was driven by altered metal availability in the host. Schematic model depicting the evolution of S. aureus camSOD (orange) under selection pressure caused by exposure of the S. aureus ancestor to host-imposed manganese-starvation mediated by calprotectin. Acquisition and evolution of camSOD enabled the ancestor of S. aureus to maintain its antioxidant defense under these conditions using its iron-dependent catalysis, and coincided with its acquisition of numerous genes (green) involved in infection. Collectively, these acquired systems resulted in increased survival of S. aureus during interaction with host immune mechanisms, creating pathogenic S. aureus. nutrient-starvation imposed by the immune system, implying that metal availability can shape enzyme metal utilization 18,52 .
Our data also demonstrate a critical role for secondary coordination sphere residues in mediating this specificity switch, with wider implications for the study of natural and synthetic metalloenzymes.
CD spectroscopy. Circular dichroism data were collected using a JASCO J-810 spectropolarimeter equipped with a PTC-4235 Peltier temperature controller. Spectral measurements were performed within the far-UV range (185-260 nm) at a protein concentration of 0.8 μM in 10 mM sodium phosphate buffer, pH 7.0 at 20°C, in a 1 mm path length quartz cuvette (Hellma), using a scan rate of 50 nm min -1 , 2 s response time, 0.2 nm data pitch and a 2 nm bandwidth. For each sample, five accumulated scans were averaged, corrected for the buffer reference, and then converted into mean residue ellipticity (MRE, θ). Deconvolution was performed using the CDSSTR program with reference dataset 4 (250-190 nm) on the DichroWeb server 56,57 . The thermal denaturation of protein was performed by following the change in ellipticity at a fixed wavelength of 222 nm as a function of temperature increasing from 20 to 90°C. Protein melting temperature (T m ) values were calculated from the first derivative of the thermal denaturation curves, converted to mean residue ellipticity (MRE 222 , θ 222 ).
UV/visible spectroscopy. UV/visible spectroscopy was performed on a Perkin-Elmer λ35 spectrophotometer, collecting data at room temperature between 200 and 800 nm using 10 mm path length quartz cuvettes. Measurements of resting spectra were performed in 100 mM potassium phosphate buffer, pH 7.5, 100 mM KCl, 1 mM EDTA, with samples prepared at~800 µM protein. Spectra were converted to extinction coefficient, ε, using the protein concentration corrected for manganese-loading, as detected by inductively coupled plasma mass spectrometry (ICP-MS). For reductive titrations, each sample was fully oxidized by incubation with 1 mole equivalent of potassium permanganate for 10 min, followed by extensive buffer exchange using centrifugal filtration devices (Amicon, 10 kDa cutoff) to remove the oxidant in the same buffer. Samples were titrated by addition of small volumes of fresh solutions of concentrated sodium dithionite, mixed vigorously by pipette and incubated for 20 min prior to acquisition of each spectrum. Spectra were corrected using the manganese content as detected by ICP-MS, and normalized to allow comparison between titrations.
Electron paramagnetic resonance (EPR) spectroscopy. The 94 GHz EPR spectra were obtained at 5-6 K using a Bruker Elexsys II 680 EPR spectrometer equipped with a "power upgrade 2" and an Oxford Instruments CF935 flow cryostat. The spectra were obtained by measuring the integrated amplitude of a standard two-pulse Hahn echo (t p (π) = 24 ns and interpulse time of 400 ns) as a function of the magnetic-field. The zero-field D and E values were determined using a modified version of the method described in our previous work 24 , because the shapes of the low-field edges of the 94 GHz spectra were different (Supplementary Note 1). To remove any arbitrariness in locating the magnetic-field of the low-field edge, the spectra were numerically differentiated and the lowest-field inflection points were taken as the edge positions. This had the effect of taking into account the distribution in the D values, which produced the different edge shapes, but also overestimated their values by 100 to 200 MHz in comparison to the previously reported values. The same approach was used for the high-field edges (E) as well.
ICP-MS elemental analysis. Metal analysis was mostly performed by ICP-MS using a Thermo x-series instrument and Thermo's PlasmaLab software in Newcastle, operating in collision cell mode as previously described 8 . For Supplementary  Fig. 9, ICP-MS analysis was performed by Durham University Bio-ICP-MS Facility. Briefly, protein samples were diluted in 2.5 % (w/v) solution of HNO 3 (Merck) containing 20 μg L -1 Co and Pt or In and Pt (Newcastle), or 10 μg L -1 Ga and Bi (Durham) as internal standards. Matrix-matched elemental standards (containing analyte metal concentrations of 0-500 μg L -1 ) were analyzed alongside each set of samples, each in triplicate, to enable metal concentrations to be determined from standard curves.
X-ray crystallography. Preparations of the iron-loaded forms of recombinant MnSOD and camSOD, and the manganese-loaded forms of both double mutant variants and of the triple mutant of camSOD (15 mg ml -1 ), were each subjected to crystallization screening and optimization as previously described 24  SOD activity assays. SOD activity was assessed qualitatively in-gel, or quantitatively using a SOD Assay Kit (Sigma-Aldrich) per the manufacturer's instructions, as previously described 8 . S. aureus cell extracts were prepared by mechanical disruption in 50 mM phosphate buffer at pH 7.8 with 0.1 mM EDTA. Protein content of lysates was quantified by bicinchoninic acid assay (Sigma). Aliquots of crude extracts (5.4 μg) or purified recombinant proteins (0.3 μg) were resolved on 10% (w/v) native polyacrylamide gels. For staining, gels were incubated in 50 mM potassium phosphate buffer pH 7.8, 1 mM EDTA, 0.25 mM nitroblue tetrazolium chloride (Sigma), 50 μM riboflavin and then exposed to light to detect SOD activity. Incubation of replica gels in 20 mM H 2 O 2 prior to activity staining was performed to assess Fe-dependent SOD activity. Note that, as the native gels used for assays were stained with a SOD-specific stain, no molecular weight markers were resolved on these gels.
Calprotectin growth assays. Assays of S. aureus growth in the presence of calprotectin and paraquat were performed as previously described 8  SOD phylogenetic analysis. SOD sequences were identified using BLASTP search 60 (E-value threshold = 0.005), with S. aureus MnSOD sequence as query, against a database of protein sequences downloaded from the NCBI server encoded in 2577 bacterial (including Candidate Phyla Radiation 61,62 ), 289 archaeal (including ASGARD Archaea 63,64 ), and 148 eukaryotic genomes. All 2691 identified SOD homologs were aligned using MAFFT 65 and trimmed with trimAl 66 (gappyout mode) resulting in a trimmed alignment of 208 positions (Supplementary Data 1). The best fitting phylogenetic model (WAG + G) was selected with PROTTEST 67 , and used to generate a SOD phylogeny in RAXML 68 with 100 bootstrap replicates. The tree was visualized and annotated with the coevolving amino acid residues ( Fig. 4 and Supplementary Fig. 9) using GraPhlAn 69 (Supplementary Data 1 and 2).
Amino acid correlation analysis. Groups of coevolving amino acids were identified (Supplementary Data 2) in the alignment of the 2,691 SOD sequences sampled across the tree of life (Supplementary Data 1), using amino acid correlation algorithm implemented in PFstats 70 following the user guide. PFstat correlation analyses parameters were as follows: Minimum Score: 60 (18.75% of the maximum positive correlation score of 320); MRsA fraction: 0.2; minimum delta: 0.3. The amino acid correlation network was visualized in Cytoscape 71 (Supplementary Fig. 9).

Protein network analyses. Protein similarity networks (Supplementary Data 3)
were generated out of 71,757 protein sequences encoded in 29 analyzed Staphylococcal genomes (downloaded from the NCBI server) with BLAST 60 , and quick edge file optimization implemented in EGN 72 using the following thresholds: BLAST (a = 12, e = 1e -05 ), simple link parameter (E-value threshold = 1e -05 , hit identity threshold = 20%, minimum hit coverage = 20%, without the best reciprocal hit condition enforcement), and with the quick edge file creation (E-value threshold = 1e -05 , hit identity threshold = 20%, minimum hit coverage = 20%, minimal hit length = 30 amino acids). Protein sequences were annotated 73 using eggNOG-mapper v1. The generated networks were analyzed using igraph 74 HKUOPL8). The Identified in all networks were defined as those networks containing only a single homolog in each of the analyzed genomes; not identified in S. aureus networks were defined as networks not containing protein homologs from any of the camSOD-positive genomes, and containing homologs from at least 11 out of 22 camSOD-negative species; S. aureus-specific networks were defined as networks containing protein homologs from 7 out of 7 genomes from camSOD-positive species, and none from the camSOD-negative species. To be defined in the group labeled as Enriched in S. aureus, each camSOD-positive species had to contain more protein homologs than any of the camSOD-negative species within the network. Heatmaps of the identified protein networks ( Fig. 5 and Supplementary Fig. 12) were generated using gplots package (https://CRAN.R-project.org/package=gplots) within R. Protein network analyses of metal acquisition systems. Networks containing homologs of proteins known to be involved in manganese and iron import were isolated using igraph 74 and dnet 75 packages in R 76 , and visualized in Cytoscape 77 ( Supplementary Fig. 12). Sequence of the proteins present in the networks were aligned using MAFFT 65 , and the alignments were trimmed with trimAl 66 and used to generate phylogenies under LG + G model in FastTree 86 . Homologs of the proteins of interest were identified based on the network and tree topologies, together with manual inspection of protein sequence alignments, and used to generate the heatmap with gplots package.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.