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Interchangeable utilization of metals: New perspectives on the impacts of metal ions employed in ancient and extant biomolecules

Open AccessPublished:October 31, 2021DOI:https://doi.org/10.1016/j.jbc.2021.101374
      Metal ions provide considerable functionality across biological systems, and their utilization within biomolecules has adapted through changes in the chemical environment to maintain the activity they facilitate. While ancient earth's atmosphere was rich in iron and manganese and low in oxygen, periods of atmospheric oxygenation significantly altered the availability of certain metal ions, resulting in ion replacement within biomolecules. This adaptation mechanism has given rise to the phenomenon of metal cofactor interchangeability, whereby contemporary proteins and nucleic acids interact with multiple metal ions interchangeably, with different coordinated metals influencing biological activity, stability, and toxic potential. The ability of extant organisms to adapt to fluctuating metal availability remains relevant in a number of crucial biomolecules, including the superoxide dismutases of the antioxidant defense systems and ribonucleotide reductases. These well-studied and ancient enzymes illustrate the potential for metal interchangeability and adaptive utilization. More recently, the ribosome has also been demonstrated to exhibit interchangeable interactions with metal ions with impacts on function, stability, and stress adaptation. Using these and other examples, here we review the biological significance of interchangeable metal ions from a new angle that combines both biochemical and evolutionary viewpoints. The geochemical pressures and chemical properties that underlie biological metal utilization are discussed in the context of their impact on modern disease states and treatments.

      Keywords

      Abbreviations:

      cam-SOD (cambialistic SOD), Co (cobalt), COVID-19 (coronavirus disease 2019), Cu (copper), Fe (iron), Fe–S (iron–sulfur), Fe-SOD (Fe-containing SOD), H2O2 (hydrogen peroxide), ISCU (Iron–Sulfur Cluster), MCO (metal-catalyzed oxidation), MCT (metal chelation therapy), Mg (magnesium), Mn (manganese), Mn-SOD (manganese-containing SOD), Ni (nickel), O2•− (superoxide), •OH (hydroxyl radical), RdRp (RNA-dependent RNA polymerase), RNR (ribonucleotide reductase), ROS (reactive oxygen species), Rpe (ribulose-5-phosphate 3-epimerase), SARS-CoV2 (severe acute respiratory syndrome coronavirus 2), SOD (superoxide dismutase), ZF (zinc finger), Zn (zinc)
      The unique chemical properties of numerous metal ions facilitate extensive interactions with biomolecules, with impacts across all areas of cellular activity, including fundamental processes, such as respiration, metabolism, nitrogen fixation, photosynthesis, DNA replication, transcription, and protein synthesis (
      • Bellenger J.P.
      • Wichard T.
      • Xu Y.
      • Kraepiel A.M.L.
      Essential metals for nitrogen fixation in a free-living N2-fixing bacterium: Chelation, homeostasis and high use efficiency.
      ,
      • Garcia-Diaz M.
      • Bebenek K.
      • Krahn J.M.
      • Pedersen L.C.
      • Kunkel T.A.
      Role of the catalytic metal during polymerization by DNA polymerase lambda.
      ,
      • Guth-Metzler R.
      • Bray M.S.
      • Frenkel-Pinter M.
      • Suttapitugsakul S.
      • Montllor-Albalate C.
      • Bowman J.C.
      • Wu R.
      • Reddi A.R.
      • Okafor C.D.
      • Glass J.B.
      • Williams L.D.
      Cutting in-line with iron: Ribosomal function and non-oxidative RNA cleavage.
      ,
      • Klein D.J.
      The contribution of metal ions to the structural stability of the large ribosomal subunit.
      ,
      • Li L.
      • Yang X.
      The essential element manganese, oxidative stress, and metabolic diseases: Links and interactions.
      ,
      • Steffens G.C.M.
      • Soulimane T.
      • Wolff G.
      • Buse G.
      Stoichiometry and redox behaviour of metals in cytochrome-c oxidase.
      ,
      • Thiele D.J.
      Metal-regulated transcription in eukaryotes.
      ,
      • Yruela I.
      Transition metals in plant photosynthesis.
      ). At least ten metal elements are considered essential for most forms of life (
      • Maret W.
      The metals in the biological periodic system of the elements: Concepts and conjectures.
      ), including six of the d-block elements of the periodic table: manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), and zinc (Zn) ((
      • Crans D.C.
      • Kostenkova K.
      Open questions on the biological roles of first-row transition metals.
      ) and Fig. 1A). These metals are characterized by the ability to form ions with partially filled d-subshells (shown in Fig. 1A, blue). This electron configuration facilitates multiple oxidation states, defining many of their chemical properties. These metals are called transition metals, with the most biologically relevant examples appearing in the first row of the d-block in the periodic table (Fig. 1A, dark cyan squares). Although biologically important, Zn is excluded from the transition metals by some definitions because of possession of a complete d-subshell (Fig. 1A).
      Figure thumbnail gr1
      Figure 1Transition metals and oxidative stress. A, transition metals. A section of the periodic table showing s-block (orange), d-block (cyan), and p-block (green) chemical elements. Transition metals of the first row, characterized by partially filled d-subshells, are shown in dark cyan squares in bolded black lettering. Zn (which possesses a complete d-subshell) is also shown in this group. The electronic structures of the d-block elements are shown in blue lettering. B, reactions of the superoxide anion; iron and redox cycling. Negatively charged free radical superoxide (O2•−, shown in red lettering) is the product of one-electron (e) reduction of dioxygen (O2). Upon protonation, O2•− can form the hydroperoxyl radical (HO2). Superoxide dismutase (SOD, cyan oval) catalyzes the dismutation (disproportionation) of O2•−, thereby generating O2 and hydrogen peroxide (H2O2). H2O2 is converted to H2O by various antioxidant enzymes, such as catalases (CAT), glutathione peroxidases (GPX), and peroxiredoxins (PRX). Redox-active Fe2+ ions are oxidized by H2O2, generating highly reactive hydroxyl radicals (OH) and Fe3+ through the Fenton reaction. Fe3+ can be reduced to Fe2+ by O2•−, resulting in redox cycling (purple arrows). By itself, O2•− can reduce Fe3+ to Fe2+ within iron–sulfur cluster proteins, resulting in enzyme inactivation and accumulation of Fe2+, which further powers Fenton chemistry. Modified from Ref. (
      • Wang Y.
      • Branicky R.
      • Noë A.
      • Hekimi S.
      Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS signaling.
      ).
      The human body contains amounts of magnesium (Mg), Fe, and Zn in the gram range, whereas milligram amounts of Mn, Cu, Co, and molybdenum are present (
      • Maret W.
      The metals in the biological periodic system of the elements: Concepts and conjectures.
      ,
      • Zoroddu M.A.
      • Aaseth J.
      • Crisponi G.
      • Medici S.
      • Peana M.
      • Nurchi V.M.
      The essential metals for humans: A brief overview.
      ). These metals perform both catalytic and structure-stabilizing roles and are predominantly available as divalent cations (possessing two fewer electrons than the neutral state). In fact, approximately 40% of enzymes with known structures depend upon at least one metal cofactor for catalytic activity (
      • Andreini C.
      • Bertini I.
      • Cavallaro G.
      • Holliday G.L.
      • Thornton J.M.
      Metal ions in biological catalysis: From enzyme databases to general principles.
      ). Analysis of metal-binding domains in the proteome suggests that metal-mediated folds are proportional to proteome size across the kingdoms of life, whereas the specific metals predicted to be utilized reveal distinct changes through evolutionary history (
      • Dupont C.L.
      • Butcher A.
      • Valas R.E.
      • Bourne P.E.
      • Caetano-Anollés G.
      History of biological metal utilization inferred through phylogenomic analysis of protein structures.
      ). The utilization of metal ions for life on earth may predate protein-oriented extant biology, as nucleic acids in a metal-rich prebiotic environment are hypothesized to generate the earliest enzymatic mechanisms. Much as in proteins, metal ions are employed as catalytic cofactors in RNA species (
      • Freisinger E.
      • Sigel R.K.O.
      From nucleotides to ribozymes-a comparison of their metal ion binding properties.
      ) and coordinated by both the anionic sugar phosphate backbone and the nucleotide bases (
      • Klein D.J.
      The contribution of metal ions to the structural stability of the large ribosomal subunit.
      ,
      • Freisinger E.
      • Sigel R.K.O.
      From nucleotides to ribozymes-a comparison of their metal ion binding properties.
      ). Metal ions similarly interact with the DNA backbone and bases (
      • Morris D.L.
      DNA-bound metal ions: Recent developments.
      ). The high charge density of the metal ions allows large RNAs to form complexes and closely packed folds and tertiary interactions, facilitating elaborate and dynamic structures, such as the ribosome—the essential protein-synthesizing machine that operates in every living cell. Transition metal ions bind throughout rRNA both loosely and at specific sites, with Mg being the major metal ion contributing to present structures of large and small ribosomal subunits (
      • Klein D.J.
      The contribution of metal ions to the structural stability of the large ribosomal subunit.
      ,
      • Freisinger E.
      • Sigel R.K.O.
      From nucleotides to ribozymes-a comparison of their metal ion binding properties.
      ,
      • Sigel R.K.O.
      • Vaidya A.
      • Pyle A.M.
      Metal ion binding sites in a group II intron core.
      ).
      Despite an effort, drawing definitive conclusions about the physiological utilization of metal ions by biomolecules within cells is a technically challenging task, hindered by several experimental issues. These include the dissociation of ions during biomolecule purification, physical properties that limit detection in structural models, and broader impacts of overexpressing metal-binding species on metal availability (
      • Högbom M.
      Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear - an intricate bioinorganic workaround to use different metals for the same reaction.
      ). Further complications in the elucidation of metal ions usage for biomolecules functionality arise from the phenomenon of metal ion interchangeability, wherein one of several different ions are able to occupy a specific biomolecular binding site. Metal ions, in many cases, self-assemble into complexes with biomolecules (
      • Griese J.J.
      • Roos K.
      • Cox N.
      • Shafaat H.S.
      • Branca R.M.M.
      • Lehtiö J.
      • Gräslund A.
      • Lubitz W.
      • Siegbahn P.E.M.
      • Högbom M.
      Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein.
      ) and, as such, can be greatly influenced by intracellular metal availability dictated by a particular physiological or environmental condition. Therefore, the flexibility in metal ion preference may be more prevalent than currently understood. Thus, the view of a single native metal for a given binding site may, at least in some cases, be an unfavorably strict categorization. In a healthy cell, cytosolic and organellar metal cation levels are tightly regulated as a means of protection against the undesired activity of certain metal elements, while metal imbalances manifest in numerous human diseased states. This identified link between the ability of certain biomolecules to interact with a non-native metal ion(s), which may abolish their correct functionality leading to mitigation of disease, supports the need for further knowledge of the molecular mechanisms governing metal ion interchangeability. Such investigation will advance our understanding of disease etiology and progression, with a potential for new therapeutical intervention strategies.
      Particular progress in revealing transition metal interchangeability in biomolecular structure and function has been made recently because of new technological developments. An emerging example of particular interest is the utilization of metals by the ribosome. While the high Mg content of contemporary ribosomes supports a preference of the metal-binding sites for Mg2+, a recently published study that replicated a prebiotic environment of anoxic earth rich in Fe and Mn and low in oxygen revealed that Mg2+ on ribosomes can be replaced by Fe2+ or Mn2+ without affecting protein-synthesizing activity (
      • Athavale S.S.
      • Petrov A.S.
      • Hsiao C.
      • Watkins D.
      • Prickett C.D.
      • Gossett J.J.
      • Lie L.
      • Bowman J.C.
      • O'Neill E.
      • Bernier C.R.
      • Hud N.V.
      • Wartell R.M.
      • Harvey S.C.
      • Williams L.D.
      RNA folding and catalysis mediated by iron (II).
      ,
      • Bray M.S.
      • Lenz T.K.
      • Haynes J.W.
      • Bowman J.C.
      • Petrov A.S.
      • Reddi A.R.
      • Hud N.V.
      • Williams L.D.
      • Glass J.B.
      Multiple prebiotic metals mediate translation.
      ). Besides an important impact on new biochemical features of a ribosome and medicine-related translational science, this technically advanced approach provided the scientific community with an elaborate biological model crucial for investigating the origin of life and evolution of biological molecules. In fact, the recapitulated conditions mimicking the anoxic earth environment supported a hypothesis that a ribosome represents an extraordinarily well-conserved RNA–protein structure that existed in a complex with Fe ions when earth's atmosphere was depleted of oxygen (
      • Guth-Metzler R.
      • Bray M.S.
      • Frenkel-Pinter M.
      • Suttapitugsakul S.
      • Montllor-Albalate C.
      • Bowman J.C.
      • Wu R.
      • Reddi A.R.
      • Okafor C.D.
      • Glass J.B.
      • Williams L.D.
      Cutting in-line with iron: Ribosomal function and non-oxidative RNA cleavage.
      ,
      • Bray M.S.
      • Lenz T.K.
      • Haynes J.W.
      • Bowman J.C.
      • Petrov A.S.
      • Reddi A.R.
      • Hud N.V.
      • Williams L.D.
      • Glass J.B.
      Multiple prebiotic metals mediate translation.
      ). These studies were corroborated with biochemical assays conducted with ribosomes from Saccharomyces cerevisiae, wherein it was demonstrated that eukaryotic ribosomes maintained an ability to interact with Fe2+ at the selected sites under normal physiological conditions. This suggests a possibility that this biological atavism might play an essential role in the regulation of protein synthesis, the faultless processivity of which contributes to protein homeostasis and protection against neurodegenerative diseases (
      • Zinskie J.A.
      • Ghosh A.
      • Trainor B.M.
      • Shedlovskiy D.
      • Pestov D.G.
      • Shcherbik N.
      Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.
      ). These recent developments in understanding ribosome biology in the context of transition metal interchangeability open up many questions requiring further investigation.
      Flexibility in metal ion interactions with biomolecules is not only limited to ribosomes but also has been documented to be prevalent in metalloproteins. This is commonly observed in in vitro assays, wherein enzyme activity is assessed in the presence of different metals to identify which confers the greatest catalytic activity. In addition, certain metal ions, including Mg, are not directly detectable in spectroscopic studies and difficult to identify in crystallographic studies (
      • Maguire M.E.
      • Cowan J.A.
      Magnesium chemistry and biochemistry.
      • Weston J.
      Biochemistry of magnesium.
      ) and can be readily substituted with alternative ions for the purposes of structural assessment (
      • Bock C.W.
      • Katz A.K.
      • Markham G.D.
      • Glusker J.P.
      Manganese as a replacement for magnesium and zinc: Functional comparison of the divalent ions.
      ,
      • Cowan J.A.
      Metal activation of enzymes in nucleic acid biochemistry.
      ), further indicating the relative ease with which certain ions can replace one another.
      Large-scale environmental changes accompanied with the accumulation of molecular oxygen in ancient earth's atmosphere, the groundbreaking event that occurred in the course of evolution dated billions of years ago, drove adaptation of biomolecules by selecting organisms that had the capability to defend against highly reactive chemical products derived from the incomplete reduction of oxygen (known as reactive oxygen species [ROS]) and utilize alternative metals to catalyze crucial biochemical reactions. One of the prominent examples of how these pressures are mirrored in extant biology is the host–pathogen interface. The innate immune system orchestrates challenging chemical assaults upon invading pathogens that in many cases display flexibility in metal utilization in their responses. For example, the connection between metal biochemistry and oxidative stress is central to the phagocytic immune response, which employs both oxidant assault and nutrient metal limitation in the defense against pathogens (
      • Jakubovics N.S.
      An ion for an iron: Streptococcal metal homeostasis under oxidative stress.
      ). Many pathogens are sensitive to metal levels on either side of a relatively narrow window, and host systems exploit this (
      • Hood M.I.
      • Skaar E.P.
      Nutritional immunity: Transition metals at the pathogen-host interface.
      ). During infection of a host organism, bacterial pathogens will commonly experience challenging conditions, including both overload and limitation of trace metal elements, as well as severe oxidative stress (
      • Imlay J.A.
      Where in the world do bacteria experience oxidative stress?.
      ,
      • Sultana S.
      • Foti A.
      • Dahl J.U.
      Bacterial defense systems against the neutrophilic oxidant hypochlorous acid.
      ,
      • Turner A.G.
      • Djoko K.Y.
      • Ong C.
      • lynn Y.
      • Barnett T.C.
      • Walker M.J.
      • McEwan A.G.
      Group A Streptococcus co-ordinates manganese import and iron efflux in response to hydrogen peroxide stress.
      ).
      Transition metals are also known to play an important role in several viruses' survival and pathogenesis. Relevant to the present time, much of the research has been conducted investigating the role of Fe and other transition metals in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)–related pathologies. Although many questions remain unanswered, it is clear that metals play an important role during SARS-CoV2 infection and propagation, whereas multiple manifestations of coronavirus disease 2019 (COVID-19), such as immune dysfunction, inflammation, hypercoagulation, hyperferritinemia, have been linked to Fe overload (
      • Habib H.M.
      • Ibrahim S.
      • Zaim A.
      • Ibrahim W.H.
      The role of iron in the pathogenesis of COVID-19 and possible treatment with lactoferrin and other iron chelators.
      ).
      The possible ambiguity in assignments of metal ion associations with biomolecules, along with emerging examples of physiologically relevant metal interchangeability, indicates an existing gap in knowledge with an impact on diverse biological topics briefly mentioned previously. To gain an up-to-date picture of the transition metal interchangeability phenomenon, here, we discuss the current progress that has been made.
      As such, we outline the deeply embedded nature of divalent metal cations in biological macromolecules and place this in the context of the risk of oxidative damage, which is present in an oxygen-rich environment. The geochemical changes that occurred on earth since the establishment of life are then discussed as they relate to metal availability and utilization. As some of the best-studied examples that have had recent developments in understanding metal interchangeability, we describe the role of metal ions in the superoxide dismutases (SODs), the R2 subunit of the ribonucleotide reductases (RNRs), and the ribosome. We choose these biomolecule examples as they remain in the frontline of scientific research, providing new information on how metals can replace each other to meet various physiological cues. Finally, we describe how flexible metal utilization impacts both bacterial and viral infections and immunity. While we focus on Fe and Mn as the most prominent examples of physiological metal cofactors, other biologically important metals are also discussed.

      Metal ions provide powerful biochemical functionality to biomolecules

      As stated previously, ∼40% of biomolecules utilize metals as cofactors, suggesting that metal ions are essential for cellular physiology, with the first-row transition metals being of particular importance (Fig. 1A). For their interactions with biomolecules, metal ions can be considered in terms of several properties, including charge density, radii, and reactivity. Redox activity is also of particular relevance to biology because it prescribes some catalytic capabilities. Redox inactive metal cations, of which Mg and Zn are the most common in biomolecules, tend to be utilized in structures to stabilize negative charges, as well as functioning as Lewis acids to activate substrates by accepting lone pair electrons with no net change in oxidation state (
      • Andreini C.
      • Bertini I.
      • Cavallaro G.
      • Holliday G.L.
      • Thornton J.M.
      Metal ions in biological catalysis: From enzyme databases to general principles.
      ,
      • Valasatava Y.
      • Rosato A.
      • Furnham N.
      • Thornton J.M.
      • Andreini C.
      To what extent do structural changes in catalytic metal sites affect enzyme function?.
      ,
      • Valdez C.E.
      • Smith Q.A.
      • Nechay M.R.
      • Alexandrova A.N.
      Mysteries of metals in metalloenzymes.
      ). Many of the transition metals are redox active, such as Fe, Mn, Cu, and Ni, as electrons of their incomplete d-subshells (Fig. 1A) can be lost, allowing for several oxidation states. While such cations can act as Lewis acids, they are commonly used in the catalysis of redox reactions, in which electron transfer results in a change of oxidation state.
      Both the intracellular availability and the stability of formed complexes are important factors in metal cofactor binding. The predicted complex stability of divalent metal cations is described by the Irving–Williams series (Mg2+ < Mn2+ < Fe2+ < Ni2+ < Co2+ < Cu2+ > Zn2+) (
      • Miličević A.
      • Branica G.
      • Raos N.
      Irving-Williams order in the framework of connectivity index 3χv enables simultaneous prediction of stability constants of bivalent transition metal complexes.
      ), which illustrates that stability of complexes increases with atomic number across the divalent metal cations until reaching Zn, which does not possess unpaired d-shell electrons and thus forms less stable interactions than Cu2+ (
      • Irving H.
      • Williams R.J.P.
      Order of stability of metal complexes.
      ). However, many biomolecules associate with less competitive cations, such as Mg2+, Fe2+, and Mn2+ (
      • Griese J.J.
      • Roos K.
      • Cox N.
      • Shafaat H.S.
      • Branca R.M.M.
      • Lehtiö J.
      • Gräslund A.
      • Lubitz W.
      • Siegbahn P.E.M.
      • Högbom M.
      Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein.
      ), as well as monovalent cations of potassium or sodium, which form even less stable interactions.
      Despite tight regulation of cellular concentrations of Mg2+, Fe2+, and Mn2+ accomplished by a coordinated effort of metal transporters and buffering chaperones in regulating free ion levels (
      • Foster A.W.
      • Osman D.
      • Robinson N.J.
      Metal preferences and metallation.
      ), the high affinity of these metals toward biomolecules ensures their activity even at low concentrations. For example, Fe is central to the heme and Fe–sulfur (Fe–S) cluster complexes, which are crucial cofactors in electron transport chain reactions, oxygen transport, translation termination, and antioxidant pathways. The background and current understanding of Fe–S cluster assembly and function have been detailed in several informative recent reviews (
      • Cardenas-Rodriguez M.
      • Chatzi A.
      • Tokatlidis K.
      Iron–sulfur clusters: From metals through mitochondria biogenesis to disease.
      • Srour B.
      • Gervason S.
      • Monfort B.
      • D'Autréaux B.
      Mechanism of iron–sulfur cluster assembly: In the intimacy of iron and sulfur encounter.
      ). The synthesis of these ancient cofactors can be catalyzed by UV light from the reduced Fe–S species, which were prevalent in the prebiotic earth, supporting an early role for Fe–S clusters in the evolution of life (
      • Bonfio C.
      • Valer L.
      • Scintilla S.
      • Shah S.
      • Evans D.J.
      • Jin L.
      • Szostak J.W.
      • Sasselov D.D.
      • Sutherland J.D.
      • Mansy S.S.
      UV-light-driven prebiotic synthesis of iron-sulfur clusters.
      ).
      The propensity for biomolecules to be flexible in their metal binding partner may reflect two features of the interactions. First, while biomolecular structures may evolve to prefer a metal cofactor by excluding similar ions, which are suboptimal, nonfunctional, or deleterious, there are overlapping characteristics of cations, which in many cases impede absolute specificity. Second, experimental evidence of selective advantages gained by retaining or acquiring the ability to exchange cofactors suggests that tolerance of alternative metal ions may be beneficial in certain circumstances. In other examples, divergent or convergent evolution has produced multiple distinct biomolecules within organisms, allowing consistent activity in environments of varying metal availability and limitations.

      Change of earth's atmosphere as a driving force of the evolution of biomolecules

      Great oxidation events, molecular oxygen, and ROS

      Early in the history of earth, volcanic processes were the major contributors to the composition of the atmosphere and oceans. This ancient earth's atmosphere was anoxic and reducing, with oceans rich in soluble divalent transition metal ions (
      • Konhauser K.O.
      • Pecoits E.
      • Lalonde S.V.
      • Papineau D.
      • Nisbet E.G.
      • Barley M.E.
      • Arndt N.T.
      • Zahnle K.
      • Kamber B.S.
      Oceanic nickel depletion and a methanogen famine before the Great Oxidation Event.
      ,
      • Robbins L.J.
      • Lalonde S.V.
      • Planavsky N.J.
      • Partin C.A.
      • Reinhard C.T.
      • Kendall B.
      • Scott C.
      • Hardisty D.S.
      • Gill B.C.
      • Alessi D.S.
      • Dupont C.L.
      • Saito M.A.
      • Crowe S.A.
      • Poulton S.W.
      • Bekker A.
      • et al.
      Trace elements at the intersection of marine biological and geochemical evolution.
      ,
      • Williams R.J.P.
      • Fraústo Da Silva J.J.R.
      Evolution was chemically constrained.
      ). While the time line of changes in earth's atmosphere remains under discussion (
      • Case A.J.
      On the origin of superoxide dismutase: An evolutionary perspective of superoxide-mediated redox signaling.
      ,
      • Nisbet E.G.
      • Grassineau N.V.
      • Howe C.J.
      • Abell P.I.
      • Regelous M.
      • Nisbet R.E.R.
      The age of Rubisco: The evolution of oxygenic photosynthesis.
      ), it is thought that 2 to 3 billion years ago, the accumulation of molecular oxygen in the atmosphere occurred in what is known as the great oxidation event. A further oxidation event is likely to have transpired less than 1 billion years ago (neoproterozoic oxidation event), which had a more significant impact on oxygen levels in the ocean (
      • Canfield D.E.
      The early history of atmospheric oxygen: Homage to Robert M. Garrels.
      ). These shifts in atmospheric composition occurred subsequent to biogenesis (
      • Nisbet E.G.
      • Grassineau N.V.
      • Howe C.J.
      • Abell P.I.
      • Regelous M.
      • Nisbet R.E.R.
      The age of Rubisco: The evolution of oxygenic photosynthesis.
      ,
      • Canfield D.E.
      The early history of atmospheric oxygen: Homage to Robert M. Garrels.
      ), involving complex fluctuations of oxygen stores with biological processes likely being the primary source of the molecular oxygen (
      • Case A.J.
      On the origin of superoxide dismutase: An evolutionary perspective of superoxide-mediated redox signaling.
      ,
      • Fischer W.W.
      • Hemp J.
      • Johnson J.E.
      Evolution of oxygenic photosynthesis.
      ,
      • Sessions A.L.
      • Doughty D.M.
      • Welander P.V.
      • Summons R.E.
      • Newman D.K.
      The continuing puzzle of the great oxidation event.
      ). It has been proposed that prevalent methanogenic archaea, which depended on Ni for metabolic catalysis, declined following a decrease in volcanic sources of Ni (
      • Thauer R.K.
      My lifelong passion for biochemistry and anaerobic microorganisms.
      ). This led to a decrease in methane production and allowed proliferation of species requiring less Ni, such as photosynthetic marine cyanobacteria (
      • Konhauser K.O.
      • Pecoits E.
      • Lalonde S.V.
      • Papineau D.
      • Nisbet E.G.
      • Barley M.E.
      • Arndt N.T.
      • Zahnle K.
      • Kamber B.S.
      Oceanic nickel depletion and a methanogen famine before the Great Oxidation Event.
      ,
      • Raymond J.
      • Segre D.
      The effect of oxygen on biochemical networks and the evolution of complex life.
      ).
      Abundant oxygen led to the expansion of organisms utilizing oxidative metabolism, with oxygen acting as an electron acceptor, as it contains two unpaired electrons with parallel spins and is metabolized by a univalent (single electron) mechanism, generating several ROS as intermediates. Among others (
      • Ghosh A.
      • Shcherbik N.
      Effects of oxidative stress on protein translation: Implications for cardiovascular diseases.
      ), ROS include the superoxide (O2•−), hydroxyl (•OH) radicals, and hydrogen peroxide (H2O2) (
      • Fridovich I.
      Oxygen: How do we stand it?.
      ). H2O2, while itself not a radical, is prone to univalent reduction by Fe and Cu ions, making it a significant contributor to oxidative damage to various biomolecules, along with the O2•− and •OH radicals. O2•− radicals are generated from the electron transport chain reactions and are converted to the less reactive H2O2 by SODs (
      • Case A.J.
      On the origin of superoxide dismutase: An evolutionary perspective of superoxide-mediated redox signaling.
      ). Other antioxidant enzymes, such as catalase, glutathione peroxidase, and/or peroxiredoxins then convert H2O2 to water (
      • Chen Y.
      • Azad M.B.
      • Gibson S.B.
      Superoxide is the major reactive oxygen species regulating autophagy.
      ). Alternatively, the •OH radical can be generated from H2O2 by high-energy radiation, or by metal ion catalysis reactions, such as the Fenton reaction that is discussed later (Fig. 1B).

      Oxidation of biomolecules and role of metal ions

      ROS, especially highly reactive •OH radical, can oxidize most biological targets, resulting in short lifetimes and very limited diffusion distances (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). ROS broadly damage proteins and amino acids via a range of modifications to amino acid side chains that lead to protein inactivation and degradation, the induction of polypeptide cleavages, and promotion of cross-linking and aggregation (
      • Stadtman E.R.
      • Levine R.L.
      Free radical-mediated oxidation of free amino acids and amino acid residues in proteins.
      ). In many cases, damage to the proteins can occur following exposure to xenobiotic metals, such as heavy metals (
      • Solenkova N.V.
      • Newman J.D.
      • Berger J.S.
      • Thurston G.
      • Hochman J.S.
      • Lamas G.A.
      Metal pollutants and cardiovascular disease: Mechanisms and consequences of exposure.
      ). Another metal-induced harmful protein modification mechanism is related to site-specific metal-catalyzed oxidation (MCO) of amino acids at metal-binding sites. Specifically, MCO systems were found to target a wide variety of essential cellular enzymes and structural proteins, including glutamine synthetase, chymotrypsin, myosin, α-synuclein, catalases, and SODs. It was found that the activity of most of the MCO systems is dependent on ions of Fe or Cu, both of which are also involved in Fenton chemistry, whereby Fe2+, and in some instances, Cu2+, react with H2O2 to produce a •OH radical and a hydroxide ion ((
      • Winterbourn C.C.
      Toxicity of iron and hydrogen peroxide: The Fenton reaction.
      ) and Fig. 1B). Fenton-generated •OH radical damages amino acids within proteins (
      • Cole N.B.
      • Murphy D.D.
      • Lebowitz J.
      • Di Noto L.
      • Levine R.L.
      • Nussbaum R.L.
      Metal-catalyzed oxidation of α-synuclein.
      ,
      • Stadtman E.R.
      Metal ion-catalyzed oxidation of proteins: Biochemical mechanism and biological consequences.
      ).
      The oxidative damage of proteins is implicated in a plethora of human pathologies, including neurodegenerative diseases, such as Alzheimer's (
      • Sultana R.
      • Perluigi M.
      • Butterfield D.A.
      Protein oxidation and lipid peroxidation in brain of subjects with Alzheimer's disease: Insights into mechanism of neurodegeneration from redox proteomics.
      ) and Parkinson's disease (
      • Rodgers K.J.
      • Hume P.M.
      • Morris J.G.L.
      • Dean R.T.
      Evidence for L-dopa incorporation into cell proteins in patients treated with levodopa.
      ), amyotrophic lateral sclerosis (
      • Hilton J.B.
      • White A.R.
      • Crouch P.J.
      Metal-deficient SOD1 in amyotrophic lateral sclerosis.
      ); muscular dystrophy (
      • Terrill J.R.
      • Radley-Crabb H.G.
      • Iwasaki T.
      • Lemckert F.A.
      • Arthur P.G.
      • Grounds M.D.
      Oxidative stress and pathology in muscular dystrophies: Focus on protein thiol oxidation and dysferlinopathies.
      ), pulmonary emphysema (
      • McGuinness A.J.A.
      • Sapey E.
      Oxidative stress in COPD: Sources, markers, and potential mechanisms.
      ), atherosclerosis (
      • Stocker R.
      • Keaney J.F.
      Role of oxidative modifications in atherosclerosis.
      ), and age-related clinical pathologies, such as age-related macular degeneration (
      • Ethen C.M.
      • Reilly C.
      • Feng X.
      • Olsen T.W.
      • Ferrington D.A.
      Age-related macular degeneration and retinal protein modification by 4-hydroxy-2-nonenal.
      ), and cataractogenesis (
      • Dean R.T.
      • Dunlop R.
      • Hume P.
      • Rodgers K.J.
      Proteolytic “defences” and the accumulation of oxidized polypeptides in cataractogenesis and atherogenesis.
      ). For example, inactivation of SODs via oxidation (
      • Hodgson E.K.
      • Fridovich I.
      The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: Inactivation of the enzyme.
      ,
      • Salo D.C.
      • Lin S.W.
      • Pacifici R.E.
      • Davies K.J.
      Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide.
      ) or mutations (
      • Hilton J.B.
      • White A.R.
      • Crouch P.J.
      Metal-deficient SOD1 in amyotrophic lateral sclerosis.
      ,
      • Kang S.W.
      Superoxide dismutase 2 gene and cancer risk: Evidence from an updated meta-analysis.
      ) results in enzymatic incompetency, degradation, or aberrant cellular localization. This leads to increased levels of ROS, causing protein damage and aggregation (which manifest in the progression of amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease (
      • Flynn J.M.
      • Melov S.
      SOD2 in mitochondrial dysfunction and neurodegeneration.
      )) and DNA lesions (which can promote tumorigenesis (
      • Hempel N.
      • Carrico P.M.
      • Melendez J.A.
      Manganese superoxide dismutase (Sod2) and redox-control of signaling events that drive metastasis.
      )). Oxidation and cleavage of protein chaperones, such as Hsp90, has also been demonstrated to occur by an Fe-mediated mechanism, compounding the direct damage of ROS to proteins by disrupting the folding and stability of the targets of this chaperone (
      • Beck R.
      • Dejeans N.
      • Glorieux C.
      • Creton M.
      • Delaive E.
      • Dieu M.
      • Raes M.
      • Levêque P.
      • Gallez B.
      • Depuydt M.
      • Collet J.F.
      • Calderon P.B.
      • Verrax J.
      Hsp90 is cleaved by reactive oxygen species at a highly conserved N-terminal amino acid motif.
      ,
      • Castro J.P.
      • Fernando R.
      • Reeg S.
      • Meinl W.
      • Almeida H.
      • Grune T.
      Non-enzymatic cleavage of Hsp90 by oxidative stress leads to actin aggregate formation: A novel gain-of-function mechanism.
      ), leading to neurodegeneration and prionopathies (
      • Bohush A.
      • Bieganowski P.
      • Filipek A.
      Hsp90 and its co-chaperones in neurodegenerative diseases.
      ). The scope of protein oxidation and the various mechanisms are reviewed in detail in Ref. (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ).
      Nucleic acids are also highly susceptible to damage by ROS, and oxidant damage to both RNA and DNA is implicated in many diseases, including neurodegenerative conditions and cancer (
      • Cooke M.S.
      • Evans M.D.
      • Dizdaroglu M.
      • Lunec J.
      Oxidative DNA damage: Mechanisms, mutation, and disease.
      ,
      • Kong Q.
      • Lin C.-L.G.
      Oxidative damage to RNA: Mechanisms, consequences, and diseases.
      ). The Fe- and/or Cu-driven Fenton reaction (Fig. 1B) has been identified as a source of oxidants leading to nucleic acid damage (
      • Imlay J.A.
      • Chin S.M.
      • Linn S.
      Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro.
      ,
      • Li Z.
      • Chen X.
      • Liu Z.
      • Ye W.
      • Li L.
      • Qian L.
      • Ding H.
      • Li P.
      • Aung L.H.H.
      Recent advances: Molecular mechanism of RNA oxidation and its role in various diseases.
      ,
      • Poulsen H.E.
      • Specht E.
      • Broedbaek K.
      • Henriksen T.
      • Ellervik C.
      • Mandrup-Poulsen T.
      • Tonnesen M.
      • Nielsen P.E.
      • Andersen H.U.
      • Weimann A.
      RNA modifications by oxidation: A novel disease mechanism?.
      ), and this is discussed in the following section. In addition, Fe and Mg can cleave RNA by a nonoxidative mechanism (termed in-line cleavage), as was recently demonstrated (
      • Guth-Metzler R.
      • Bray M.S.
      • Frenkel-Pinter M.
      • Suttapitugsakul S.
      • Montllor-Albalate C.
      • Bowman J.C.
      • Wu R.
      • Reddi A.R.
      • Okafor C.D.
      • Glass J.B.
      • Williams L.D.
      Cutting in-line with iron: Ribosomal function and non-oxidative RNA cleavage.
      ).
      Oxidation of free RNA species can cause strand cleavages and oxidative base modifications. While oxidized mRNAs are recognized by ribosomes, the lesions are associated with decreased translational efficiency and an increase in truncated or misfolded protein products (
      • Simms C.L.
      • Hudson B.H.
      • Mosior J.W.
      • Rangwala A.S.
      • Zaher H.S.
      An active role for the ribosome in determining the fate of oxidized mRNA.
      ,
      • Tanaka M.
      • Chock P.B.
      • Stadtman E.R.
      Oxidized messenger RNA induces translation errors.
      ). Besides damages to the transcripts, the translational machinery, which is constructed of proteins and RNAs, is also susceptible to oxidant-induced impairments. Ribosomes are large ribonucleotide–protein complexes that are at the center of the protein synthesis machinery. Ribosomes are composed of the small subunit (30S for prokaryotes and 40S for eukaryotes) and large subunit (50S for prokaryotes and 60S for eukaryotes). The subunits are assembled as intricately folded rRNAs for which Mg2+ ions play a critical role by coordinating rRNA folding and interaction with ribosomal proteins. Eukaryotes also have distinct ribosomes in the plastids and mitochondria, where they are likely to be particularly exposed to oxidative species derived from electron transport chain reactions (
      • Shcherbik N.
      • Pestov D.G.
      The impact of oxidative stress on ribosomes: From injury to regulation.
      ). RNA oxidation leads to guanine modification (8-oxo-7,8-dihydroguanine), which is associated with wide range of pathologies, such as neurodegeneration, neuropsychiatric disorders, and atherosclerosis (
      • Shcherbik N.
      • Pestov D.G.
      The impact of oxidative stress on ribosomes: From injury to regulation.
      ). Thus, oxidative stress negatively impacts translational processes (
      • Shenton D.
      • Smirnova J.B.
      • Selley J.N.
      • Carroll K.
      • Hubbard S.J.
      • Pavitt G.D.
      • Ashe M.P.
      • Grant C.M.
      Global translational responses to oxidative stress impact upon multiple levels of protein synthesis.
      ), while maintenance of translational function can promote adaptation and survival responses (
      • Blevins W.R.
      • Tavella T.
      • Moro S.G.
      • Blasco-Moreno B.
      • Closa-Mosquera A.
      • Díez J.
      • Carey L.B.
      • Albà M.M.
      Extensive post-transcriptional buffering of gene expression in the response to severe oxidative stress in baker's yeast.
      ,
      • Zhu M.
      • Dai X.
      Maintenance of translational elongation rate underlies the survival of Escherichia coli during oxidative stress.
      ).

      Fenton chemistry and role of Fe and Cu

      Fe can act as an electron donor and acceptor owing to its reduced Fe2+ and oxidized Fe3+ states, which are important for its biological utility. The employment of Fe in biomolecules became established in biology in an aqueous earth environment, which was Fe rich and contained little oxygen.
      The mononuclear or dinuclear ions of Fe are employed in the catalytically active centers of many enzymes, including the SODs and RNRs, both of which are susceptible to metal interchange and are the focus of this review. Much of the toxicity associated with Fe is a result of the Fenton reaction (Fig. 1B). In the reducing environment of the cytosol, O2•− can oxidize and destabilize Fe–S cluster complexes with crucial activity in the citric acid cycle, thereby blocking aerobic metabolism and generating free Fe, which can further participate in Fenton chemistry (Fig. 1B). The term “redox cycling” is used to refer to this propagation of ROS by a combination of the Fenton reaction and the activity of the O2•− anion in the regeneration of free Fe2+ (
      • Fridovich I.
      Oxygen: How do we stand it?.
      ,
      • Murray K.F.
      • Messner D.J.
      • Kowdley K.V.
      Mechanisms of hepatocyte detoxification.
      ).
      Similar to Fe, ions of Cu that exist in oxidized (Cu2+) and reduced (Cu+) states play roles in electron transport and many redox enzyme–driven mechanisms (
      • Festa R.A.
      • Thiele D.J.
      Copper: An essential metal in biology.
      ). Importantly, Cu+ also participates in Fenton-like reactions, and there is a partial overlap of biological activities and interactions between homeostatic mechanisms between Cu and Fe. Both are highly redox active, facilitating roles in many enzymatic reactions (
      • Gulec S.
      • Collins J.F.
      Molecular mediators governing iron-copper interactions.
      ). Because of the oxidative damage associated with loss of homeostasis of these metals, regulation of free Fe and Cu in cells is important for protection from metal-dependent oxidative damage. Cu is especially toxic because of subsequent reactions involving the Cu2+ ion, including binding to thiol groups, generating large amounts of •OH radicals, and promoting further Fe2+-mediated Fenton reactions (
      • Hong Enriquez R.P.
      • Do T.N.
      Bioavailability of metal ions and evolutionary adaptation.
      ). For these reasons, free Cu is extremely limited within cells, being almost entirely bound by highly conserved Cu-binding proteins (
      • Rae A.T.D.
      • Schmidt P.J.
      • Pufahl R.A.
      • Culotta V.C.
      • Halloran T.V.O.
      Undetectable intracellular free copper: The requirement of a copper chaperone for superoxide dismutase.
      ). Toxicity of Cu is mitigated in cells by metallothioneins that sequester Cu as well as Zn and non-nutrient heavy metals cadmium and mercury (
      • Calvo J.
      • Jung H.
      • Meloni G.
      Copper metallothioneins.
      ). Cu was likely not employed in primitive biomolecules because of limited availability but became required later, for example, in the catalytic center of certain SOD enzymes (Table 1), as an alternative to Fe or Mn (
      • Festa R.A.
      • Thiele D.J.
      Copper: An essential metal in biology.
      ).
      Table 1Metal ions interchangeability within SODs
      Kingdom/orderSpeciesSODNative Me cofactorActive site's Me replacementDismutation enzymatic activityCambialismReference
      Bacteria/BacillalesStaphylococcus aureusSodAMn2+Active(
      • Garcia Y.M.
      • Barwinska-Sendra A.
      • Tarrant E.
      • Skaar E.P.
      • Waldron K.J.
      • Kehl-Fie T.E.
      A superoxide dismutase capable of functioning with iron or manganese promotes the resistance of Staphylococcus aureus to calprotectin and nutritional immunity.
      )
      SodM
      Highlight SODs with cambialistic properties.
      Mn2+/Fe2+
      Highlight SODs with cambialistic properties.
      Mn2+ or Fe2+
      Highlight SODs with cambialistic properties.
      Active
      Highlight SODs with cambialistic properties.
      Yes
      Highlight SODs with cambialistic properties.
      (
      • Garcia Y.M.
      • Barwinska-Sendra A.
      • Tarrant E.
      • Skaar E.P.
      • Waldron K.J.
      • Kehl-Fie T.E.
      A superoxide dismutase capable of functioning with iron or manganese promotes the resistance of Staphylococcus aureus to calprotectin and nutritional immunity.
      )
      Highlight SODs with cambialistic properties.
      smSod
      Highlight SODs with cambialistic properties.
      Mn2+
      Highlight SODs with cambialistic properties.
      Mn2+ or Fe2+
      Highlight SODs with cambialistic properties.
      Active
      Highlight SODs with cambialistic properties.
      Yes
      Highlight SODs with cambialistic properties.
      (
      • De Vendittis A.
      • Amato M.
      • Mickniewicz A.
      • Parlato G.
      • De Angelis A.
      • Castellano I.
      • Rullo R.
      • Riccitiello F.
      • Rengo S.
      • Masullo M.
      • De Vendittis E.
      Regulation of the properties of superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans by iron- and manganese-bound co-factor.
      ,
      • Russo Krauss I.
      • Merlino A.
      • Pica A.
      • Rullo R.
      • Bertoni A.
      • Capasso A.
      • Amato M.
      • Riccitiello F.
      • De Vendittis E.
      • Sica F.
      Fine tuning of metal-specific activity in the Mn-like group of cambialistic superoxide dismutases.
      )
      Highlight SODs with cambialistic properties.
      stSod
      Highlight SODs with cambialistic properties.
      Mn2+
      Highlight SODs with cambialistic properties.
      Mn2+ or Fe2+
      Highlight SODs with cambialistic properties.
      Active
      Highlight SODs with cambialistic properties.
      Yes
      Highlight SODs with cambialistic properties.
      (
      • Russo Krauss I.
      • Merlino A.
      • Pica A.
      • Rullo R.
      • Bertoni A.
      • Capasso A.
      • Amato M.
      • Riccitiello F.
      • De Vendittis E.
      • Sica F.
      Fine tuning of metal-specific activity in the Mn-like group of cambialistic superoxide dismutases.
      ,
      • Andrus J.M.
      • Bowen S.W.
      • Klaenhammer T.R.
      • Hassan H.M.
      Molecular characterization and functional analysis of the manganese-containing superoxide dismutase gene (sodA) from Streptococcus thermophilus AO54.
      )
      Highlight SODs with cambialistic properties.
      Bacteria/EnterobacteralesEscherichia coliSodAMn2+Active(
      • Vance C.K.
      • Miller A.F.
      Novel insights into the basis for Escherichia coli superoxide dismutase's metal ion specificity from mn-substituted FeSOD and its very high Em.
      ,
      • Privalle C.T.
      • Fridovich I.
      Transcriptional and maturational effects of manganese and iron on the biosynthesis of manganese-superoxide dismutase in Escherichia coli.
      )
      Fe2+Gain of function: peroxidase
      Highlight SODs with peroxidase activity gained upon metal ion replacement.
      No(
      • Garcia Y.M.
      • Barwinska-Sendra A.
      • Tarrant E.
      • Skaar E.P.
      • Waldron K.J.
      • Kehl-Fie T.E.
      A superoxide dismutase capable of functioning with iron or manganese promotes the resistance of Staphylococcus aureus to calprotectin and nutritional immunity.
      )
      Mutant SodAG165T
      Highlight SODs with cambialistic properties.
      Highlight SODs with cambialistic properties.
      Fe2+
      Highlight SODs with cambialistic properties.
      Active
      Highlight SODs with cambialistic properties.
      Yes
      Highlight SODs with cambialistic properties.
      (
      • Osawa M.
      • Yamakura F.
      • Mihara M.
      • Okubo Y.
      • Yamada K.
      • Hiraoka B.Y.
      Conversion of the metal-specific activity of Escherichia coli Mn-SOD by site-directed mutagenesis of Gly165Thr.
      )
      Highlight SODs with cambialistic properties.
      SodAMn2+Mn2+, Fe2+ hybrid
      Highlight hybrid SODs, whereby two different metal ions (indicated in the table) occupy two distinct enzyme dimer's subunits.
      Partially active
      Highlight hybrid SODs, whereby two different metal ions (indicated in the table) occupy two distinct enzyme dimer's subunits.
      No
      Highlight hybrid SODs, whereby two different metal ions (indicated in the table) occupy two distinct enzyme dimer's subunits.
      (
      • Privalle C.T.
      • Fridovich I.
      Inductions of superoxide dismutases in Escherichia coli under anaerobic conditions. Accumulation of an inactive form of the manganese enzyme.
      )
      Highlight hybrid SODs, whereby two different metal ions (indicated in the table) occupy two distinct enzyme dimer's subunits.
      SodBFe2+Active(
      • Vance C.K.
      • Miller A.F.
      Novel insights into the basis for Escherichia coli superoxide dismutase's metal ion specificity from mn-substituted FeSOD and its very high Em.
      )
      Mn2+InactiveNo(
      • Vance C.K.
      • Miller A.F.
      Novel insights into the basis for Escherichia coli superoxide dismutase's metal ion specificity from mn-substituted FeSOD and its very high Em.
      )
      Mutant SodBT165G
      Highlight SODs with cambialistic properties.
      Highlight SODs with cambialistic properties.
      Mn2+
      Highlight SODs with cambialistic properties.
      Active
      Highlight SODs with cambialistic properties.
      Yes
      Highlight SODs with cambialistic properties.
      (
      • Yamakura F.
      • Sugio S.
      • Hiraoka B.Y.
      • Ohmori D.
      • Yokota T.
      Pronounced conversion of the metal-specific activity of superoxide dismutase from Porphyromonas gingivalis by the mutation of a single amino acid (Gly155Thr) located apart from the active site.
      )
      Highlight SODs with cambialistic properties.
      SodCCu2+Zn2+Active(
      • Gabbianelli R.
      • D'Orazio M.
      • Pacello F.
      • O'Neill P.
      • Nicolini L.
      • Rotilio G.
      • Battistoni A.
      Distinctive functional features in prokaryotic and eukaryotic Cu,Zn superoxide dismutases.
      )
      Bacteria/StreptomycetalesStreptomyces coelicolorSodNNi2+Ni2+Active(
      • Kim F.J.
      • Kim H.P.
      • Hah Y.C.
      • Roe J.H.
      Differential expression of superoxide dismutases containing Ni and Fe/Zn in Streptomyces coelicolor.
      ,
      • Youn H.D.
      • Kim E.J.
      • Roe J.H.
      • Hah Y.C.
      • Kang S.O.
      A novel nickel-containing superoxide dismutase from Streptomyces spp.
      )
      Fe2+InactiveNo(
      • Youn H.D.
      • Kim E.J.
      • Roe J.H.
      • Hah Y.C.
      • Kang S.O.
      A novel nickel-containing superoxide dismutase from Streptomyces spp.
      )
      Zn2+InactiveNo(
      • Youn H.D.
      • Kim E.J.
      • Roe J.H.
      • Hah Y.C.
      • Kang S.O.
      A novel nickel-containing superoxide dismutase from Streptomyces spp.
      )
      Fe2+, Zn2+ hybrid
      Highlight hybrid SODs, whereby two different metal ions (indicated in the table) occupy two distinct enzyme dimer's subunits.
      Active
      Highlight hybrid SODs, whereby two different metal ions (indicated in the table) occupy two distinct enzyme dimer's subunits.
      No
      Highlight hybrid SODs, whereby two different metal ions (indicated in the table) occupy two distinct enzyme dimer's subunits.
      (
      • Kim F.J.
      • Kim H.P.
      • Hah Y.C.
      • Roe J.H.
      Differential expression of superoxide dismutases containing Ni and Fe/Zn in Streptomyces coelicolor.
      )
      Highlight hybrid SODs, whereby two different metal ions (indicated in the table) occupy two distinct enzyme dimer's subunits.
      Bacteria/BacteroidalesPorphyromonas gingivalispgSod
      Highlight SODs with cambialistic properties.
      Fe2+
      Highlight SODs with cambialistic properties.
      Mn2+ or Fe2+
      Highlight SODs with cambialistic properties.
      Active
      Highlight SODs with cambialistic properties.
      Yes
      Highlight SODs with cambialistic properties.
      (
      • Russo Krauss I.
      • Merlino A.
      • Pica A.
      • Rullo R.
      • Bertoni A.
      • Capasso A.
      • Amato M.
      • Riccitiello F.
      • De Vendittis E.
      • Sica F.
      Fine tuning of metal-specific activity in the Mn-like group of cambialistic superoxide dismutases.
      )
      Highlight SODs with cambialistic properties.
      Bacteria/ActinobacteriaPropionibacterium shermaniipsSod
      Highlight SODs with cambialistic properties.
      Fe2+
      Highlight SODs with cambialistic properties.
      Mn2+ or Fe2+
      Highlight SODs with cambialistic properties.
      Active
      Highlight SODs with cambialistic properties.
      Yes
      Highlight SODs with cambialistic properties.
      (
      • Russo Krauss I.
      • Merlino A.
      • Pica A.
      • Rullo R.
      • Bertoni A.
      • Capasso A.
      • Amato M.
      • Riccitiello F.
      • De Vendittis E.
      • Sica F.
      Fine tuning of metal-specific activity in the Mn-like group of cambialistic superoxide dismutases.
      )
      Highlight SODs with cambialistic properties.
      Archaea/SulfolobalesAcidianus ambivalensFeSodFe2+Active(
      • Schäfer G.
      • Kardinahl S.
      Iron superoxide dismutases: Structure and function of an archaic enzyme.
      )
      Co2+InactiveNo(
      • Schäfer G.
      • Kardinahl S.
      Iron superoxide dismutases: Structure and function of an archaic enzyme.
      )
      Ni2+InactiveNo(
      • Schäfer G.
      • Kardinahl S.
      Iron superoxide dismutases: Structure and function of an archaic enzyme.
      )
      Mn2+InactiveNo(
      • Schäfer G.
      • Kardinahl S.
      Iron superoxide dismutases: Structure and function of an archaic enzyme.
      )
      Fungi/SaccharomycetalesSaccharomyces cerevisiaeSod1Cu2+Zn2+Active(
      • Chang E.C.
      • Crawford B.F.
      • Hong Z.
      • Bilinski T.
      • Kosman D.J.
      Genetic and biochemical characterization of Cu,Zn superoxide dismutase mutants in Saccharomyces cerevisiae.
      )
      Sod2Mn2+Active(
      • Luk E.
      • Yang M.
      • Jensen L.T.
      • Bourbonnais Y.
      • Culotta V.C.
      Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae.
      )
      Fe2+InactiveNo(
      • Kang Y.
      • He Y.X.
      • Zhao M.X.
      • Li W.F.
      Structures of native and Fe-substituted SOD2 from Saccharomyces cerevisiae.
      ,
      • Yang M.
      • Cobine P.A.
      • Molik S.
      • Naranuntarat A.
      • Lill R.
      • Winge D.R.
      • Culotta V.C.
      The effects of mitochondrial iron homeostasis on cofactor specificity of superoxide dismutase 2.
      )
      Animalia/MammaliaMammalsSOD1Cu2+Zn2+Active(
      • Robinett N.G.
      • Peterson R.L.
      • Culotta V.C.
      Eukaryotic copper-only superoxide dismutases (SODs): A new class of SOD enzymes and SOD-like protein domains.
      )
      Cu2+Cu2+InactiveNo(
      • Nedd S.
      • Redler R.L.
      • Proctor E.A.
      • Dokholyan N.V.
      • Alexandrova A.N.
      Cu,Zn-superoxide dismutase without Zn is folded but catalytically inactive.
      )
      SOD2Mn2+Active(
      • Weisiger R.A.
      • Fridovich I.
      Superoxide dismutase. Organelle specificity.
      ,
      • Zelko I.N.
      • Mariani T.J.
      • Folz R.J.
      Superoxide dismutase multigene family: A comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression.
      )
      Fe2+Gain of function: peroxidase
      Highlight SODs with peroxidase activity gained upon metal ion replacement.
      No(
      • Ganini D.
      • Santos J.H.
      • Bonini M.G.
      • Mason R.P.
      Switch of mitochondrial superoxide dismutase into a prooxidant peroxidase in manganese-deficient cells and mice.
      )
      SOD3Cu2+Zn2+Active(
      • Marklund S.L.
      Human copper-containing superoxide dismutase of high molecular weight.
      ,
      • Zelko I.N.
      • Mariani T.J.
      • Folz R.J.
      Superoxide dismutase multigene family: A comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression.
      ,
      • Antonyuk S.V.
      • Strange R.W.
      • Marklund S.L.
      • Hasnain S.S.
      The structure of human extracellular copper-zinc superoxide dismutase at 1.7 A resolution: Insights into heparin and collagen binding.
      ,
      • Shrestha P.
      • Yun J.-H.
      • Kim W.T.
      • Kim T.-Y.
      • Lee W.
      Cloning, purification, and characterization of recombinant human extracellular superoxide dismutase in SF9 insect cells.
      )
      Plantae/lauralesCinnamomum camphoraFeSOD
      Highlight SODs with cambialistic properties.
      Fe2+
      Highlight SODs with cambialistic properties.
      Mn2+ or Fe2+
      Highlight SODs with cambialistic properties.
      Active
      Highlight SODs with cambialistic properties.
      Yes
      Highlight SODs with cambialistic properties.
      (
      • Chen H.-Y.
      • Hu R.-G.
      • Wang B.-Z.
      • Chen W.-F.
      • Liu W.-Y.
      • Schröder W.
      • Frank P.
      • Ulbrich N.
      Structural studies of an eukaryotic cambialistic superoxide dismutase purified from the mature seeds of camphor tree.
      )
      Highlight SODs with cambialistic properties.
      a Highlight SODs with cambialistic properties.
      b Highlight hybrid SODs, whereby two different metal ions (indicated in the table) occupy two distinct enzyme dimer's subunits.
      c Highlight SODs with peroxidase activity gained upon metal ion replacement.

      Chemical properties of Fe, Mn, and Mg dictate their interchangeability within biomolecules

      Conserved and ancient biomolecules are associated with Mn and Fe as a result of the availability of these cations in the early earth environment, as well as the catalytic capabilities supplied by their redox activities, which provide essential functions. The utilization of either Fe or Mn within closely related SODs (Table 1) from the most ancient group of these enzymes reflects the chemical similarities of these two divalent ions for use in redox mechanisms. These transition metal elements are adjacent in the periodic table and have similar radii, ligand affinities, and coordination preferences, presenting a challenge for biomolecules to choose between (
      • Foster A.W.
      • Osman D.
      • Robinson N.J.
      Metal preferences and metallation.
      ,
      • Dudev T.
      • Lim C.
      Competition among metal ions for protein binding sites: Determinants of metal ion selectivity in proteins.
      ).
      Unlike Fe, Mn does not participate in Fenton chemistry because of possessing a higher reduction potential (
      • Aguirre J.D.
      • Culotta V.C.
      Battles with iron: Manganese in oxidative stress protection.
      ), and as such, does not present the same toxicity risks in an oxidative environment. Mn also functions in enzymes as a Lewis acid in mechanisms that are more comparable to those catalyzed by Mg or Zn (
      • Schmidt S.B.
      • Husted S.
      The biochemical properties of manganese in plants.
      ). Coordinated Mg ions can often be replaced with Mn (
      • Garcia-Diaz M.
      • Bebenek K.
      • Krahn J.M.
      • Pedersen L.C.
      • Kunkel T.A.
      Role of the catalytic metal during polymerization by DNA polymerase lambda.
      ,
      • Harty M.L.
      • Sharma A.N.
      • Bearne S.L.
      Catalytic properties of the metal ion variants of mandelate racemase reveal alterations in the apparent electrophilicity of the metal cofactor.
      ,
      • Hohle T.H.
      • O'Brian M.R.
      Magnesium-dependent processes are targets of bacterial manganese toxicity.
      ), as they have similar binding site requirements (
      • Khrustalev V.V.
      • Barkovsky E.V.
      • Khrustaleva T.A.
      Magnesium and manganese binding sites on proteins have the same predominant motif of secondary structure.
      ). While Mg is an alkaline metal possessing no d-electrons, the Mg2+ ion shares a relatively similar ionic radius to Mn2+ and Fe2+. When forming complexes, all three cations prefer to coordinate six ligands (
      • Bock C.W.
      • Katz A.K.
      • Markham G.D.
      • Glusker J.P.
      Manganese as a replacement for magnesium and zinc: Functional comparison of the divalent ions.
      ) in an octahedral liganding geometry (Fig. 2A), and, in several examples, can occupy the same binding sites (
      • Athavale S.S.
      • Petrov A.S.
      • Hsiao C.
      • Watkins D.
      • Prickett C.D.
      • Gossett J.J.
      • Lie L.
      • Bowman J.C.
      • O'Neill E.
      • Bernier C.R.
      • Hud N.V.
      • Wartell R.M.
      • Harvey S.C.
      • Williams L.D.
      RNA folding and catalysis mediated by iron (II).
      ,
      • Maguire M.E.
      • Cowan J.A.
      Magnesium chemistry and biochemistry.
      ,
      • Cowan J.A.
      Metal activation of enzymes in nucleic acid biochemistry.
      ,
      • Moon W.J.
      • Liu J.
      Replacing Mg2+ by Fe2+ for RNA-cleaving DNAzymes.
      ). Mg forms very few covalent interactions with its ligands, making it able to be rapidly exchanged. The d-electrons of Mn2+ contribute to electrophilic interaction with ligands, allowing it to tolerate greater distortions of the bonding geometry than Mg2+, thereby lending itself better to catalytic mechanisms (
      • Kehres D.G.
      • Maguire M.E.
      Emerging themes in manganese transport, biochemistry and pathogenesis in bacteria.
      ). While the Irving–Williams series predicts greater stability of Mn or Fe complexes, the substitution of Mg with either transition metal may be limited by the vastly greater intracellular availability of Mg. In support of this, it has been reported that disruption of Mg2+-dependent processes occurs with increased Mn2+ import under conditions of low environmental Mg (
      • Hohle T.H.
      • O'Brian M.R.
      Magnesium-dependent processes are targets of bacterial manganese toxicity.
      ).
      Figure thumbnail gr2
      Figure 2The six- and five-coordinate geometries. Diagrams (2D on the left; 3D on the right) of the six-coordinate octahedral geometry (A) and the atypical five-coordinate trigonal bipyramidal geometry (B) of various metal ions within biomolecules discussed in the article. M represents the ion of iron or manganese; L represents a ligand. Modified from Ref. (
      • Borgstahl G.E.
      • Pokross M.
      • Chehab R.
      • Sekher A.
      • Snell E.H.
      Cryo-trapping the six-coordinate, distorted-octahedral active site of manganese superoxide dismutase.
      ).
      Mg is fundamental in stabilizing protein, lipid, and nucleic acid structures (
      • Piovesan D.
      • Profiti G.
      • Martelli P.L.
      • Casadio R.
      The human “magnesome”: Detecting magnesium binding sites on human proteins.
      ) and is involved in many catalytic mechanisms (
      • Cowan J.A.
      Metal activation of enzymes in nucleic acid biochemistry.
      ). It is the most common metal found in enzymes according to systematic analyses of reported protein structures, appearing in 16% of all enzymes (
      • Andreini C.
      • Bertini I.
      • Cavallaro G.
      • Holliday G.L.
      • Thornton J.M.
      Metal ions in biological catalysis: From enzyme databases to general principles.
      ,
      • Piovesan D.
      • Profiti G.
      • Martelli P.L.
      • Casadio R.
      The human “magnesome”: Detecting magnesium binding sites on human proteins.
      ). For comparison, Mn is identified as a cofactor in approximately 6% of all enzymes with known structure, whereas Fe is a cofactor in 8% (
      • Andreini C.
      • Bertini I.
      • Cavallaro G.
      • Holliday G.L.
      • Thornton J.M.
      Metal ions in biological catalysis: From enzyme databases to general principles.
      ,
      • Waldron K.J.
      • Rutherford J.C.
      • Ford D.
      • Robinson N.J.
      Metalloproteins and metal sensing.
      ). As Mg is known to form less stable interactions than other metal ions and is readily replaced, it is possible that structural reports underestimate the physiological occupation of binding sites by this element (
      • Waldron K.J.
      • Rutherford J.C.
      • Ford D.
      • Robinson N.J.
      Metalloproteins and metal sensing.
      ). Zn is also utilized extensively in cells and appears in more enzyme structures than Mn or Fe (
      • Waldron K.J.
      • Rutherford J.C.
      • Ford D.
      • Robinson N.J.
      Metalloproteins and metal sensing.
      ). Divalent ions of Zn and Mn have very similar radii (0.74 and 0.75 Å, respectively) but otherwise have dissimilar binding profiles and biochemical behavior and do not typically replace one another (
      • Bock C.W.
      • Katz A.K.
      • Markham G.D.
      • Glusker J.P.
      Manganese as a replacement for magnesium and zinc: Functional comparison of the divalent ions.
      ).

      Geochemical shifts drove extant metal biology: SOD as a prominent example

      With the proliferation of oxygen and the potential for oxidative damage to cellular components by ROS, organisms that had evolved antioxidant defenses would have had a major advantage (
      • Case A.J.
      On the origin of superoxide dismutase: An evolutionary perspective of superoxide-mediated redox signaling.
      ). Phylogenetic analysis suggests that ROS scavenging enzymes, such as SODs, peroxiredoxins, and catalases, had already emerged prior to the great oxidation event in response to the presence of low-level or localized oxygen (
      • Inupakutika M.A.
      • Sengupta S.
      • Devireddy A.R.
      • Azad R.K.
      • Mittler R.
      The evolution of reactive oxygen species metabolism.
      ). Antioxidant systems, thus, became increasingly valuable as oxygenation increased (
      • Case A.J.
      On the origin of superoxide dismutase: An evolutionary perspective of superoxide-mediated redox signaling.
      ), whereas broad fluctuations in metal availability occurred concomitantly. The transition metals Mn, Fe, Co, and Ni were in solutions at relatively high levels in the early ocean environments because of the high sulfur content and low levels of oxygen, whereas precipitation of Cu and Zn would have rendered them unavailable for biochemistry (
      • Saito M.A.
      • Sigman D.M.
      • Morel F.M.M.
      The bioinorganic chemistry of the ancient ocean: The co-evolution of cyanobacterial metal requirements and biogeochemical cycles at the Archean-Proterozoic boundary?.
      ). The increase in environmental oxygen led to the precipitation of Mn, Fe, Co, and Ni, and a major increase in the amount of available Zn, effectively reversing the availability of these metals (
      • Dupont C.L.
      • Butcher A.
      • Valas R.E.
      • Bourne P.E.
      • Caetano-Anollés G.
      History of biological metal utilization inferred through phylogenomic analysis of protein structures.
      ,
      • Williams R.J.P.
      • Fraústo Da Silva J.J.R.
      Evolution was chemically constrained.
      ,
      • Saito M.A.
      • Sigman D.M.
      • Morel F.M.M.
      The bioinorganic chemistry of the ancient ocean: The co-evolution of cyanobacterial metal requirements and biogeochemical cycles at the Archean-Proterozoic boundary?.
      ,
      • Feig A.L.
      • Uhlenbeck O.C.
      12 the role of metal ions in RNA biochemistry.
      ). These shifts in metal solubility were reflected by changes in their biological utilization. As such, comparison of structural motifs in the proteomes of organisms across the three domains of life supports the hypothesis that bioavailability of metals presented an evolutionary pressure resulting in the differences in their utilization (
      • Dupont C.L.
      • Yang S.
      • Palenik B.
      • Bourne P.E.
      Modern proteomes contain putative imprints of ancient shifts in trace metal geochemistry.
      ).
      One prominent example of geochemical shift–driven evolution of biomolecules is the appearance of alternatives to the primitive Fe-containing SOD (Fe-SOD), which utilize other metal cofactors (Fig. 3 and Table 1). SODs are the major enzymes responsible for the removal of O2•− anions that are unavoidably generated during aerobic metabolic reactions (
      • Fridovich I.
      Oxygen: How do we stand it?.
      ). British biochemist and writer Dr Lane characterized discovery of SODs as “the most important discovery of modern biology never to win a Nobel prize.” In fact, being discovered in 1969, SODs from various organisms remain of high interest for over last 5 decades.
      Figure thumbnail gr3
      Figure 3Schematic representation of concentrations of oxygen and selected metal cations in earth oceans. Estimates of the appearance of metal-utilizing biomolecules are shown above the graph in gradient-colored horizontal bars, indicating the early appearance of the ribosome. The most ancient superoxide dismutases (SODs), likely Fe-SODs, gave rise to Mn-SODs as oxygenation increased, with Cu/Zn-SODs appearing subsequently as copper and zinc became available. Oxygen levels (red line, right axis) are given as pO2 (partial pressure of oxygen) relative to PAL (present atmospheric level). The dotted line indicates the time of the great oxidation event (GOE). Modified from Refs. (
      • Saito M.A.
      • Sigman D.M.
      • Morel F.M.M.
      The bioinorganic chemistry of the ancient ocean: The co-evolution of cyanobacterial metal requirements and biogeochemical cycles at the Archean-Proterozoic boundary?.
      ,
      • Jones C.
      • Nomosatryo S.
      • Crowe S.A.
      • Bjerrum C.J.
      • Canfield D.E.
      Iron oxides, divalent cations, silica, and the early earth phosphorus crisis.
      ,
      • Moore E.K.
      • Jelen B.I.
      • Giovannelli D.
      • Raanan H.
      • Falkowski P.G.
      Metal availability and the expanding network of microbial metabolisms in the Archaean eon.
      ).
      The SODs catalyze a dismutation (disproportionation) reaction to produce O2 and H2O2 from two O2•− via a cyclic oxidation–reduction electron transfer (
      • Miller A.-F.
      Superoxide dismutases: Active sites that save, but a protein that kills.
      ):
      Mox-SOD+O2+H+Mred-SOD(H+)+O2
      (1)


      Mred-SOD(H+)+O2+H+Mox-SOD+H2O2
      (2)


      In this two-step reaction, the oxidized form of the metal (M) center ions (Mox-SOD) are first converted to the reduced form (Mred-SOD) with the formation of O2 (reaction 1), followed by oxidation of the reduced form of the metal ions into their oxidized form by O2•− with the release of H2O2 (reaction 2). Thus, one O2•− reduces the SOD, whereas another O2•− oxidizes the SOD in a so-called “ping–pong” mechanism (
      • Miller A.F.
      Superoxide dismutases: Ancient enzymes and new insights.
      ). Several metal ion cofactors (such as Fe, Mn, Cu, Zn, and Ni) can be employed in the SOD active sites, which perform this mechanism, as summarized in Table 1. In this catalytic mechanism, the metal ion is utilized by the SODs as a source of protons by employing structural aspects of the metal binding site to adjust the redox potential, which also acts to regulate the access of anions (
      • Herbst R.W.
      • Guce A.
      • Bryngelson P.A.
      • Higgins K.A.
      • Ryan K.C.
      • Cabelli D.E.
      • Garman S.C.
      • Maroney M.J.
      Role of conserved tyrosine residues in NiSOD catalysis: A case of convergent evolution.
      ). The activity of SODs requires them to exert tight control over the reactivity of the bound metals. The reduction midpoint potential (Em, a measure of the propensity of a chemical species to gain electrons in a redox reaction) of the metal cofactors is manipulated to around 300 mV in all known examples, despite the variety of metals employed across the enzyme families. The calculated reduction midpoint potential in aqueous solution compared with normal hydrogen electrode (a standard for zero redox potential as defined by the potential of a platinum electrode in 1 M acid solution) for the M3+/M2+ transition of Fe, Mn, and Ni are considerably different (0.77, 1.5, and 2.4 V, respectively) (
      • Uudsemaa M.
      • Tamm T.
      Density-functional theory calculations of aqueous redox potentials of fourth-period transition metals.
      ), requiring structural adaptation to bring the value into effective catalytic range (
      • Herbst R.W.
      • Guce A.
      • Bryngelson P.A.
      • Higgins K.A.
      • Ryan K.C.
      • Cabelli D.E.
      • Garman S.C.
      • Maroney M.J.
      Role of conserved tyrosine residues in NiSOD catalysis: A case of convergent evolution.
      ,
      • Miller A.F.
      Redox tuning over almost 1 V in a structurally conserved active site: Lessons from Fe-containing superoxide dismutase.
      ). The SODs demonstrate both the power of redox-active inorganic cofactors and the need to control their redox activity for function.
      SODs are found in the archaea, prokarya, and eukarya ((
      • Case A.J.
      On the origin of superoxide dismutase: An evolutionary perspective of superoxide-mediated redox signaling.
      ) and Table 1). Originally derived independently as three distinct families, abundance of SODs has undergone shift during course of evolution because of changes in earth's environment (Fig. 3). The first Mn/Fe-SOD arose in the low oxygen/high Fe environment (Fig. 3, orange and green curves). Thus, SODs represent a powerful and informative model to investigate metal ions interchangeability within biomolecules.
      The Mn/Fe-SOD family utilizes Fe or Mn at their catalytic center, reflecting the high levels of these metals in the preoxidation earth in which they originated. Fe-SODs are found in some primitive eukaryotes, plants, and bacteria, whereas Mn-containing SODs (Mn-SODs) are widespread. Within bacteria, aerobes tend to contain Mn-SODs or both Mn-SOD and Fe-SOD, whereas strict anaerobes may have one Fe-SOD, or none at all (
      • Takao M.
      • Yasui A.
      • Oikawa A.
      Unique characteristics of superoxide dismutase of a strictly anaerobic archaebacterium Methanobacterium thermoautotrophicum.
      ). Mn-SOD (SodA) from Escherichia coli is flexible in binding Mn2+ or Fe2+ depending on growth conditions. As such, Mn-SOD prefers cognate Mn ion when cultivated in the presence of oxygen; whereas under anaerobic conditions, bacterial Mn-SOD accommodates ions of Fe resulting in dismutation-inactive enzyme. Excess Fe has also been linked to the formation of partially active Fe-Mn-SOD (hybrid SOD), wherein a dimer's subunits contain Mn2+ and Fe2+ in their active site resulting in partial activity (summarized in Table 1). Thus, it was proposed that the selectivity of a metal ion cofactor in the bacterial Fe/Mn-SODs is defined by the bioavailability of Mn or Fe (
      • Mizuno K.
      • Whittaker M.M.
      • Bächinger H.P.
      • Whittaker J.W.
      Calorimetric studies on the tight binding metal interactions of Escherichia coli manganese superoxide dismutase.
      ).
      The most recently evolved family, the Cu/Zn-SODs, utilize Cu along with Zn. The Cu/Zn-SODs are found only in certain bacterial species (
      • Benov L.T.
      • Fridovich I.
      Escherichia coli expresses a copper- and zinc-containing superoxide dismutase.
      ,
      • Schnell S.
      • Steinman H.M.
      Function and stationary-phase induction of periplasmic copper-zinc superoxide dismutase and catalase/peroxidase in Caulobacter crescentus.
      ) but are ubiquitous among higher eukaryotes (
      • Bermingham-McDonogh O.
      • Gralla E.B.
      • Valentine J.S.
      The copper, zinc-superoxide dismutase gene of Saccharomyces cerevisiae: Cloning, sequencing, and biological activity.
      ,
      • Crapo J.D.
      • Oury T.
      • Rabouille C.
      • Slot J.W.
      • Chang L.Y.
      Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells.
      ,
      • McCord J.M.
      • Fridovich I.
      Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein).
      ). Humans express three Cu/Zn-SOD isoforms; the cytosolic SOD1 (
      • McCord J.M.
      • Fridovich I.
      Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein).
      ), the extracellular SOD3 (
      • Marklund S.L.
      Human copper-containing superoxide dismutase of high molecular weight.
      ), and the mitochondrial Mn-SOD (SOD2) (
      • Mondola P.
      • Damiano S.
      • Sasso A.
      • Santillo M.
      The Cu, Zn superoxide dismutase: Not only a dismutase enzyme.
      ,
      • Weisiger R.A.
      • Fridovich I.
      Superoxide dismutase. Organelle specificity.
      ). They are absent in archaeal species and are considered to have appeared long after the evolution of the Mn/Fe-SOD family (
      • Miller A.F.
      Superoxide dismutases: Ancient enzymes and new insights.
      ), correlating with the increase in bioavailable Cu and Zn (
      • Case A.J.
      On the origin of superoxide dismutase: An evolutionary perspective of superoxide-mediated redox signaling.
      ,
      • Saito M.A.
      • Sigman D.M.
      • Morel F.M.M.
      The bioinorganic chemistry of the ancient ocean: The co-evolution of cyanobacterial metal requirements and biogeochemical cycles at the Archean-Proterozoic boundary?.
      ). The Ni-SODs are found in algae and predominantly marine species of bacteria and likely appeared after Mn/Fe-SODs, presenting a selective advantage in marine environments, as levels of Fe and Mn have diminished and concentrations of Ni remained relatively consistent (
      • Saito M.A.
      • Sigman D.M.
      • Morel F.M.M.
      The bioinorganic chemistry of the ancient ocean: The co-evolution of cyanobacterial metal requirements and biogeochemical cycles at the Archean-Proterozoic boundary?.
      • Dupont C.L.
      • Neupane K.
      • Shearer J.
      • Palenik B.
      Diversity, function and evolution of genes coding for putative Ni-containing superoxide dismutases.
      ).
      These examples illustrate that the appearance and accumulation of molecular oxygen throughout earth's evolution accompanied by a shift in the availability of transition metals induced a switch from Fe-containing SODs to new variants of this enzyme that adapted alternative metal ions as cofactors, as illustrated in Figure 3 (
      • Nisbet E.G.
      • Grassineau N.V.
      • Howe C.J.
      • Abell P.I.
      • Regelous M.
      • Nisbet R.E.R.
      The age of Rubisco: The evolution of oxygenic photosynthesis.
      ,
      • Tripp B.C.
      • Bell C.B.
      • Cruz F.
      • Krebs C.
      • Ferry J.G.
      A role for iron in an ancient carbonic anhydrase.
      ). Thus, the utilization of metal ions throughout extant biological systems is intrinsically linked to the employment of molecular oxygen for fundamental processes in aerobic biology and the management of the associated toxicity of ROS. In addition, extant systems can be informative of the chemical environments in which they arose, even when these are drastically different from those in which they now function.

      Cation replacement examples: Focus on Fe, Mn, and Mg

      The exchange of Fe and Mn in functional enzymes is illustrated by a few prominent examples. The mononuclear SODs demonstrate the flexible utilization of either Mn or Fe within an isoform, whereas the essential RNR enzyme family, which employs a dinuclear cation pair, includes a further interesting example of an ancient and conserved enzyme, which utilizes different metal ions, and the expression of distinct forms with differing specificities. Finally, recent studies have identified an ability of the ribosome to undergo interchange of bound metal ions (
      • Bray M.S.
      • Lenz T.K.
      • Haynes J.W.
      • Bowman J.C.
      • Petrov A.S.
      • Reddi A.R.
      • Hud N.V.
      • Williams L.D.
      • Glass J.B.
      Multiple prebiotic metals mediate translation.
      ,
      • Smethurst D.G.J.
      • Kovalev N.
      • McKenzie E.R.
      • Pestov D.G.
      • Shcherbik N.
      Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese.
      ). These examples, discussed in detail later, suggest that the transition metal interchangeability occurs across different molecular structures and can be informative about the conditions under which they developed.

      Mn-SOD, Fe-SOD, and cambialistic Mn/Fe-SOD

      The Fe-SODs and Mn-SODs are highly homologous in sequence and three-dimensional structure (
      • Stallings W.C.
      • Pattridge K.A.
      • Strong R.K.
      • Ludwig M.L.
      Manganese and iron superoxide dismutases are structural homologs.
      ,
      • Vance C.K.
      • Miller A.F.
      Novel insights into the basis for Escherichia coli superoxide dismutase's metal ion specificity from mn-substituted FeSOD and its very high Em.
      ), whereby the amino acid residues and the funnel that allows a substrate (O2•−) access to the metal ion are identical. Catalytically active sites of both SODs contain three histidines and one aspartic acid that bind the metal ion cofactor (Fig. 4A, left and middle panels). In addition, the Fe and Mn ions also bind to a solvent molecule (water or hydroxide) that is engaged in the formation of hydrogen bonds and, together with histidines and aspartic acid, participates in the coordination of either cation in an unusual distorted trigonal bipyramidal geometry around the metal center, as depicted in Figure 2B (
      • Miller A.F.
      • Wang T.
      A single outer-sphere mutation stabilizes apo-Mn superoxide dismutase by 35 °C and disfavors Mn binding.
      ,
      • Tabares L.C.
      • Gätjens J.
      • Un S.
      Understanding the influence of the protein environment on the Mn(II) centers in superoxide dismutases using high-field electron paramagnetic resonance.
      ,
      • Borgstahl G.E.
      • Pokross M.
      • Chehab R.
      • Sekher A.
      • Snell E.H.
      Cryo-trapping the six-coordinate, distorted-octahedral active site of manganese superoxide dismutase.
      ).
      Figure thumbnail gr4
      Figure 4Structural properties of Mn-specific, Fe-specific, and cambialistic Mn/Fe-SODs. A, comparison of the crystal structures (top) and the metal-bound active sites (bottom) of Fe-SOD from Escherichia coli, Mn-SOD (SodA) and cambialistic Mn/Fe-SOD (cam-SOD, SodM) from Staphylococcus aureus. Metal ions (shown as colored spheres; orange for iron ion and green for manganese ion) are coordinated by three histidine (His) residues and one aspartic acid (Asp) residue and a solvent molecule (not shown). The figure is generated using PyMol; Protein Data Bank IDs are indicated in the figure. B, superimposed ribbon representation of Mn-specific SodA (orange) and cambialistic Mn/Fe-SodM (cyan) monomer structures from Staphylococcus aureus is shown on the left. Superimposed structures of active centers of SodA and SodM are shown on the right. Four residues from the primary coordination sphere (His27, His81, Asp161, and His165, black lettering) coordinate a metal ion (Mn is shown as a green sphere). Two residues from the secondary coordination sphere (SodA: Gly159, Leu160, orange lettering; SodM: Leu159, Phe160, cyan lettering) provide cambialistic properties to SodM. SOD, superoxide dismutase.
      Despite this remarkable similarity, most Fe-SOD and Mn-SOD enzymes are only functional when bound to their cognate metal ion (Table 1), illustrating their high specificity to metal cofactor (
      • Kwasigroch J.M.
      • Wintjens R.
      • Gilis D.
      • Rooman M.
      SODa: An Mn/Fe superoxide dismutase prediction and design server.
      ,
      • Wintjens R.
      • Noël C.
      • May A.C.W.
      • Gerbod D.
      • Dufernez F.
      • Capron M.
      • Viscogliosi E.
      • Rooman M.
      Specificity and phenetic relationships of iron- and manganese-containing superoxide dismutases on the basis of structure and sequence comparisons.
      ,
      • Whittaker J.W.
      The irony of manganese superoxide dismutase.
      ). Moreover, it has been revealed that the replacement of Mn with Fe in Mn-SODs from mammals (SOD2) and E. coli (SodA) results in the generation of an alternative isoform (Fe-SOD2s) with peroxidase prooxidant activity, thereby promoting oxidative stress, presumably via utilization of H2O2 ((
      • Ganini D.
      • Petrovich R.M.
      • Edwards L.L.
      • Mason R.P.
      Iron incorporation into MnSOD A (bacterial Mn-dependent superoxide dismutase) leads to the formation of a peroxidase/catalase implicated in oxidative damage to bacteria.
      ,
      • Ganini D.
      • Santos J.H.
      • Bonini M.G.
      • Mason R.P.
      Switch of mitochondrial superoxide dismutase into a prooxidant peroxidase in manganese-deficient cells and mice.
      ) and Table 1). These findings demonstrate that, in some cases, incorporation of a noncognate metal ion does not disable an enzyme but switches its function, highlighting the biological significance of the metal selectivity process.
      What is the mechanism behind the metal ions selectivity of SODs? Being structural components of SODs, Fe and Mn ions cycle between +2 and +3 oxidation states during O2•− turnover; however, these oxidation states correspond to different d-electron configurations for Mn and Fe, resulting in distinct +3 to +2 reduction characteristics. It is believed that these differences are compensated by protein components, such as specific amino acids of a secondary coordination sphere of Fe-SOD or Mn-SOD. Of note, the first (inner) coordination sphere refers to the array of direct interactions of a ligand with a metal ion, whereas the secondary (outer) coordination sphere consists of ions that interact with the first coordination sphere, without direct binding to a metal ion. Although Mn-SODs and Fe-SODs share identical metal ion–containing catalytically active sites (Fig. 4A, left and middle panels), different amino acid residues lie adjacent to it and are responsible for metal ion specificity and activity that occur without major structural reorganization of the active center. For example, glutamine from the second coordination sphere of SODs was found to be involved in determining the redox potential of the active site, thus, impacting the metal cofactor selection process. As such, Gln69/Fe-SOD and Gln146/Mn-SOD from E. coli promote metal ion specificity (
      • Wintjens R.
      • Noël C.
      • May A.C.W.
      • Gerbod D.
      • Dufernez F.
      • Capron M.
      • Viscogliosi E.
      • Rooman M.
      Specificity and phenetic relationships of iron- and manganese-containing superoxide dismutases on the basis of structure and sequence comparisons.
      ,
      • Whittaker J.W.
      The irony of manganese superoxide dismutase.
      ,
      • Parker M.W.
      • Blake C.C.
      Iron- and manganese-containing superoxide dismutases can be distinguished by analysis of their primary structures.
      ,
      • Whittaker J.W.
      Prokaryotic manganese superoxide dismutases.
      ), and a similar observation was made of Sod2 (Mn-Sod) from S. cerevisiae (
      • Kang Y.
      • He Y.X.
      • Zhao M.X.
      • Li W.F.
      Structures of native and Fe-substituted SOD2 from Saccharomyces cerevisiae.
      ). Furthermore, the crystal structures of native Mn-Sod2 and artificial Fe-Sod2 from S. cerevisiae at 2.05 and 1.79 Å resolution, respectively, demonstrated no significant alteration in the active site or overall structure upon binding the non-native metal Fe, and identified Asp163 and Lys80 as those responsible for the metal specificity of Mn-SOD (
      • Kang Y.
      • He Y.X.
      • Zhao M.X.
      • Li W.F.
      Structures of native and Fe-substituted SOD2 from Saccharomyces cerevisiae.
      ). Another residue important for the SOD specificity is Thr165 present in Fe-SODs from E. coli (whereas a Gly residue occurs at this position in the majority of Mn-SODs (
      • Yamakura F.
      • Sugio S.
      • Hiraoka B.Y.
      • Ohmori D.
      • Yokota T.
      Pronounced conversion of the metal-specific activity of superoxide dismutase from Porphyromonas gingivalis by the mutation of a single amino acid (Gly155Thr) located apart from the active site.
      ,
      • Bachega J.F.R.
      • Navarro M.V.A.S.
      • Bleicher L.
      • Bortoleto-Bugs R.K.
      • Dive D.
      • Hoffmann P.
      • Viscogliosi E.
      • Garratt R.C.
      Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity.
      )), whereby swapping Thr165 and Gly165 in Fe-SOD (SodB) and Mn-SOD (SodA) changes the metal cofactor preference ((
      • Yamakura F.
      • Sugio S.
      • Hiraoka B.Y.
      • Ohmori D.
      • Yokota T.
      Pronounced conversion of the metal-specific activity of superoxide dismutase from Porphyromonas gingivalis by the mutation of a single amino acid (Gly155Thr) located apart from the active site.
      ,
      • Osawa M.
      • Yamakura F.
      • Mihara M.
      • Okubo Y.
      • Yamada K.
      • Hiraoka B.Y.
      Conversion of the metal-specific activity of Escherichia coli Mn-SOD by site-directed mutagenesis of Gly165Thr.
      ) and Table 1).
      Despite the high metal ion specificity of Mn-SODs and Fe-SODs, some organisms have acquired an alternative form of an enzyme that can accommodate Mn or Fe ions in the catalytic center and retain enzymatic activity. These metal cofactor flexible SODs have been termed cambialistic or cam-SOD (
      • Meier B.
      • Barra D.
      • Bossa F.
      • Calabrese L.
      • Rotilio G.
      Synthesis of either Fe- or Mn-superoxide dismutase with an apparently identical protein moiety by an anaerobic bacterium dependent on the metal supplied.
      ). Cam-SODs were found in microorganisms adapted to different growth conditions, including microaerophiles, aerobes, obligate anaerobes, and thermophiles. This interesting phenomenon raises questions of how and why some of these enzymes developed cambialistic properties.
      In this regard, SodM (cam-SOD) from the Gram-positive opportunistic pathogen Staphylococcus aureus has become an informative experimental model, as it allowed direct comparison with the strictly Mn-dependent SodA (Mn-SOD) isoform (
      • Garcia Y.M.
      • Barwinska-Sendra A.
      • Tarrant E.
      • Skaar E.P.
      • Waldron K.J.
      • Kehl-Fie T.E.
      A superoxide dismutase capable of functioning with iron or manganese promotes the resistance of Staphylococcus aureus to calprotectin and nutritional immunity.
      ,
      • Valderas M.W.
      • Gatson J.W.
      • Wreyford N.
      • Hart M.E.
      The superoxide dismutase gene sodM is unique to Staphylococcus aureus: Absence of sodM in coagulase-negative staphylococci.
      ,
      • Karavolos M.H.
      • Horsburgh M.J.
      • Ingham E.
      • Foster S.J.
      Role and regulation of the superoxide dismutases of Staphylococcus aureus.
      ). Thus, it has been shown that Mn- and Fe-bound cam-SOD exhibits comparable enzymatic activities (
      • Garcia Y.M.
      • Barwinska-Sendra A.
      • Tarrant E.
      • Skaar E.P.
      • Waldron K.J.
      • Kehl-Fie T.E.
      A superoxide dismutase capable of functioning with iron or manganese promotes the resistance of Staphylococcus aureus to calprotectin and nutritional immunity.
      ). Moreover, the X-ray diffraction analysis performed with Mn-loaded or Fe-loaded SodA and SodM demonstrated only minor deviations in the catalytic center architecture and metal binding physicochemical properties, whereby metal ions (Mn or Fe) are coordinated by His27, His81, Asp161, and His165 ((
      • Barwinska-Sendra A.
      • Garcia Y.M.
      • Sendra K.M.
      • Baslé A.
      • Mackenzie E.S.
      • Tarrant E.
      • Card P.
      • Tabares L.C.
      • Bicep C.
      • Un S.
      • Kehl-Fie T.E.
      • Waldron K.J.
      An evolutionary path to altered cofactor specificity in a metalloenzyme.
      ) and Figure 4A, middle and right panels). These structural similarities suggest that cambialism is not provided by the inner sphere coordination geometry but relies on differences in the secondary coordination sphere. Mutational analysis has identified two amino acids present in positions 159 and 160 (Fig. 4B) that vary between SodA (possesses Gly159 and Leu160) and SodM (Leu159 and Phe160) and make no direct contact but are in close proximity (<10 Å) to a metal cofactor (
      • Barwinska-Sendra A.
      • Garcia Y.M.
      • Sendra K.M.
      • Baslé A.
      • Mackenzie E.S.
      • Tarrant E.
      • Card P.
      • Tabares L.C.
      • Bicep C.
      • Un S.
      • Kehl-Fie T.E.
      • Waldron K.J.
      An evolutionary path to altered cofactor specificity in a metalloenzyme.
      ). Significantly, swapping these amino acids between SodA and SodM did not affect active center structures but enabled cambialistic properties to SodA (
      • Barwinska-Sendra A.
      • Garcia Y.M.
      • Sendra K.M.
      • Baslé A.
      • Mackenzie E.S.
      • Tarrant E.
      • Card P.
      • Tabares L.C.
      • Bicep C.
      • Un S.
      • Kehl-Fie T.E.
      • Waldron K.J.
      An evolutionary path to altered cofactor specificity in a metalloenzyme.
      ). It was proposed that amino acid side chains in positions 159 and 160 are responsible for changes in the reduction potential of the metal ions, likely underlying the mechanism of catalysis governed by Mn and Fe ions (
      • Barwinska-Sendra A.
      • Garcia Y.M.
      • Sendra K.M.
      • Baslé A.
      • Mackenzie E.S.
      • Tarrant E.
      • Card P.
      • Tabares L.C.
      • Bicep C.
      • Un S.
      • Kehl-Fie T.E.
      • Waldron K.J.
      An evolutionary path to altered cofactor specificity in a metalloenzyme.
      ). This example indicates that subtle sequence alterations near the active site impose metal specificity on one isoform or allow flexibility in the other. One possible explanation for the impact of the amino acid residues equivalent to 159 and 160 of S. aureus is their role in assembling the appropriate hydrogen-bonding network that includes a metal-coordinated solvent, as described in Ref. (
      • Osawa M.
      • Yamakura F.
      • Mihara M.
      • Okubo Y.
      • Yamada K.
      • Hiraoka B.Y.
      Conversion of the metal-specific activity of Escherichia coli Mn-SOD by site-directed mutagenesis of Gly165Thr.
      ). However, other studies demonstrated that solvent proton positions are similar in the structure of Mn-SOD and cam-SOD (
      • Barwinska-Sendra A.
      • Baslé A.
      • Waldron K.J.
      • Un S.
      A charge polarization model for the metal-specific activity of superoxide dismutases.
      ). These discrepancies call for further evaluation of the mechanisms by which the secondary sphere amino acids control redox tuning in cooperation with Mn and Fe ions.
      The key Leu159 and Phe160 residues of cam-SODs are highly conserved within the lineages of the S. aureus tree, including Staphylococcus argenteus and Staphylococcus schweitzeri (
      • Tong S.Y.C.
      • Schaumburg F.
      • Ellington M.J.
      • Corander J.
      • Pichon B.
      • Leendertz F.
      • Bentley S.D.
      • Parkhill J.
      • Holt D.C.
      • Peters G.
      • Giffard P.M.
      Novel staphylococcal species that form part of a Staphylococcus aureus-related complex: The non-pigmented Staphylococcus argenteus sp. nov. and the non-human primate-associated Staphylococcus schweitzeri sp. nov.
      ). Thus, it has been proposed that cam-SOD arose from a redundant gene encoding a second Mn-SOD via evolutionary-enforced mutagenesis (
      • Barwinska-Sendra A.
      • Garcia Y.M.
      • Sendra K.M.
      • Baslé A.
      • Mackenzie E.S.
      • Tarrant E.
      • Card P.
      • Tabares L.C.
      • Bicep C.
      • Un S.
      • Kehl-Fie T.E.
      • Waldron K.J.
      An evolutionary path to altered cofactor specificity in a metalloenzyme.
      ,
      • Suzuki H.
      • Lefébure T.
      • Bitar P.P.
      • Stanhope M.J.
      Comparative genomic analysis of the genus Staphylococcus including Staphylococcus aureus and its newly described sister species Staphylococcus simiae.
      ). Such an Fe-to-Mn switch likely occurred in response to diminished Fe bioavailability during oxygenation of the atmosphere (
      • Dupont C.L.
      • Butcher A.
      • Valas R.E.
      • Bourne P.E.
      • Caetano-Anollés G.
      History of biological metal utilization inferred through phylogenomic analysis of protein structures.
      ), accompanied by Fe engagement in Fenton chemistry with O2•−. Furthermore, the appearance of modern cam-SODs that are able to utilize both Mn and Fe allows adaptation to a plethora of stresses, including oxidative stress and nutrient starvation, such as Mn scarcity (
      • Garcia Y.M.
      • Barwinska-Sendra A.
      • Tarrant E.
      • Skaar E.P.
      • Waldron K.J.
      • Kehl-Fie T.E.
      A superoxide dismutase capable of functioning with iron or manganese promotes the resistance of Staphylococcus aureus to calprotectin and nutritional immunity.
      ,
      • Kehl-Fie T.E.
      • Chitayat S.
      • Hood M.I.
      • Damo S.
      • Restrepo N.
      • Garcia C.
      • Munro K.A.
      • Chazin W.J.
      • Skaar E.P.
      Nutrient metal sequestration by calprotectin inhibits bacterial superoxide defense, enhancing neutrophil killing of Staphylococcus aureus.
      ), representing elegant stress-resistance strategies (
      • Wessling-Resnick M.
      Nramp1 and other transporters involved in metal withholding during infection.
      ) that are discussed later.

      The R2 subunit of class I RNRs

      The RNRs are an ancient enzyme group uniquely responsible for the production of deoxyribonucleotides (dNTPs) from ribonucleotide precursors (
      • Lundin D.
      • Berggren G.
      • Logan D.T.
      • Sjöberg B.M.
      The origin and evolution of ribonucleotide reduction.
      ,
      • Torrents E.
      • Aloy P.
      • Gibert I.
      • Rodríguez-Trelles F.
      Ribonucleotide reductases: Divergent evolution of an ancient enzyme.
      ). Different cation pair requirements have evolved in isoforms of the RNR R2 subunits. Fe or Mn cations are coordinated by this subunit, with the different metal ions facilitating activity in differing oxidative conditions. A high level of functional interchangeability between Fe or Mn cations has complicated the elucidation of the precise mechanism of the R2 subunit function. The chemically demanding redox reaction catalyzed by RNRs is essential in most organisms as a central controller of DNA replication, positioning these enzymes as potent targets for anticancer and antiviral drugs (
      • Aye Y.
      • Li M.
      • Long M.J.C.
      • Weiss R.S.
      Ribonucleotide reductase and cancer: Biological mechanisms and targeted therapies.
      ,
      • Zimanyi C.M.
      • Chen P.Y.T.
      • Kang G.
      • Funk M.A.
      • Drennan C.L.
      Molecular basis for allosteric specificity regulation in class ia ribonucleotide reductase from Escherichia coli.
      ). The implications of metal selection in these enzymes in pathogens are discussed later.
      Three classes of RNR enzymes exist, all of which involve transition metal cofactors for radical generation and differ in their activity in aerobic or anaerobic environments. Class I RNRs, which are discussed here, are oxygen dependent and generate the catalytic radical via their dimetal binding R2 subunit.
      The R2 subunits of class I RNRs are ferritin-like proteins (
      • Andrews S.C.
      The ferritin-like superfamily: Evolution of the biological iron storeman from a rubrerythrin-like ancestor.
      ,
      • Hintze K.J.
      • Theil E.C.
      Cellular regulation and molecular interactions of the ferritins.
      ). These subunits contain a dinuclear metal binding site where the radical species are generated (
      • Högbom M.
      Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear - an intricate bioinorganic workaround to use different metals for the same reaction.
      ,
      • Kolberg M.
      • Strand K.R.
      • Graff P.
      • Kristoffer Andersson K.
      Structure, function, and mechanism of ribonucleotide reductases.
      ). Metals are incorporated as divalent cations and oxidized to higher oxidation states as part of the activation mechanism. In the prototypic E. coli enzyme of the RNR family, the R2 subunit coordinates two Fe ions, defining class Ia subgroup, which also includes the human and yeast RNRs. The mechanism utilizes the electrons of the bound di-Fe in the reduction of molecular oxygen, resulting in an active state with a tyrosine radical (Tyr122) and an oxidized Fe3+ ion pair (
      • Högbom M.
      Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear - an intricate bioinorganic workaround to use different metals for the same reaction.
      ). While Mn2+ can bind in place of Fe2+, it does not support catalytic activity (
      • Pierce B.S.
      • Hendrich M.P.
      Local and global effects of metal binding within the small subunit of ribonucleotide reductase.
      ).
      R2 proteins from other organisms have since been found to employ a pair of Mn ions (class Ib) or a “heterobimetallic” mixed Mn/Fe cofactor (class Ic) (
      • Cotruvo J.A.
      • Stubbe J.
      Escherichia coli class Ib ribonucleotide reductase contains a dimanganese(III)-tyrosyl radical cofactor in vivo.
      ,
      • Cox N.
      • Ogata H.
      • Stolle P.
      • Reijerse E.
      • Auling G.
      • Lubitz W.
      A tyrosyl-dimanganese coupled spin system is the native metalloradical cofactor of the R2F subunit of the ribonucleotide reductase of Corynebacterium ammoniagenes.
      ,
      • Dassama L.M.K.
      • Krebs C.
      • Bollinger J.M.
      • Rosenzweig A.C.
      • Boal A.K.
      Structural basis for assembly of the MnIV/FeIII cofactor in the class Ic ribonucleotide reductase from Chlamydia trachomatis.
      ). While the di-Fe R2 proteins are damaged by H2O2, the Mn/Fe-binding R2 protein from the human pathogen Chlamydia trachomatis is resistant, and, in fact, becomes oxidized and activated in the presence of H2O2 (
      • Jiang W.
      • Xie J.
      • Nørgaard H.
      • Bollinger J.M.
      • Krebs C.
      Rapid and quantitative activation of Chlamydia trachomatis ribonucleotide reductase by hydrogen peroxide.
      ). Interestingly, the C. trachomatis R2 subunit also has significant adaptations to the residues involved in the transfer of the radical from the metal site to the active site, lacking the highly conserved tyrosine, which usually harbors the radical following oxidation. It was proposed that because of the differing redox properties of the Mn in the metal ion pair, Mn can exist in an Mn2+ state upon oxidation and fulfill the oxidant function of the tyrosine radical. At the same time, Fe is unable to participate (
      • Roos K.
      • Siegbahn P.E.M.
      Density functional theory study of the manganese-containing ribonucleotide reductase from Chlamydia trachomatis: Why manganese is needed in the active complex.
      ).
      An interesting structural feature of the R2 proteins from the Mn/Fe or Mn/Mn binding RNRs allows selective self-assembly with the appropriate cations, despite the generally higher concentration of Fe in cells and the higher predicted stability of Fe2+ complexes over Mn2+ complexes (
      • Dassama L.M.K.
      • Krebs C.
      • Bollinger J.M.
      • Rosenzweig A.C.
      • Boal A.K.
      Structural basis for assembly of the MnIV/FeIII cofactor in the class Ic ribonucleotide reductase from Chlamydia trachomatis.
      ,
      • Grāve K.
      • Griese J.J.
      • Berggren G.
      • Bennett M.D.
      • Högbom M.
      The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron.
      ). Minor changes to coordinating residues can switch the binding preference between the two transition metals. A single mutation of a residue in the second coordination sphere alters the specificity of class Ib R2 subunit of Bacillus anthracis, such that under aerobic conditions, the protein is populated with an Mn/Fe ion pair (
      • Grāve K.
      • Griese J.J.
      • Berggren G.
      • Bennett M.D.
      • Högbom M.
      The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron.
      ).
      The investigation of R2 proteins, which natively coordinate a di-Mn cofactor, proved problematic. Despite in vivo evidence that Mn was required, attempts to reconstitute the enzyme with Mn in vitro produced no activity, whereas introducing Fe was able to restore some enzymatic capacity. This is due to a requirement of the di-Mn mechanism for an additional cofactor, a flavin-mononucleotide coenzyme NrdI, which is present in all organisms expressing class Ib RNR. The oxidation of bound Mn2+ or Fe2+ is required for the activation of the R2 protein, and whereas Fe2+ reacts directly with O2 to become oxidized, Mn2+ requires NrdI to produce the oxidizing radical (
      • Cotruvo J.A.
      • Stich T.A.
      • Britt R.D.
      • Stubbe J.
      Mechanism of assembly of the dimanganese-tyrosyl radical cofactor of class Ib ribonucleotide reductase: Enzymatic generation of superoxide is required for tyrosine oxidation via a Mn(III)Mn(IV) intermediate.
      ).

      Ribosomal Mg is a subject to replacement

      As discussed earlier, the geological data indicate that the changes in earth's atmosphere and metal availability correlate with the biological utilization of metal ions by various biomolecules, including ribosomes (Fig. 3). Similarly to ancient SOD enzymes that appeared early during earth's evolution and underwent a significant divergence over time, the ribosomal core serves remarkably well as a tool to investigate early molecular biology and biochemistry, as it remained largely invariant since the last universal common ancestor (
      • Bernier C.R.
      • Petrov A.S.
      • Kovacs N.A.
      • Penev P.I.
      • Williams L.D.
      Translation: The universal structural core of life.
      ), yet appeared to go through significant changes in respect of the utilization of metal ions (
      • Bray M.S.
      • Lenz T.K.
      • Haynes J.W.
      • Bowman J.C.
      • Petrov A.S.
      • Reddi A.R.
      • Hud N.V.
      • Williams L.D.
      • Glass J.B.
      Multiple prebiotic metals mediate translation.
      ,
      • Bray M.S.
      • Lenz T.K.
      • Bowman J.C.
      • Petrov A.S.
      • Reddi A.R.
      • Hud N.V.
      • Williams L.D.
      • Glass J.B.
      Ferrous iron folds rRNA and mediates translation.
      ).
      The contemporary ribosome coordinates Mg2+ extensively for structural stabilization, with X-ray analysis indicating ∼200 Mg2+ ions are associated with the large subunit alone (
      • Klein D.J.
      The contribution of metal ions to the structural stability of the large ribosomal subunit.
      ), and further studies indicating as many as 1000 Mg2+ sites on the entire ribosome (
      • Zheng H.
      • Shabalin I.G.
      • Handing K.B.
      • Bujnicki J.M.
      • Minor W.
      Magnesium-binding architectures in RNA crystal structures: Validation, binding preferences, classification and motif detection.
      ). At least six distinct Mg2+ binding structures were evident (
      • Bray M.S.
      • Lenz T.K.
      • Haynes J.W.
      • Bowman J.C.
      • Petrov A.S.
      • Reddi A.R.
      • Hud N.V.
      • Williams L.D.
      • Glass J.B.
      Multiple prebiotic metals mediate translation.
      ,
      • Zheng H.
      • Shabalin I.G.
      • Handing K.B.
      • Bujnicki J.M.
      • Minor W.
      Magnesium-binding architectures in RNA crystal structures: Validation, binding preferences, classification and motif detection.
      ), aiding in folding and assembly of the rRNA (
      • Petrov A.S.
      • Bowman J.C.
      • Harvey S.C.
      • Williams L.D.
      Bidentate RNA-magnesium clamps: On the origin of the special role of magnesium in RNA folding.
      ), mediating interactions with tRNA, mRNA, and stabilizing the intersubunit interface (
      • Selmer M.
      Structure of the 70S ribosome complexed with mRNA and tRNA.
      ). Mg ions maintain a kink between the P-site and the A-site of the ribosome (
      • Demeshkina N.
      • Jenner L.
      • Westhof E.
      • Yusupov M.
      • Yusupova G.
      A new understanding of the decoding principle on the ribosome.
      ), and microclusters of Mg2+ pairs within the large subunit stabilize the peptidyl transfer center (
      • Hsiao C.
      • Williams L.D.
      A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center.
      ).
      The high level of conservation of the ribosome since its evolution 3 to 4 billion years ago (
      • Fox G.E.
      Origin and evolution of the ribosome.
      ) leads to the idea that Mg2+ may not have been the original cation utilized in ribosomal structures, which first appeared prior to oxidation of the environment with less abundant Mg2+ and more prevalent ions of Mn2+ or Fe2+ (
      • Bray M.S.
      • Lenz T.K.
      • Haynes J.W.
      • Bowman J.C.
      • Petrov A.S.
      • Reddi A.R.
      • Hud N.V.
      • Williams L.D.
      • Glass J.B.
      Multiple prebiotic metals mediate translation.
      ,
      • Jones C.
      • Nomosatryo S.
      • Crowe S.A.
      • Bjerrum C.J.
      • Canfield D.E.
      Iron oxides, divalent cations, silica, and the early earth phosphorus crisis.
      ). Direct proof for this hypothesis came from a recent study by Bray et al. (
      • Bray M.S.
      • Lenz T.K.
      • Haynes J.W.
      • Bowman J.C.
      • Petrov A.S.
      • Reddi A.R.
      • Hud N.V.
      • Williams L.D.
      • Glass J.B.
      Multiple prebiotic metals mediate translation.
      ), wherein an ancient earth's atmosphere was replicated in an anoxic chamber with a 98% Ar and 2% H2, and lyophilized ribosomes were reassembled in the presence of Mn2+ or Fe2+ instead of Mg2+ ions. This elegant approach demonstrated that ribosomes retained their translational competency when their structure was rebuilt in vitro in the presence of alternative rRNA-stabilizing cations, suggesting metals' interchangeability within a prokaryotic ribosome (
      • Bray M.S.
      • Lenz T.K.
      • Haynes J.W.
      • Bowman J.C.
      • Petrov A.S.
      • Reddi A.R.
      • Hud N.V.
      • Williams L.D.
      • Glass J.B.
      Multiple prebiotic metals mediate translation.
      ). The conservation of the ribosome may mean that Mn2+ or Fe2+ represent ancient binding partners, which remain functional in extant organisms. However, the physiological relevance is dependent on metal availability within the cell. In addition, the presence of oxidant species in the present environment, which would not have been a concern in an anoxic archaean earth, produces the risk of damage to RNA structures closely associating with Fe2+ by Fenton-induced ROS.
      Indeed, a genetic screen of the S. cerevisiae deletion strains conducted in our laboratory has identified grx5Δ and yfh1Δ strains as highly susceptible to oxidant-induced rRNA scissions (
      • Zinskie J.A.
      • Ghosh A.
      • Trainor B.M.
      • Shedlovskiy D.
      • Pestov D.G.
      • Shcherbik N.
      Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.
      ). Both these deletion strains contain a high level of labile Fe (
      • Zinskie J.A.
      • Ghosh A.
      • Trainor B.M.
      • Shedlovskiy D.
      • Pestov D.G.
      • Shcherbik N.
      Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.
      ,
      • Smethurst D.G.J.
      • Kovalev N.
      • McKenzie E.R.
      • Pestov D.G.
      • Shcherbik N.
      Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese.
      ), which prompted investigation of the possibility that rRNA hydrolysis is accomplished via the site-specific Fenton reaction. By devising an in vitro assay, the rRNA cleavage pattern observed in oxidant-treated grx5Δ cells was recapitulated in vitro with ribosomes purified from wildtype cells grown under normal nutrient-rich conditions, Fe(NH4)2(SO4)2 and ascorbic acid used as a prooxidant. The intensity and number of rRNA cleavage events were dependent on a concentration of Fe present in the in vitro reaction, as well as in strains carrying various levels of labile Fe, and correlated with cell viability. Interestingly, treatment of ribosomes with ascorbic acid alone still resulted in low-level cleavage within the expansion segment 7 of the large ribosomal subunit 60S (ES7L) of 25S rRNA (Fig. 5A), suggesting that even under normal growth conditions, ribosomes retained an ability to replace Mg2+ with Fe2+ at the selected sites. Similar low-intensity ES7L 25S rRNA cleavage has been detected upon treatment of cells with low doses of H2O2 (a condition, wherein H2O2 functions as a signaling molecule (
      • Veal E.A.
      • Day A.M.
      • Morgan B.A.
      Hydrogen peroxide sensing and signaling.
      )), thus promoting resistance to subsequent acute oxidative stress (
      • Shedlovskiy D.
      • Zinskie J.A.
      • Gardner E.
      • Pestov D.G.
      • Shcherbik N.
      Endonucleolytic cleavage in the expansion segment 7 of 25S rRNA is an early marker of low-level oxidative stress in yeast.
      ). The Fe-dependent ES7L cleavage did not affect the translational activity of ribosomes, further suggesting a role of Mg2+-to-Fe2+ replacement within ES7L in the adaptive response to stress (
      • Shedlovskiy D.
      • Zinskie J.A.
      • Gardner E.
      • Pestov D.G.
      • Shcherbik N.
      Endonucleolytic cleavage in the expansion segment 7 of 25S rRNA is an early marker of low-level oxidative stress in yeast.
      ). Structural data (
      • Zinskie J.A.
      • Ghosh A.
      • Trainor B.M.
      • Shedlovskiy D.
      • Pestov D.G.
      • Shcherbik N.
      Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.
      ) demonstrated that 2 Mg2+ ions exposed to the solvent side are located ∼6 to 8 Å away from the ES7L cleavage site (A611↓U612), implying that •OH radical generated during the Fenton reaction is in close proximity to the sugar phosphate backbone (Fig. 5A). Furthermore, we identified that Fe2+-mediated oxidant-dependent rRNA hydrolysis (
      • Shedlovskiy D.
      • Zinskie J.A.
      • Gardner E.
      • Pestov D.G.
      • Shcherbik N.
      Endonucleolytic cleavage in the expansion segment 7 of 25S rRNA is an early marker of low-level oxidative stress in yeast.
      ) was mitigated by increased Mn2+ availability, which seemingly can compete with Fe2+ for binding sites, resists induction of the Fenton reaction and, thus, accomplishes a protective role (
      • Smethurst D.G.J.
      • Kovalev N.
      • McKenzie E.R.
      • Pestov D.G.
      • Shcherbik N.
      Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese.
      ). The impact of Mn and Fe binding to ribosomes is outlined in Figure 5B. These results were consistent with the protective effect of replacing Fe2+ with Mn2+ described in E. coli under oxidative stress (
      • Anjem A.
      • Imlay J.A.
      Mononuclear iron enzymes are primary targets of hydrogen peroxide stress.
      • Sobota J.M.
      • Imlay J.A.
      Iron enzyme ribulose-5-phosphate 3-epimerase in Escherichia coli is rapidly damaged by hydrogen peroxide but can be protected by manganese.
      ).
      Figure thumbnail gr5
      Figure 5rRNA is a substrate for the site-specific Fenton reaction. A, the highly conserved part of the expansion segment 7 (ES7L) of the large ribosomal subunit 60S from Saccharomyces cerevisiae. The depicted fragment corresponds to nucleotide pairs A501:U612 and U502:A611 of the ES7L and is shown in colors, surrounding bases are shown in gray. Dashed lines indicate predicted polar contacts between the four conserved bases (A501:U612 and U502:A611). Mg2+ ions are shown as green spheres. The Protein Data Bank file 4V88 was used. Replacement of Mg2+ with Fe2+ (shown as an orange sphere) powers the rRNA-localized Fenton reaction under oxidative conditions, whereupon the hydroxyl radical (•OH) is formed. •OH hydrolyses the sugar phosphate backbone at the specific site between A611 and U612. Modified from Ref. (
      • Zinskie J.A.
      • Ghosh A.
      • Trainor B.M.
      • Shedlovskiy D.
      • Pestov D.G.
      • Shcherbik N.
      Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.
      ). B, Mg2+, Fe2+, and Mn2+ interchangeability on the ribosome. Unstressed cells contain active ribosomes, which bind divalent metal cations throughout their structure (bottom). These are predominantly Mg2+ but include a number of Fe2+ ions. In oxidative stress conditions, Fe2+ participates in Fenton reactions generating •OH radicals, which cleave the rRNA and fragment the ribosome (top left). However, if sufficient Mn2+ is available, the Fe2+ is displaced and Mn2+ occupies these sites. Under oxidative stress, a ribosome that coordinates Mn2+ in place of Fe2+ is not subjected to the generation of hydrolytic radical species and, thereby, is resistant to the stress-induced damages (top right). Modified from Ref. (
      • Smethurst D.G.J.
      • Kovalev N.
      • McKenzie E.R.
      • Pestov D.G.
      • Shcherbik N.
      Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese.
      ).
      Taken together, it seems reasonable to propose that the translation machinery maintained an ability to associate with the transition metal cations that stabilized its structure. Given that eukaryotic ribosomes retained the capability to replace Fenton-resistant Mg2+ with Fenton-active Fe2+ through the course of evolution, it is possible that this newly identified quality of the protein-translating machinery plays a regulatory role during gene expression as a means of adjustment to environmental changes.
      The work described in this section was conducted and published recently, and, therefore, many questions have remained unanswered. It will be important to map other cleavage sites, dissect the molecular mechanics of stress adaptation accomplished by Fenton-cleaved ribosomes, investigate site-specific Fenton cleavages within human ribosomes, and elucidate human disease relevance. Furthermore, we would like to highlight that the discovery of metal interchangeability within the ancient molecular structure of the ribosome identifies an overlooked yet promising model molecule to study the evolution of biomolecules. Such observations recontextualize the ribosome, which is often considered as a stable entity resistant to changes or environment-induced perturbations, as being subject to metal ion replacement depending on genetically or environmentally induced changes in metal homeostasis. Thus, researchers should take special considerations while studying fundamental processes, such as translation and translational control.
      The largely invariant core of the ribosome and the persistence of the translation machinery since the last universal common ancestor are central to many hypotheses regarding cellular evolution (
      • Fox G.E.
      Origin and evolution of the ribosome.
      ,
      • Root-Bernstein R.
      • Root-Bernstein M.
      The ribosome as a missing link in prebiotic evolution III: Over-representation of tRNA-and rRNA-like sequences and plieofunctionality of ribosome-related molecules argues for the evolution of primitive genomes from ribosomal RNA modules.
      ). The oldest and most conserved part of the ribosome is free of protein and supports an early phase of biology, which was dominated by RNA. It has been demonstrated that RNA species are capable of carrying out Fe and Mn-mediated redox mechanisms (
      • Hamm M.L.
      • Nikolic D.
      • Van Breemen R.B.
      • Piccirilli J.A.
      Unconventional origin of metal ion rescue in the hammerhead ribozyme reaction: Mn2+-assisted redox conversion of 2′-mercaptocytidine to cytidine.
      ,
      • Hsiao C.
      • Chou I.-C.
      • Okafor C.D.
      • Bowman J.C.
      • O'Neill E.B.
      • Athavale S.S.
      • Petrov A.S.
      • Hud N.V.
      • Wartell R.M.
      • Harvey S.C.
      • Williams L.D.
      RNA with iron(II) as a cofactor catalyses electron transfer.
      ). Under anoxic conditions, a single-electron transfer reaction, much like those fundamental to metabolism, can be performed by RNA coordinating Fe2+ instead of Mg2+ (
      • Hsiao C.
      • Chou I.-C.
      • Okafor C.D.
      • Bowman J.C.
      • O'Neill E.B.
      • Athavale S.S.
      • Petrov A.S.
      • Hud N.V.
      • Wartell R.M.
      • Harvey S.C.
      • Williams L.D.
      RNA with iron(II) as a cofactor catalyses electron transfer.
      ). It is possible to substitute Mn2+ for Mg2+ in the hammerhead ribozyme, which is a small self-cleaving RNA that catalyzes reversible cleavage and ligation reactions at a specific site. Using 2′-mercaptonucleosides as biochemical probe and the “metal specificity switch” approach, it was found that Mn2+-to-Mg2+ substitution enhances enzymatic activity of the hammerhead ribozyme (
      • Hamm M.L.
      • Nikolic D.
      • Van Breemen R.B.
      • Piccirilli J.A.
      Unconventional origin of metal ion rescue in the hammerhead ribozyme reaction: Mn2+-assisted redox conversion of 2′-mercaptocytidine to cytidine.
      ). These findings indicate the capability of RNA molecules to associate with a greater range of metal ions and possess more diverse catalytic abilities than are frequently observed in extant biology.

      Interchangeability with other metal ions

      While much of the research on this topic focuses on Mn and Mg replacement by Fe at metal binding sites, flexible binding certainly extends to other biologically relevant metal ions. Zn is a widely utilized metal ion in proteins and may have replaced Fe as a cofactor in many enzymes where it presents less of a risk of toxicity (
      • Cardenas-Rodriguez M.
      • Chatzi A.
      • Tokatlidis K.
      Iron–sulfur clusters: From metals through mitochondria biogenesis to disease.
      ).
      The Zn finger (ZF) domain represents one of the most widespread and diverse structural motifs in biology, interacting with nucleic acid, protein, and lipid targets (
      • Kluska K.
      • Adamczyk J.
      • Krężel A.
      Metal binding properties, stability and reactivity of zinc fingers.
      ), and the bound Zn cation can be subject to replacement, such as in the estrogen receptor (
      • Koch K.A.
      • Peña M.M.O.
      • Thiele D.J.
      Copper-binding motifs in catalysis, transport, detoxification and signaling.
      ,
      • Predki P.F.
      • Sarkar B.
      Effect of replacement of “zinc finger” zinc on estrogen receptor DNA interactions.
      ). In this enzyme, Cu or Ni substitution results in loss of function, whereas DNA binding capacity is retained with Co or cadmium (
      • Predki P.F.
      • Sarkar B.
      Effect of replacement of “zinc finger” zinc on estrogen receptor DNA interactions.
      ). In the case of Cu, this example illustrates the potential for damage caused by uncontrolled Cu availability as it can deactivate enzymes by displacement of Zn, in addition to its contribution to the generation of •OH radicals. In addition, replacement of Zn2+ with Fe2+ in the ZF domain of the estrogen receptor does not abolish DNA binding but can induce generation of oxidative radicals and cause DNA damage (
      • Conte D.
      • Narindrasorasak S.
      • Sarkar B.
      In vivo and in vitro iron-replaced zinc finger generates free radicals and causes DNA damage.