Michael J. Maroney

Superoxide dismutases and superoxide reductases.

Superoxide, O2•–, is formed in all living organisms that come in contact with air, and, depending upon its biological context, it may act as a signaling agent, a toxic species, or a harmless intermediate that decomposes spontaneously. Its levels are limited in vivo by two different types of enzymes, superoxide reductase (SOR) and superoxide dismutase (SOD). Although superoxide has long been an important factor in evolution, it was not so when life first emerged on Earth at least 3.5 billion years ago. At that time, the early biosphere was highly reducing and lacking in any significant concentrations of dioxygen (O2), very different from what it is today. Consequently, there was little or no O2•– and therefore no reason for SOR or SOD enzymes to evolve. Instead, the history of biological O2•– probably commences somewhere around 2.4 billion years ago, when the biosphere started to experience what has been termed the “Great Oxidation Event”, a transformation driven by the increase in O2 levels, formed by cyanobacteria as a product of oxygenic photosynthesis.1 The rise of O2 on Earth caused a reshaping of existing metabolic pathways, and it triggered the development of new ones.2 Its appearance led to the formation of the so-called “reactive oxygen species” (ROS), for example, superoxide, hydrogen peroxide, and hydroxyl radical, and to a need for antioxidant enzymes and other antioxidant systems to protect against the growing levels of oxidative damage to living systems. Dioxygen is a powerful four-electron oxidizing agent, and the product of this reduction is water. 1 When O2 is reduced in four sequential one-electron steps, the intermediates formed are the three major ROS, that is, O2•–, H2O2, and HO•. 2 3 4 5 Each of these intermediates is a potent oxidizing agent. The consequences of their presence to early life must have been an enormous evolutionary challenge. In the case of superoxide, we find the SOD and SOR enzymes to be widely distributed throughout current living organisms, both aerobic and anaerobic, suggesting that, from the start of the rise of O2 on Earth, the chemistry of superoxide has been an important factor during evolution. The SORs and three very different types of SOD enzymes are redox-active metalloenzymes that have evolved entirely independently from one another for the purpose of lowering superoxide concentrations. SORs catalyze the one-electron reduction of O2•– to give H2O2, a reaction requiring two protons per superoxide reacted as well as an external reductant to provide the electron (eq 6). SODs catalyze the disproportionation of superoxide to give O2 and H2O2, a reaction requiring one proton per superoxide reacted, but no external reductant (eq 7). 6 7 All of the SOR enzymes contain only iron, while the three types of SODs are the nickel-containing SODs (NiSOD), the iron- or manganese-containing SODs (FeSOD and MnSOD), and the copper- and zinc-containing SODs (CuZnSOD). Although the structures and other properties of these four types of metalloenzymes are quite different, they all share several characteristics, including the ability to react rapidly and selectively with the small anionic substrate O2•–. Consequently, there are some striking similarities between these otherwise dissimilar enzymes, many of which can be explained by considering the nature of the chemical reactivity of O2•– (see below). Numerous valuable reviews describing the SOD and SOR enzymes have appeared over the years, but few have covered and compared all four classes of these enzymes, as we attempt to do here. Thus, the purpose of this Review is to describe, compare, and contrast the properties of the SOR and the four SOD enzymes; to summarize what is known about their evolutionary pathways; and to analyze the properties of these enzymes in light of what is known of the inherent chemical reactivity of superoxide.

Ni(II) and Co(II) sensing by Escherichia coli RcnR

Escherichia coli RcnR and Mycobacterium tuberculosis CsoR are the founding members of a recently identified, large family of bacterial metal-responsive DNA-binding proteins. RcnR controls the expression of the metal efflux protein RcnA only in response to Ni(II) and Co(II) ions. Here, the interaction of Ni(II) and Co(II) with wild-type and mutant RcnR proteins is examined to understand how these metals function as allosteric effectors. Both metals bind to RcnR with nanomolar affinity and stabilize the protein to denaturation. X-ray absorption and electron paramagnetic resonance spectroscopies reveal six-coordinate high-spin sites for each metal that contains a thiolate ligand. Experimental data support a tripartite N-terminal coordination motif (NH2-Xaa-NH-His) that is common for both metals. However, the Ni(II)- and Co(II)-RcnR complexes are shown to differ in the remaining coordination environment. Each metal coordinates a conserved Cys ligand but with distinct M-S distances. Co(II)-thiolate coordination has not been observed previously in Ni(II)-/Co(II)-responsive metalloregulators. The ability of RcnR to recruit ligands from the N-terminal region of the protein distinguishes it from CsoR, which uses a lower coordination geometry to bind Cu(I). These studies facilitate comparisons between Ni(II)-RcnR and NikR, the other Ni(II)-responsive transcriptional regulator in E. coli, to provide a better understanding how different nickel levels are sensed in E. coli. The characterization of the Ni(II)- and Co(II)-binding sites in RcnR, in combination with bioinformatics analysis of all RcnR/CsoR family members, identified a four amino acid fingerprint that likely defines ligand-binding specificity, leading to an emerging picture of the similarities and differences between different classes of RcnR/CsoR proteins.

Nickel Ions Inhibit Histone Demethylase JMJD1A and DNA Repair Enzyme ABH2 by Replacing the Ferrous Iron in the Catalytic Centers

Iron- and 2-oxoglutarate-dependent dioxygenases are a diverse family of non-heme iron enzymes that catalyze various important oxidations in cells. A key structural motif of these dioxygenases is a facial triad of 2-histidines-1-carboxylate that coordinates the Fe(II) at the catalytic site. Using histone demethylase JMJD1A and DNA repair enzyme ABH2 as examples, we show that this family of dioxygenases is highly sensitive to inhibition by carcinogenic nickel ions. We find that, with iron, the 50% inhibitory concentrations of nickel (IC50 [Ni(II)]) are 25 μm for JMJD1A and 7.5 μm for ABH2. Without iron, JMJD1A is 10 times more sensitive to nickel inhibition with an IC50 [Ni(II)] of 2.5 μm, and approximately one molecule of Ni(II) inhibits one molecule of JMJD1A, suggesting that nickel causes inhibition by replacing the iron. Furthermore, nickel-bound JMJD1A is not reactivated by excessive iron even up to a 2 mm concentration. Using x-ray absorption spectroscopy, we demonstrate that nickel binds to the same site in ABH2 as iron, and replacement of the iron by nickel does not prevent the binding of the cofactor 2-oxoglutarate. Finally, we show that nickel ions target and inhibit JMJD1A in intact cells, and disruption of the iron-binding site decreases binding of nickel ions to ABH2 in intact cells. Together, our results reveal that the members of this dioxygenase family are specific targets for nickel ions in cells. Inhibition of these dioxygenases by nickel is likely to have widespread impacts on cells (e.g. impaired epigenetic programs and DNA repair) and may eventually lead to cancer development.

Biochemical characterization of purified OmcS, a c-type cytochrome required for insoluble Fe(III) reduction in Geobacter sulfurreducens

Previous studies with Geobacter sulfurreducens have demonstrated that OmcS, an abundant c-type cytochrome that is only loosely bound to the outer surface, plays an important role in electron transfer to Fe(III) oxides as well as other extracellular electron acceptors. In order to further investigate the function of OmcS, it was purified from a strain that overproduces the protein. Purified OmcS had a molecular mass of 47015 Da, and six low-spin bis-histidinyl hexacoordinated heme groups. Its midpoint redox potential was -212 mV. A thermal stability analysis showed that the cooperative melting of purified OmcS occurs in the range of 65-82 °C. Far UV circular dichroism spectroscopy indicated that the secondary structure of purified OmcS consists of about 10% α-helix and abundant disordered structures. Dithionite-reduced OmcS was able to transfer electrons to a variety of substrates of environmental importance including insoluble Fe(III) oxide, Mn(IV) oxide and humic substances. Stopped flow analysis revealed that the reaction rate of OmcS oxidation has a hyperbolic dependence on the concentration of the studied substrates. A ten-fold faster reaction rate with anthraquinone-2,6-disulfonate (AQDS) (25.2 s⁻¹) was observed as compared to that with Fe(III) citrate (2.9 s⁻¹). The results, coupled with previous localization and gene deletion studies, suggest that OmcS is well-suited to play an important role in extracellular electron transfer.

Nonredox Nickel Enzymes

ion as well as other steps contributed to the mechanism Figure 11. Sequence alignments of selected class I and class II glyoxalase I enzymes created using Clustal W2. Amino acids are colored by property (hydrophobic (red), acidic (blue), basic (purple), other (green)). Metal binding residues are highlighted in yellow. Residues marked with an asterisk (∗) are invariant; thosemarked by other symbols represent low (:) andmoderate (.) variability. TheN-terminal extension and additional loops found in class I enzymes are highlighted in blue. The S. cerevisiae sequence was truncated after 226 of 326 residues. Chemical Reviews Review dx.doi.org/10.1021/cr4004488 | Chem. Rev. 2014, 114, 4206−4228 4218 of product formation. One possible explanation for the difference between the Ni(II)and Cd(II)-bound enzyme kinetics is that differences in the extent of polarization of the substrate result from different metals in the active site, with Cd(II) being less polarizing and therefore less efficient at lowering the energy barrier for proton abstraction. Differences in aqua ligand exchange rates may also play a role, particularly for the Ni(II) enzyme where a slower on-rate for the substrate might suppress the observation of the KIE. Once formed, the enediol(ate) can be protonated at the alternate C atom to complete the isomerization. Based in part on crystal structures of inhibitor complexes (vide supra), mechanisms involving coordination of the enediolate have been proposed (Figure 14), as have mechanisms that involve activatingmetal-bound water molecules to serve as catalytic bases without binding the substrate to the metal. The crystal structures reveal the apparent importance of two cis-aqua ligands in the active site, which could indicate the need for two ciscoordination positions, or a mechanism that employs proton transfers involving both aqua ligands. Coordination of the substrate with loss of a carboxylate ligand has the attractive feature that the Ni(II) site is always charge neutral, and consistent with model chemistry. Efforts to distinguish these mechanisms have employed kinetics and XAS studies of the E. coli Ni(II) enzyme in solution in the presence of various inhibitors, mutations, etc., and the crystal structure of human Glo I obtained in the presence of the transition state analogue, S(N-hydroxy-N-p-iodophenylcarbamoyl)glutathione. The latter structure reveals a five-coordinate Zn center in which both water molecules and Glu172 are displaced upon binding the inhibitor, which binds in a bidentate fashion. Glu172 was proposed to play a role as the catalytic base in the deprotonation and reprotonation of the substrate, a role that is consistent with computational models. Both Rand S-enantiomers of substrates are bound and stereospecifically reprotonated. The presence of two chemically equivalent Glu ligands suggests that one (Glu172) might be the catalytic base for the S-enantiomer, while the other (Glu99) serves that role for the Renantiomer. The corresponding residues in the E. coli enzyme are Glu122 and Glu56, and XAS studies of a hydroxamate inhibitor (L-γ-glutamyl-N-hydroxy-N-methyl-L-glutaminylglycine) complex are consistent with substitution of both aqua ligands, although evidence for the release of a carboxylate was not specifically observed. Although changes in metal ligands greatly affect metal binding affinities, mutation of Glu56 in E56A-E. coli Glo I resulted in the reduction of enzymatic activity to <4% of wild type under conditions of metal saturation, consistent with similar mutations of class I enzymes and the putative role for the carboxylate as a catalytic base. Figure 12. Ribbon diagram of the crystal structure of E. coliGlo I, (PDB code 1F9Z) showing the two subunits of the homo dimer in cyan and gray and the location of the two Ni sites (green spheres) at subunit interfaces. Figure 13.Comparison of the metal site structure of the Ni(II) complex (panel A, PDB code 1F9Z) and the Zn(II) complex (panel B, 1FA5) of E. coli Glo I showing the change in coordination number and geometry for the two metals. The nickel and zinc ions are represented in gray and dark blue, respectively, while CPK coloring is used for all other atoms. WAT = solvent molecules. Protein residues are distinguished by letters indicating the two different subunits of the enzyme. Chemical Reviews Review dx.doi.org/10.1021/cr4004488 | Chem. Rev. 2014, 114, 4206−4228 4219

Structure/function relationships in nickel metallobiochemistry

Among the many highlights of nickel metallobiochemistry in 1998 were the discoveries that Escherichia coli glyoxalase I is the first example of a nickel isomerase, and that the superoxide dismutase isolated from Streptomyces seoulensis is a new structural class of superoxide dismutase that features thiolate ligation.

Octopine and nopaline strains of Agrobacterium tumefaciens differ in virulence; molecular characterization of the virF locus

Octopine and nopaline strains of Agrobacterium tumefaciens were found to differ in virulence on Nicotiana glauca. This difference is due to the absence of a functional virF locus, which is necessary for efficient tumorigenesis on N. glauca, from the nopaline Ti plasmids. Genetic studies and DNA sequence analysis of the virF locus revealed that virF embraces one open reading frame coding for a hydrophilic protein with a molecular mass of 22437 Da. Transcription of virF is directed from left to right, towards the T region, and is strongly induced by the phenolic compound acetosyringone. We established that virA and virG, two genes known to be essential for induction of the vir regulon, are necessary for acetosyringoneinduced virF expression, implying that virF is a member of this vir regulon. Agrobacterium virF mutants can be complemented for tumor induction by co-infection with avirulent Agrobacterium ‘helper’ strains. We found that such ‘helper’ strains must express not only the virF gene but also the vir operons virA, virB, virD and virG.

Modeling and experiment yields the structure of acireductone dioxygenase from Klebsiella pneumoniae

Here we report the structure of acireductone dioxygenase (ARD), the first determined for a new family of metalloenzymes. ARD represents a branch point in the methionine salvage pathway leading from methylthioadenosine to methionine and has been shown to catalyze different reactions depending on the type of metal ion bound in the active site. The solution structure of nickel-containing ARD (Ni-ARD) was determined using NMR methods. X-ray absorption spectroscopy, assignment of hyperfine shifted NMR resonances and conserved domain homology were used to model the metal-binding site because of the paramagnetism of the bound Ni2+. Although there is no structure in the Protein Data Bank within 3 Å r.m.s deviation of that of Ni-ARD, the enzyme active site is located in a conserved double-stranded b-helix domain. Furthermore, the proposed Ni-ARD active site shows significant post-facto structural homology to the active sites of several metalloenzymes in the cupin superfamily.

Direct Evidence of Active-Site Reduction and Photodriven Catalysis in Sensitized Hydrogenase Assemblies

We report photocatalytic H(2) production by hydrogenase (H(2)ase)-quantum dot (QD) hybrid assemblies. Quenching of the CdTe exciton emission was observed, consistent with electron transfer from the quantum dot to H(2)ase. GC analysis showed light-driven H(2) production in the presence of a sacrificial electron donor with an efficiency of 4%, which is likely a lower limit for these hybrid systems. FTIR spectroscopy was employed for direct observation of active-site reduction in unprecedented detail for photodriven H(2)ase catalysis with sensitivity toward both H(2)ase and the sacrificial electron donor. Photosensitization with Ru(bpy)(3)(2+) showed distinct FTIR photoreduction properties, generating all of the states along the steady-state catalytic cycle with minimal H(2) production, indicating slow, sequential one-electron reduction steps. Comparing the H(2)ase activity and FTIR results for the two systems showed that QDs bind more efficiently for electron transfer and that the final enzyme state is different for the two sensitizers. The possible origins of these differences and their implications for the enzymatic mechanism are discussed.

Nickel coordination is regulated by the DNA-bound state of NikR

The uptake of nickel in Escherichia coli and other microorganisms is transcriptionally regulated by the NikR repressor or its homologs. Here we report the structure of the high-affinity nickel-binding site in NikR and show that it responds dramatically to DNA binding. X-ray absorption spectroscopy reveals that nickel in the holo-NikR protein is bound in a novel four-coordinate planar site consisting of two histidines, one additional O- or N-donor ligand and one S-donor ligand. Site-directed mutation of His87, His89, Cys95 or Glu97 in NikR to alanine eliminates high-affinity nickel binding and abolishes DNA binding but maintains stable protein folding. An unanticipated feature of the NikR structure is that the nickel coordination responds to DNA binding. A six-coordinate nickel site composed of O- or N-donor ligands, but lacking cysteine, forms when NikR binds to operator DNA. Because nickel binding and DNA binding are mediated by different domains within NikR, a communication link between the two domains is implicated, consistent with the finding that the nickel-binding site in a fragment corresponding to the C-terminal domain of NikR is structurally distinct from that found in holo-NikR.