Ni Superoxide Dismutase Research Articles

Outstanding Superoxide Dismutase Catalytic Activity Of Simple Peptide‐Based Nickel(II) Complexes

AbstractWe present here the most active synthetic Ni superoxide dismutase (NiSOD) mimic reported to date. Reactive oxygen species are aggressive compounds, which concentrations are tightly regulated in vivo. Among them, the superoxide anion, O2⋅−, is controlled by superoxide dismutases. Capitalizing on the versatility of the Amino‐Terminal CuII‐ and NiII‐binding (ATCUN) peptide motif, we introduced positive charges around the NiII center to favor the interaction with the superoxide radical anion. At physiological pH, the pentapeptide H−Cys−His−Cys−Arg−Arg−NH2 coordinates NiII after the deprotonation of one thiol, two amides, and either the second thiol or the N‐terminal ammonium, leading to an equilibrium between the two N3S1 and N2S2 coordination modes. Under catalytic conditions, a kcat value of 8.6(4)×106 L.mol−1.s−1 was measured. Within the first second, the catalyst remained undegraded with quantitative consumption of O2⋅− (completed up to 37 catalytic cycles). An extra arginine (Arg) was introduced at the peptide C‐terminus to increase the global charge of the NiII complex from +1 to +2. This had no effect on the catalytic performance, highlighting the critical role of charge distribution in space as a determining factor influencing the reactivity.

Outstanding Superoxide Dismutase Catalytic Activity Of Simple Peptide-Based Nickel(II) Complexes.

We present here the most active synthetic Ni superoxide dismutase (NiSOD) mimic reported to date. Reactive oxygen species are aggressive compounds, which concentrations are tightly regulated in vivo. Among them, the superoxide anion, O2⋅-, is controlled by superoxide dismutases. Capitalizing on the versatility of the Amino-Terminal CuII- and NiII-binding (ATCUN) peptide motif, we introduced positive charges around the NiII center to favor the interaction with the superoxide radical anion. At physiological pH, the pentapeptide H-Cys-His-Cys-Arg-Arg-NH2 coordinates NiII after the deprotonation of one thiol, two amides, and either the second thiol or the N-terminal ammonium, leading to an equilibrium between the two N3S1 and N2S2 coordination modes. Under catalytic conditions, a kcat value of 8.6(4)×106 L.mol-1.s-1 was measured. Within the first second, the catalyst remained undegraded with quantitative consumption of O2⋅- (completed up to 37 catalytic cycles). An extra arginine (Arg) was introduced at the peptide C-terminus to increase the global charge of the NiII complex from +1 to +2. This had no effect on the catalytic performance, highlighting the critical role of charge distribution in space as a determining factor influencing the reactivity.

Nickel stress-tolerance in plant-bacterial associations.

Nickel (Ni) is an essential element for plant growth and is a constituent of several metalloenzymes, such as urease, Ni-Fe hydrogenase, Ni-superoxide dismutase. However, in high concentrations, Ni is toxic and hazardous to plants, humans and animals. High levels of Ni inhibit plant germination, reduce chlorophyll content, and cause osmotic imbalance and oxidative stress. Sustainable plant-bacterial native associations are formed under Ni-stress, such as Ni hyperaccumulator plants and rhizobacteria showed tolerance to high levels of Ni. Both partners (plants and bacteria) are capable to reduce the Ni toxicity and developed different mechanisms and strategies which they manifest in plant-bacterial associations. In addition to physical barriers, such as plants cell walls, thick cuticles and trichomes, which reduce the elevated levels of Ni entrance, plants are mitigating the Ni toxicity using their own antioxidant defense mechanisms including enzymes and other antioxidants. Bacteria in its turn effectively protect plants from Ni stress and can be used in phytoremediation. PGPR (plant growth promotion rhizobacteria) possess various mechanisms of biological protection of plants at both whole population and single cell levels. In this review, we highlighted the current understanding of the bacterial induced protective mechanisms in plant-bacterial associations under Ni stress.

Evolution of Superoxide Dismutases and Catalases in Cyanobacteria: Occurrence of the Antioxidant Enzyme Genes before the Rise of Atmospheric Oxygen.

Knowledge on the evolution of antioxidant systems in cyanobacteria is crucial for elucidating the cause and consequence of the rise of atmospheric oxygen in the Earth's history. In this study, to elucidate the origin and evolution of cyanobacterial antioxidant enzymes, we analyzed the occurrence of genes encoding four types of superoxide dismutases and three types of catalases in 85 complete cyanobacterial genomes, followed by phylogenetic analyses. We found that Fe superoxide dismutase (FeSOD), Mn superoxide dismutase (MnSOD), and Mn catalase (MnCat) are widely distributed among modern cyanobacteria, whereas CuZn superoxide dismutase (CuZnSOD), bifunctional catalase (KatG), and monofunctional catalase (KatE) are less common. Ni superoxide dismutase (NiSOD) is distributed among marine Prochlorococcus and Synechococcus species. Phylogenetic analyses suggested that bacterial MnSOD evolved from cambialistic Fe/MnSOD before the diversification of major bacterial lineages. The analyses suggested that FeSOD evolved from MnSOD before the origin of cyanobacteria. MnCat also evolved in the early stages of bacterial evolution, predating the emergence of cyanobacteria. KatG, KatE, and NiSOD appeared 2.3-2.5 billion years ago. Thus, almost all cyanobacterial antioxidant enzymes emerged before or during the rise of atmospheric oxygen. The loss and appearance of these enzymes in marine cyanobacteria may be also related to the change in the metal concentration induced by the increased oxygen concentration in the ocean.

Structure, function, and biosynthesis of nickel‐dependent enzymes

Nickel enzymes, present in archaea, bacteria, plants, and primitive eukaryotes are divided into redox and nonredox enzymes and play key functions in diverse metabolic processes, such as energy metabolism and virulence. They catalyze various reactions by using active sites of diverse complexities, such as mononuclear nickel in Ni-superoxide dismutase, glyoxylase I and acireductone dioxygenase, dinuclear nickel in urease, heteronuclear metalloclusters in [NiFe]-carbon monoxide dehydrogenase, acetyl-CoA decarbonylase/synthase and [NiFe]-hydrogenase, and even more complex cofactors in methyl-CoM reductase and lactate racemase. The presence of metalloenzymes in a cell necessitates a tight regulation of metal homeostasis, in order to maintain the appropriate intracellular concentration of nickel while avoiding its toxicity. As well, the biosynthesis and insertion of nickel active sites often require specific and elaborated maturation pathways, allowing the correct metal to be delivered and incorporated into the target enzyme. In this review, the phylogenetic distribution of nickel enzymes will be briefly described. Their tridimensional structures as well as the complexity of their active sites will be discussed. In view of the latest findings on these enzymes, a special focus will be put on the biosynthesis of their active sites and nickel activation of apo-enzymes.

Role of Nickel in Microbial Pathogenesis

Nickel is an essential cofactor for some pathogen virulence factors. Due to its low availability in hosts, pathogens must efficiently transport the metal and then balance its ready intracellular availability for enzyme maturation with metal toxicity concerns. The most notable virulence-associated components are the Ni-enzymes hydrogenase and urease. Both enzymes, along with their associated nickel transporters, storage reservoirs, and maturation enzymes have been best-studied in the gastric pathogen Helicobacter pylori, a bacterium which depends heavily on nickel. Molecular hydrogen utilization is associated with efficient host colonization by the Helicobacters, which include both gastric and liver pathogens. Translocation of a H. pylori carcinogenic toxin into host epithelial cells is powered by H2 use. The multiple [NiFe] hydrogenases of Salmonella enterica Typhimurium are important in host colonization, while ureases play important roles in both prokaryotic (Proteus mirabilis and Staphylococcus spp.) and eukaryotic (Cryptoccoccus genus) pathogens associated with urinary tract infections. Other Ni-requiring enzymes, such as Ni-acireductone dioxygenase (ARD), Ni-superoxide dismutase (SOD), and Ni-glyoxalase I (GloI) play important metabolic or detoxifying roles in other pathogens. Nickel-requiring enzymes are likely important for virulence of at least 40 prokaryotic and nine eukaryotic pathogenic species, as described herein. The potential for pathogenic roles of many new Ni-binding components exists, based on recent experimental data and on the key roles that Ni enzymes play in a diverse array of pathogens.

Ni complexes of N 2 S ligands with amine/imine and amine/amide donors with relevance to the active site of Ni superoxide dismutase

Abstract A series of Ni complexes of the form [Ni(NNImS)(RS)] (NNImS = (2-(N-(dimethylaminoethyl)iminomethyl)benzenethiolate; R = phenyl, 4-nitrophenyl, 4-fluorophenyl, 4-chlorophenyl, 4-t-butylphenyl, t-butyl), as well as two complexes of the form [Ni(NNAmS)(RS)]− (NNAmS = 2-(N-(dimethylaminoethyl)aminocarbonyl)benzenethiolate; R = phenyl, 4-nitrophenyl) have been synthesized using 2,2′-dithiodibenzaldehyde and 2,2′-dithiodibenzoylchloride, respectively, as synthons for introducing the thiolate into the tridentate ligands. The two ligands and series of complexes feature the N2S2 coordination environment found at the active site of Ni superoxide dismutase and differ only in the identity of one of the N donors (imine vs. amide). Those with the amide N donor contain the exact donor set present in reduced Ni superoxide dismutase. The complexes have been characterized by X-ray crystallography, electronic spectroscopy, and electrochemistry.

NiFe]-hydrogenases revisited: nickel-carboxamido bond formation in a variant with accrued O2-tolerance and a tentative re-interpretation of Ni-SI states.

[NiFe]-hydrogenases are well-studied enzymes capable of oxidizing molecular hydrogen and reducing protons. EPR and FTIR spectroscopic studies have shown that these enzymes can be isolated in several redox states that include paramagnetic oxidized inactive Ni-A and Ni-B species and a reduced Ni-C form. The latter and the diamagnetic respectively more oxidized Ni-SI and more reduced Ni-R forms are generally thought to be involved in the catalytic cycle of [NiFe]-hydrogenases. With the exception of Ni-SI, these different stable states have been well characterized. Here, based on the crystal structure of a partially reduced Desulfovibrio fructosovorans (Df) enzyme and data from the literature we propose that at least one of the Ni-SI sub-states contains an unexpected combination of hydride and sulfenic acid moieties. We have also determined the structure of the less oxygen-sensitive Df [NiFe]-hydrogenase V74C mutant and found that more than half of the active site nickel occupies a novel position, called Ni'. In this new position, the metal ion is coordinated by two cysteine thiolates, a bridging species modeled as SH(-) and a main chain carboxamido N atom. The Ni' coordination is similar to the one found in Ni superoxide dismutase, an enzyme that operates at significantly more positive potentials than [NiFe]-hydrogenases. We propose that the oxygen-tolerance of the V74C variant results from a high potential stabilization of a Ni'(iii) species induced by the change in the metal ion coordination sphere. We also propose that transient Ni'(iii) species can rapidly attract successive electrons from the Fe4S4 proximal cluster accelerating the reduction of oxygen to water and hydroxide. The naturally occurring oxygen-tolerant [NiFe]-hydrogenases have an unusual proximal cluster that has been shown to be exceptionally plastic and capable of undergoing two successive one-electron oxidations. This double oxidation is modulated by the migration of one of the iron atoms in the cluster to the main chain where, as Fe(iii), it forms a bond with a carboxamido N ligand. Like in the Df V74C variant the electrons from the proximal cluster help reducing O2 to H2O and OH(-). In conclusion, in both cases a metal-carboxamido bond may explain, at least partially, the observed oxygen tolerance.

Superoxide dismutase inspired immobilised Ni(II)–protected amino acid catalysts—Synthesis, characterisation, and catalytic activity

Covalently anchored Ni(II)–C-protected amino acid (l-histidine, l-cysteine, and l-cystine) complexes inspired by the active site of the Ni-superoxide dismutase enzyme were synthesised using chloropropylated silica gel as support. The structural features of the surface complexes were studied by the Kjeldahl method and ICP-MS, mid/far IR, UV–vis diffuse reflectance, and X-ray absorption spectroscopies. The enzyme-like activities of the materials were determined in a biochemical test reaction. Covalent grafting and building the complex onto the surface of the support were successful in all cases. It was found that in many instances the structures obtained and the coordinating groups substantially varied upon changing the conditions of the syntheses. All the covalently immobilised Ni(II)-complexes displayed enzyme-like activity. They also were active in the liquid-phase oxidation of cyclohexene, providing the epoxide with high selectivity.

Trace metals and nutrients in Baltic Sea cyanobacteria: Internal and external fractions and potential use in nitrogen fixation

Even though the availability of trace metals influences nitrogen fixation and growth of cyanobacteria, field data on their cellular metal composition are scarce. In this study, contents of Al, Ti, V, Mn, Fe, Co, Ni, Cu, Zn, Mo, Cd, and the major elements C, N, P and Si were studied in filamentous, nitrogen-fixing cyanobacteria sampled over the growth season March–October at two locations in the Baltic proper (years 2004 and 2007) and one location in the Bothnian Sea (2006). The Al and Ti contents indicated that lithogenic Fe was an important Fe fraction associated with Nodularia spumigena, but not with Aphanizomenon sp. Treatment with an oxalate–EDTA solution indicated that less than 5% of total Fe was adsorbed as oxides, but relatively high adsorbed fractions were found for Mn and Cu. Despite the large variation in biomass and dissolved Fe concentrations, the Fe:C ratio of Aphanizomenon was highly consistent within years and across sampling stations (76±13μmolmol−1 C, average ±1SD), indicating growth controls other than Fe. Species-mixed samples corrected for lithogenic metals indicate similar Fe content in Nodularia as in Aphanizomenon. Calculations based on the use efficiency of Mo for N2 fixation indicate that most Mo in Nodularia and at least a third of the Mo in Aphanizomenon are used in nitrogenase, corresponding to 5–24% of the Fe content. The high Ni content suggests excess storage or extensive use in enzymes such as Ni superoxide dismutase or in Fe-dependent Ni-hydrogenases. The trace metal composition of the investigated Baltic cyanobacteria was similar to that reported for the oceanic genus Trichodesmium, suggesting common physiological requirements of these filamentous nitrogen-fixing cyanobacteria.

Genetic Identification of a High-Affinity Ni Transporter and the Transcriptional Response to Ni Deprivation in Synechococcus sp. Strain WH8102

One biological need for Ni in marine cyanobacteria stems from the utilization of the Ni metalloenzyme urease for the assimilation of urea as a nitrogen source. In many of the same cyanobacteria, including Synechococcus sp. strain WH8102, an additional and obligate nutrient requirement for Ni results from usage of a Ni superoxide dismutase (Ni-SOD), which is encoded by sodN. To better understand the effects of Ni deprivation on WH8102, parallel microarray-based analysis of gene expression and gene knockout experiments were conducted. The global transcriptional response to Ni deprivation depends upon the nitrogen source provided for growth; fewer than 1% of differentially expressed genes for Ni deprivation on ammonium or urea were concordantly expressed. Surprisingly, genes for putative Ni transporters, including one colocalized on the genome with sodN, sodT, were not induced despite an increase in Ni transport. Knockouts of the putative Ni transporter gene sodT appeared to be lethal in WH8102, so the genes for sodT and sodN in WH8102 were interrupted with the gene for Fe-SOD, sodB, and its promoter from Synechococcus sp. strain WH7803. The sodT::sodB exconjugants were unable to grow at low Ni concentrations, confirming that SodT is a Ni transporter. The sodN::sodB exconjugants displayed higher growth rates at low Ni concentrations than did the wild type, presumably due to a relaxed competition between urease and Ni-SOD for Ni. Both sodT::sodB and sodN::sodB lines exhibited an impaired ability to grow at low Fe concentrations. We propose a posttranslational allosteric SodT regulation involving the binding of Ni to a histidine-rich intracellular protein loop.

Tridentate N 2S ligand from 2,2′-dithiodibenzaldehyde and N,N-dimethylethylenediamine: Synthesis, structure, and characterization of a Ni(II) complex with relevance to Ni Superoxide Dismutase

Nickel Superoxide Dismutase (NiSOD) and the A-cluster of Carbon Monoxide Dehydrogenase/Acetyl Coenzyme A Synthase (CODH/ACS) both feature active sites with Ni coordinated by thiolate and amide donors. It is likely that the particular set of donors is important in tuning the redox potential of the Ni center(s). We report herein an expansion of our efforts involving the use of 2,2′-dithiodibenzaldehyde (DTDB) as a synthon for metal–thiolate complexes to reactions with Ni complexes of N,N-dimethylethylenediamine (dmen). In the presence of coordinating counterions, these reactions result in monomeric square-planar complexes of the tridentate N 2S donor ligand derived from the Schiff-base condensation of dmen and DTDB. In the absence of a coordinating counterion, we have isolated a Ni(II) complex with an asymmetric N 2S 2 donor set involving one amine and one imine N donor in addition to two thiolate donors. This latter complex is discussed with respect to its relevance to the active site of NiSOD.

MAPping the Chiral Inversion and Structural Transformation of a Metal-Tripeptide Complex Having Ni-Superoxide Dismutase Activity

The metal abstraction peptide (MAP) tag is a tripeptide sequence capable of abstracting a metal ion from a chelator and binding it with extremely high affinity at neutral pH. Initial studies on the nickel-bound form of the complex demonstrate that the tripeptide asparagine-cysteine-cysteine (NCC) binds metal with 2N:2S, square planar geometry and behaves as both a structural and functional mimic of Ni superoxide dismutase (Ni-SOD). Electronic absorption, circular dichroism (CD), and magnetic CD (MCD) data collected for Ni-NCC are consistent with a diamagnetic Ni(II) center. It is apparent from the CD signal of Ni-NCC that the optical activity of the complex changes over time. Mass spectrometry data show that the mass of the complex is unchanged. Combined with the CD data, this suggests that chiral rearrangement of the complex occurs. Following incubation of the nickel-containing peptide in D(2)O and back-exchange into H(2)O, incorporation of deuterium into non-exchangeable positions is observed, indicating chiral inversion occurs at two of the α carbon atoms in the peptide. Control peptides were used to further characterize the chirality of the final nickel-peptide complex, and density functional theory (DFT) calculations were performed to validate the hypothesized position of the chiral inversions. In total, these data indicate Ni-SOD activity is increased proportionally to the degree of structural change in the complex over time. Specifically, the relationship between the change in CD signal and change in SOD activity is linear.

Development of a Functional cis‐Prolyl Bond Biomimetic and Mechanistic Implications for Nickel Superoxide Dismutase

During recent years several peptide-based Ni superoxide dismutase (NiSOD) models have been developed. These NiSOD models show an important structural difference compared to the native NiSOD enzyme, which could cause a completely different mechanism of superoxide dismutation. In the native enzyme the peptide bond between Leu4 and Pro5 is cis-configured, while the NiSOD models exhibit a trans-configured peptide bond between these two residues. To shed light on how the configuration of this single peptide bond influences the activity of the NiSOD model peptides, a new cis-prolyl bond surrogate was developed. As surrogate we chose a leucine/alanine-based disubstituted 1,2,3-triazole, which was incorporated into the NiSOD model peptide replacing residues Leu4 and Pro5. The yielded 1,5-disubstituted triazole nickel peptide exhibited high SOD activity, which was approximately the same activity as its parent trans-configured analogue. Hence, the conformation of the prolyl peptide bond apparently has of minor importance for the catalytic activity of the metallopeptides as postulated in literature. Furthermore, it is shown that the triazole metallopeptide is forming a stable cyanide adduct as a substrate analogue model complex.

Reversible Apical Coordination of Imidazole between the Ni(III) and Ni(II) Oxidation States of a Dithiolate Complex: A Process Related to the Ni Superoxide Dismutase

A bisamine aliphatic dithiolate [Ni(II)N(2)S(2)] complex that does yield a metal-based oxidation has been synthesized. A square pyramidal [Ni(III)N(3)S(2)](+) complex is generated by electrochemical oxidation in the presence of imidazole, mimicking the redox structural changes of NiSOD. In addition, EPR measurements coupled to DFT calculations demonstrate that the metal character in the redox active orbital increases drastically upon imidazole binding, implicating that these geometrical modifications are crucial for the stabilization of the Ni(III) state.

Spectroscopic and Computational Studies of atrans-μ-1,2-Disulfido-Bridged Dinickel Species, [{(tmc)Ni}2(S2)](OTf)2: Comparison of End-on Disulfido and Peroxo Bonding in (NiII)2and (CuII)2Species

A powerful means of enhancing our understanding of the structures and functions of enzymes that contain nickel-sulfur bonds, such as Ni superoxide dismutase, acetyl-coenzyme A synthase/carbon monoxide dehydrogenase, [NiFe] hydrogenase, and methyl-CoM reductase, involves the investigation of model compounds with similar structural and/or electronic properties. In this study, we have characterized a trans-mu-1,2-disulfido-bridged dinickel(II) species, [{(tmc)Ni}(2)(S(2))](2+) (1, tmc = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) by using electronic absorption, magnetic circular dichroism (MCD), and resonance Raman (rR) spectroscopic techniques, as well as density functional theory (DFT) and time-dependent DFT computational methods. Our computational results, validated on the basis of the experimental MCD data and previously reported (1)H NMR spectra, reveal that 1 is best described as containing two antiferromagnetically coupled high-spin Ni(II) centers. A normal coordinate analysis of the rR vibrational data was performed to quantify the core bond strengths, yielding force constants of k(Ni-S) = 2.69 mdyn/A and k(S-S) = 2.40 mdyn/A. These values provide a useful basis for a comparison of metal-S/O bonding in 1 and related Ni(2)(O(2)), Cu(2)(O(2)), and Cu(2)(S(2)) dimers. In both the disulfido and the peroxo species, the lower effective nuclear charge of Ni(II) as compared to Cu(II) results in a decreased covalency, and thus relatively weaker metal-S/O bonding interactions in the Ni(2) dimers than in the Cu(2) complexes.

Periodic trends within a series of five-coordinate thiolate-ligated [MII(SMe2N4(tren))]+ (M = Mn, Fe, Co, Ni, Cu, Zn) complexes, including a rare example of a stable CuII-thiolate.

A series of five-coordinate thiolate-ligated complexes [M(II)(tren)N4S(Me2)]+ (M = Mn, Fe, Co, Ni, Cu, Zn; tren = tris(2-aminoethyl)amine) are reported, and their structural, electronic, and magnetic properties are compared. Isolation of dimeric [Ni(II)(SN4(tren)-RS(dang))]2 ("dang"= dangling, uncoordinated thiolate supported by H bonds), using the less bulky [(tren)N4S](1-) ligand, pointed to the need for gem-dimethyls adjacent to the sulfur to sterically prevent dimerization. All of the gem-dimethyl derivatized complexes are monomeric and, with the exception of [Ni(II)(S(Me2)N4(tren)]+, are isostructural and adopt a tetragonally distorted trigonal bipyramidal geometry favored by ligand constraints. The nickel complex uniquely adopts an approximately ideal square pyramidal geometry and resembles the active site of Ni-superoxide dismutase (Ni-SOD). Even in coordinating solvents such as MeCN, only five-coordinate structures are observed. The MII-S thiolate bonds systematically decrease in length across the series (Mn-S > Fe-S > Co-S > Ni-S approximately Cu-S < Zn-S) with exceptions occurring upon the occupation of sigma* orbitals. The copper complex, [Cu(II)(S(Me2)N4(tren)]+, represents a rare example of a stable CuII-thiolate, and models the perturbed "green" copper site of nitrite reductase. In contrast to the intensely colored, low-spin Fe(III)-thiolates, the M(II)-thiolates described herein are colorless to moderately colored and high-spin (in cases where more than one spin-state is possible), reflecting the poorer energy match between the metal d- and sulfur orbitals upon reduction of the metal ion. As the d-orbitals drop in energy proceeding across the across the series M(2+) (M= Mn, Fe, Co, Ni, Cu), the sulfur-to-metal charge-transfer transition moves into the visible region, and the redox potentials cathodically shift. The reduced M(+1) oxidation state is only accessible with copper, and the more oxidized M(+4) oxidation state is only accessible for manganese.

A DFT study of the mechanism of Ni superoxide dismutase (NiSOD): Role of the active site cysteine‐6 residue in the oxidative half‐reaction

In the present DFT study, the catalytic mechanism of H2O2 formation in the oxidative half-reaction of NiSOD, E-Ni(II) + O2- + 2H+ --> E-Ni(III) + H2O2, has been investigated. The main objective of this study is to investigate the source of two protons required in this half-reaction. The proposed mechanism consists of two steps: superoxide coordination and H2O2 formation. The effect of protonation of Cys6 and the proton donating roles of side chains (S) and backbones (B) of His1, Asp3, Cys6, and Tyr9 residues in these two steps have been studied in detail. For protonated Cys6, superoxide binding generates a Ni(III)-O2H species in a process that is exothermic by 17.4 kcal/mol (in protein environment using the continuum model). From the Ni(III)-O2H species, H2O2 formation occurs through a proton donation by His1 via Tyr9, which relative to the resting position of the enzyme is exothermic by 4.9 kcal/mol. In this pathway, a proton donating role of His1 residue is proposed. However, for unprotonated Cys6, a Ni(II)-O2- species is generated in a process that is exothermic by 11.3 kcal/mol. From the Ni(II)-O2- species, the only feasible pathway for H2O2 formation is through donation of protons by the Tyr9(S)-Asp3(S) pair. The results discussed in this study elucidate the role of the active site residues in the catalytic cycle and provide intricate details of the complex functioning of this enzyme.

Spectroscopic and Computational Studies of Ni Superoxide Dismutase: Electronic Structure Contributions to Enzymatic Function

Ni-containing superoxide dismutase (NiSOD) is the most recently discovered member of the class of metalloenzymes that detoxify the superoxide radical in aerobic organisms. In this study, we have employed a variety of spectroscopic and computational methods to probe the electronic structure of the NiSOD active site in both its oxidized (NiSOD(ox), possessing a low-spin (S = (1)/(2)) Ni(3+) center) and reduced (NiSOD(red), containing a diamagnetic Ni(2+) center) states. Our experimentally validated computed electronic-structure description for NiSOD(ox) reveals strong sigma-bonding interactions between Ni and the equatorial S/N ligands, which give rise to intense charge-transfer transitions in the near-UV region of the absorption spectrum. Resonance Raman (rR) spectra obtained with laser excitation in this region exhibit two features at 349 and 365 cm(-)(1) that are assigned to Ni-S(Cys) stretching modes. The NiSOD(red) active site also exhibits a high degree of metal-ligand bond covalency as well as filled/filled pi-interactions between Ni and S/N orbitals, which serve to adjust the redox potential of the Ni(2+) center. Comparison of our computational results for NiSOD(red) with those obtained in parallel studies of synthetic [NiS(2)N(2)] complexes reveals that the presence of an anionic N-donor ligand is crucial for promoting metal-based (versus S-based) oxidation of the active site. The implications of our electronic-structure descriptions with respect to the function of NiSOD are discussed, and a comparison of M-S(Cys) bonding in NiSOD and other metalloenzymes with sulfur ligation is provided.

Nickel-binding proteins.

Nickel enzymes are a relatively new class of metalloenzymes. The seven known nickel enzymes are urease, hydrogenase, CO-dehydrogenase, methyl-coenzyme M reductase, Ni-superoxide dismutase, glyoxalase I and cis-trans isomerase. The requirement for nickel implies the presence of a nickel-processing system, since free transition metals are harmful to the cell. A nickel-processing system involves the recognition and transport of nickel into the cell and the handling of the nickel once it enters the cell until it is inserted into the nickel enzyme. Several mechanisms for nickel transport have been identified and will be reviewed here. Accessory proteins required for the biosynthesis of the nickel active site have been identified. Accessory proteins bind the nickel when it enters the cell and are proposed to assist with the insertion of nickel into the enzyme. The function of the characterized nickel-processing proteins is described, and models for nickel insertion into the nickel enzymes are presented.