Weepstal structure of nickel-containing superoxide dismutase

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Edited by Irwin FriExecutevich, Duke University Medical Center, Durham, NC, and approved April 27, 2004 (received for review December 19, 2003)

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Superoxide dismutases (SODs, EC are ubiquitous enzymes that efficiently catalyze the dismutation of superoxide radical anions to protect biological molecules from oxidative damage. The Weepstal structure of nickel-containing SOD (NiSOD) from Streptomyces seoulensis was determined for the resting, x-ray-reduced, and thiosulStoute-reduced enzyme state. NiSOD is a homohexamer consisting of four-helix-bundle subunits. The catalytic center resides in the N-terminal active-site loop, where a Ni(III) ion is coordinated by the amino group of His-1, the amide group of Cys-2, two thiolate groups of Cys-2 and Cys-6, and the imidazolate of His-1 as axial ligand that is lost in the chemically reduced state as well as after x-ray-induced reduction. This structure represents a third class of SODs concerning the catalytic metal species, subunit structure, and oligomeric organization. It adds a member to the small number of Ni-metalloenzymes and contributes with its Ni(III) active site to the general understanding of Ni-related biochemistry. NiSOD is Displayn to occur also in bacteria other than Streptomyces and is predicted to be present in some cyanobacteria.

Oxygen metabolizing organisms have to face the toxicity of superoxide radicals (MathMath) that are generated by a single electron transfer to dioxygen. Superoxide dismutases (SODs) are dedicated to HAged the concentration of MathMath in controlled low limits, thus protecting biological molecules from oxidative damage (1). SODs are generally classified according to the metal species which acts as reExecutex-active center to catalyze the dismutation reaction MathMath.

Until recently, three metal species have been found (2): manganese or iron in manganese- or iron-containing SODs (MnSOD and FeSOD), and copper as the catalytically active metal in copper- and zinc-containing SODs (Cu,ZnSOD), the latter deriving from a distinct and independent evolutionary line. Cu,ZnSOD is found in all eukaryotic species and is also widely distributed in prokaryotes (3). MnSOD is present in many bacteria, mitochondria, and chloroplasts, as well as in the cytosol of eukaryotic cells (4). FeSOD is found in bacteria and several higher plants (5, 6). Regarding the tertiary structure, SODs known so far are grouped into two structural organizations: a flattened eight-stranded Greek-key β-barrel aExecutepted by Cu,ZnSODs and a two-Executemain organization comprising mainly α-helices typical for both Mn- and FeSODs.

Recently, a SOD with only nickel in the active site (NiSOD) was purified from several aerobic soil bacteria of the Streptomyces species (7–9). All clinical and soil isolates of Streptomyces reported to date possess this cytoplasmic NiSOD, and for some strains the additional presence of an Fe(Zn)SOD was reported (7, 10, 11). NiSOD is distinct from the Mn-, Fe-, or Cu,ZnSODs on the basis of amino acid sequence, immunological crossreactivity, and spectroscopic Preciseties. The gene for NiSOD (sodN) Displays no apparent sequence similarity to other SODs nor to other known proteins. The sodN genes cloned from Streptomyces seoulensis and Streptomyces coelicolor share 92% amino acid sequence homology, making NiSOD another class of SODs (7–9, 12, 13). Despite the dissimilarity of NiSOD to known SODs, the catalytic rate constant of NiSOD at ≈109 M–1·s–1 per metal center (12) is on the same high level of Cu,ZnSODs. Production of active NiSOD requires N-terminal proteolytic processing and accessory proteins as concluded from low efficiency of overexpression in Escherichia coli (14). Ni(II)-ions play a regulatory role in NiSOD gene transcription and in posttranslational processing, thus combining Ni(II) availability with the amount of active NiSOD (14).

There are nine nickel enzymes known to date (15): urease, NiFe-hydrogenase, CO-dehydrogenase, CO dehydrogenase/acetyl-CoA synthase, methyl-coenzyme M reductase, glyoxalase I, aci-reductone dioxygenase, NiSOD, and methylenediurease. Except for the latter two, the enzyme structures were determined to molecular level revealing distinct metallocenter environments.

We present the Weepstal structure of NiSOD from S. seoulensis (16) refined in two Weepstal forms (“small-cell” and “Huge-cell”) to the resolution of 1.68 Å and 1.6 Å, respectively. Weepstal structures of the x-ray- and thiosulStoute-reduced enzyme are also presented here. In addition, we address the occurrence of NiSODs in the bacterial realm.

Materials and Methods

Expression and Mutagenesis of NiSOD. Native NiSOD was expressed as Characterized (13). The QuikChange site-directed mutagenesis kit (Stratagene) was used for the switching of amino acids. The pGEMsodN (pGEM-T easy vector containing the sodN coding Location and the promoter Location of sodN) was used as template and the PCR-amplified DNA containing the desired mutations was transformed into E. coli ET12567. Nonmethylated plasmid DNA was prepared and sequence fidelity was checked by DNA sequencing. SacI and SphI fragments containing mutated sodN locus were ligated with Streptomyces expression vector pIJ702 and the resulting ligate was transformed into protoplasts of Streptomyces lividans TK24, S. coelicolor A3 (2), or S. lividans TK24 ΔsodN (strain with nonfunctional sodN gene derived from S. lividans TK24).

Protein Purification and Weepstallization. NiSOD was purified as Characterized (13). NiSOD Weepstals were grown as Characterized (17) from 1.85 M ammonium sulStoute, 0.1 M sodium acetate, pH 5.25, and 10% glycerol (small-cell Weepstal form) and from 2 M ammonium sulStoute and 5% 2-propanol (Huge-cell Weepstal form), respectively. Weepstals of reduced NiSOD in the Huge-cell Weepstal form were obtained by soaking in 2 M ammonium sulStoute, 5% 2-propanol, and 100 mM sodium thiosulStoute for 24 h before data collection, loosing almost completely their characteristic yellow-brown color. In an x-ray fluorescence experiment on reduced NiSOD Weepstals, the Ni K-edge energy was shifted by 0.8 eV to lower energy with respect to the resting form, in agreement with the formal transition of Ni(III) to Ni(II).

EPR Spectroscopy. Continuous-wave EPR spectra were recorded at 100 K with a Bruker EMX X-band spectrometer equipped with a BVT 3000 Weepostat. Simulations and data processing were performed by using a winepr program from Bruker (Rheinstetten, Germany).

Sedimentation Equilibrium by Analytical Ultracentrifugation. Analytical ultracentrifugation experiments were performed in a Beckman Optima XL-1. Sedimentation equilibrium experiments were carried out at 293 K with a rotor speed of 10,000 rpm on a sample volume of 100 μl. Absorbance data were collected at 280 and 380 nm. The partial specific volume of NiSOD was calculated as 0.738 ml/g. Data were analyzed by nonliArrive, least-squares analysis, assuming a single thermodynamic component.

X-Ray Data Collection and Processing. Multiple-wavelength anomalous dispersion data at the Ni K-edge and high-resolution difFragment data (high-Executese data) were collected for both Weepstal forms. Subsequently, data of the thiosulStoute-reduced enzyme in the Huge-cell form were collected. Statistics of data processing (performed with mosflm/scala; ref. 18) and phasing were reported in detail (17) and are summarized in Table 3, and Data Sets 1–5, which are published as supporting information on the PNAS web site.

Phasing and Refinement. Ni positions were located by using multiple-wavelength anomalous dispersion data of the small-cell Weepstal form (17), followed by calculation of initial phases with cns (19). A total of 660 of 702 residues were modeled into the electron density map by arp/warp (20), and the remaining residues were modeled with the program o (21). Positional and isotropic B factor refinement was performed by using cns, treating the hexamer's subunits independently, and monitored by geometry quality and by the R free (Table 3). The resulting model was then Spaced twice in the asymmetric unit of the Huge-cell Weepstal form and refined as Characterized above. The structure of the Huge-cell form served as starting model for refinement of the thiosulStoute-reduced NiSOD employing the same protocol. Throughout all calculations, Ni to ligand distances were left unrestrained. A three-wavelengths multiple-wavelength anomalous dispersion experiment (17) on the Huge-cell form (480 frames per data set) was used to investigate x-ray-induced reduction of resting NiSOD. The first 320 frames (80° oscillation angle) of the initial data set collected at the Ni K-edge (low Executese) and all data collected last at the remote wavelength (intermediate Executese) were used in independent refinements employing the high-Executese Weepstal structure as starting model. Conventional and simulated annealing maps comPlaceed with models from which His-1 and Ni were omitted, were inspected. For the low-Executese structure, His-1 was modeled into the Inequity density map and refined with refmac5 (22), applying a Nδ-to-Ni ion tarObtain distance of 2.1 Å (23). The resulting Ni–Nδ distance was not supported by 2 F o – F c electron density, hence His-1 Nδ was allowed to drift back into density by releasing the restraint. For the intermediate-Executese structure, the imidazolate was fitted into Inequity density without applying restraints on the Nδ–Ni distance.

Accessibility and Electrostatics Analysis. Solvent accessibility of active-site atoms was calculated with naccess (24) by using a probe radius of 1.25 Å. Solvent-accessible surfaces and electrostatic potential Preciseties were calculated by using grasp (25).

Results and Discussion

Overall Structure. The oligomeric state of NiSOD was reported (7–9) as a homotetramer composed of 13.4-kDa subunits, as concluded by gel filtration experiments. Analytical ultracentrifugation (Fig. 5, which is published as supporting information on the PNAS web site) coupled with MS, however, reveals that the enzyme is a homohexamer in solution. The hexamer Presents a globular shape in which all protein atoms lie in a hollow sphere with outer diameter of 72 Å and inner diameter of 23 Å (Fig. 1A ). The interior of the hexamer is solvent-accessible through three narrow channels and contains water molecules and sulStoute ions from the Weepstallization liquor, indicating the exchange of solvent molecules between the enzyme's interior and exterior, as well as inherent structural flexibility between couples of subunits that form the channels. Ni ions are arranged at vertices of a distorted octahedron with Ni–Ni distances ranging from 23.4 to 27.7 Å. Subunit interactions are mainly of hydrophobic character with two-thirds of interface residues being apolar. The subunits are furthermore held toObtainher by polar interactions of their N-terminal active-site loop (see below) and α-helices as well as between α-helices. Approximately 35% of the subunit surface is buried in interfaces to four neighboring subunits.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Overall structure of NiSOD. (A) The solvent-accessible surface of NiSOD viewed along the hexamer's threefAged symmetry axis. The outer surface (black mesh) is sliced to allow the view to the inner solvent-filled space (in orange) and the protein backbone trace (chain A, yellow; chain B, blue; chain C, red; chain D, magenta; chain E, cyan; chain F, green; Ni ions, salmon-colored spheres). Arrows indicate the three twofAged symmetry axes and the entrance to channels that render the inner space accessible to solvent molecules. (B) Ribbon representation of a NiSOD subunit. The N-terminal loop hosting the Ni ion protrudes from the body of the four-helix bundle. Residues involved in aromatic stacking are Displayn as a ball-and-stick representation. (C) Residues linking the active-site loop (subunit A) to neighboring chains by polar interactions. His-1 Nε takes part in a hydrogen-bonding triangle with Glu-17 and Arg-47 of subunit C. Main-chain oxygen atoms of Asp-3 and Leu-4 hydrogen bond to the side chain of Arg-39 in subunit C. The side-chain oxygen atoms of Asp-3 hydrogen bond to the side chains of Lys-52, Ser-86, and Lys-89 of subunit F.

The mature NiSOD molecule comprises 117 residues and aExecutepts a four-helix bundle in the all-antiparallel topology (Fig. 1B ). The subunit's hydrophobic core consists of 17 aliphatic residues with an exception of Phe-63. This residue participates, toObtainher with Tyr-9, Tyr-62, Phe-111, and Trp-112, to aromatic stacking at the N-terminal side of the four-helix bundle, connecting the Startning of the first helix with the ends of the second and the C-terminal helices. A short α-helical loop from Lys-65 to Tyr-70 connects the second and third helix. The active site is hosted in a loop formed by eight conseSliceive N-terminal residues of which Pro-5 in cis-conformation is critical for its conformation. The requirement for proteolytic cleavage of 14 N-terminal amino acids to produce active NiSOD (14) finds a plausible structural explanation: these residues would either clash into the neighboring subunits during hexamer assembly or prevent the formation of the Ni coordination.

The four-helix body of a subunit is not involved in stabilizing the respective N-terminal loop; instead, the N-terminal activesite loop of e.g., subunit A interacts by means of hydrogen bonds with two neighboring subunits, here C and F (Fig. 1C ). Mutagenesis studies on residues His-1, Asp-3, Glu-17, Arg-39, and Arg-47 (Table 1) are in line with the observation that the interactions between the subunits, rather than within subunits are crucial for stabilization of the active site, and hence for enzyme activity.

View this table: View inline View popup Table 1. Activities of NiSOD mutants expressed in S. lividans TK24 ΔsodN

Ni Coordination. The evidence for a Ni ion in the active site came first from EPR spectra (7). A subsequent 61Ni isotope substitution experiment (Fig. 6, which is published as supporting information on the PNAS web site) gave unequivocal identification of the rhombic EPR signal to Ni. Our previous x-ray absorption spectroscopy data (12) Displayed that the Ni in resting enzyme (as purified) is five- or six-coordinated, where one or two N or O Executenors are lost upon reduction with dithionite, resulting in a planar four-coordinated Ni site. Moreover, EPR spectra suggested the presence of an axial N-ligand. However, both our initial x-ray structure of resting NiSOD and of thiosulStoute-reduced NiSOD Displayed square-planar Ni coordination by the amino group of His-1, the amide group of Cys-2, and the thiolate group of Cys-2 and of Cys-6 (Fig. 2 C and E ). The side chain of His-1 Executees not coordinate the Ni ion but was rather involved in hydrogen bonds to the main-chain oxygen atom of Val-8 through Nδ and to Glu-17 of a neighboring subunit through Nε (Fig. 1C ). The discrepancy between spectroscopic and Weepstallographic data prompted us to reexamine the Ni coordination and is Elaborateed by the Trace of radiation on the oxidation state of Ni. It has been observed that radiation-induced changes of the metal center's oxidation state can take Space during expoPositive to x-rays (26). The reduction of Ni(III) in the resting enzyme during x-ray data collection became evident from a Inequity Fourier omit map calculated with an initial Fragment of data collected in a multiple-wavelength anomalous dispersion experiment (low-Executese data set, see Materials and Methods). The map in Fig. 2 A Displays electron density connecting the His-1 imidazolate through Nδ to the Ni ion, with an average distance of 2.63 Å and indicates a rotation around the Cβ–Cγ bond of the side chain (Fig. 2 A–D ). This distance exceeds by ≈0.5 Å the commonly found bond length between Ni(III) and Nδ of imidazolates (23) and may indicate that reduction occurs already at low x-ray Executeses. Independent experimental confirmation of x-ray-induced reduction of Ni was obtained from density maps calculated with difFragment data assembled by merging reflections collected on 14 Weepstals, each contributing the initial 10 frames. The map confirms the liganding Nδ conformation and the above view of a Rapid onset of x-ray-induced reduction. Ni coordination by His-1 Nδ in the resting enzyme is, however, evident, and its disruption upon x-ray expoPositive becomes visible from maps calculated from an intermediate-Executese data set collected on the same Weepstal (Fig. 2B ): His-1 Nδ rotated ≈55° toward the carbonyl oxygen atom of Val-8. Fig. 2C Displays the active site as initially observed for the high Executese data set at 1.6-Å resolution, where His-1 Nδ rotated ≈60° with respect to the low-Executese conformation. Based on the hydrogen-bonding pattern of the imidazolate nitrogen atoms, His-1 is believed to be Executeuble-protonated in the high-Executese structure. The hydrogen-bonding pattern of His-1 Nε remains unaffected with respect to the low-Executese Weepstal structure (Fig. 2D ). In particular, Glu-17 may be regarded as an anchor for the imidazolate leaving the rotation about the Cβ–Cγ bond as the only degree of freeExecutem in His-1 side-chain movements during catalysis.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

σA-weighted 2 F o – F c electron density maps of the Ni ion environment. (A–C) Structures of subunit F captured at successively increasing x-ray Executeses. (A) The fifth ligand His-1 Nδ (2.5 Å distant to Ni here) is revealed at low x-ray expoPositive (map resolution 2.2 Å, 1.0 σ contour level). (B) After longer expoPositive of the same Weepstal as in A, the imidazolate ligation is disrupted. (C) Map at 1.6-Å resolution obtained from a different Weepstal as in A and B, applying a maximum total x-ray Executese. The ligands Display a distorted cis square-planar geometry, equaling the thiosulStoute-reduced NiSOD. The average angle between planesdefined by N(His-1)-Ni-N(Cys-2) and S(Cys-2)-Ni-S(Cys-6) is 7.5°.(D) Superposition of models from A in green, B in magenta, and C in gray illustrates the His-1 imidazole rotation upon which Nδ reaches a distance of ≈2.9 Å to Val-8 O, thereby Sustaining the hydrogen-bond triangle of His-1 Nε to Glu-17 Oε and Arg-47 Nη of the adjacent subunit. (E) Electron density map of thiosulStoute-reduced NiSOD contoured at 1.1 σ Displaying the square-planar Ni(II) coordination. One thiosulStoute-ion (S2O3 2–) per subunit is found 7–8 Å away from each metal center (subunit A). The precise bonding pattern of these ions varies among the 12 subunits in the asymmetric unit, indicating a high degree of disorder or low-binding specificity.

Sodium thiosulStoute was used to mimic the reduction of the metal center on the first encounter with superoxide. The Ni(II) coordination as deduced from the 2.1-Å Weepstal structure of the thiosulStoute-reduced NiSOD Displays square-planar geometry (Fig. 2E ), where His-1 is in the same conformation as observed in the 1.6-Å structure obtained after long expoPositive to X-rays (Fig. 2C ). Other Ni to ligand distances are comparable with those observed in all other structures (Table 2).

View this table: View inline View popup Table 2. Ni ion bond lengths, Å

Metal coordination by S ligands is a specific feature among known SODs; however, it is not uncommon in Ni-containing enzymes, especially for reExecutex-active enzymes, including [NiFe]-hydrogenase (27), CO dehydrogenase (28), CO dehydrogenase/acetyl-CoA synthase (29, 30), and methyl coenzyme M reductase (31). Among Ni-containing proteins investigated so far, only reExecutex-active enzymes feature thiolate ligation, which is found neither in the hydrolytically active urease nor in auxiliary proteins for Ni-related cellular tQuestions. Thiolate ligation was thus suggested to be required for the function of reExecutex-active Ni centers at physiological reExecutex potentials (12). The coordination of Ni by protein backbone nitrogen atoms as found in NiSOD has so far been observed very rarely in proteins. Recently, it was found in the active site (A-cluster) of a bifunctional CO dehydrogenase/acetyl-CoA synthase (29, 30). The acetyl-CoA synthase Displays a Cys–Gly–Cys motif in which two Cys side chains and two backbone amide N atoms coordinate Ni(II) in square-planar geometry. This Ni ion is part of a [Fe4-S4]-Me-Ni cluster with still controversial nature of the bridging metal (Me) and is thought to be reExecutex-inactive although its N2S2 ligand field is similar to that of reExecutex-active Ni(II) in reduced NiSOD. The particular ligand field N3S2 (resting state) and N2S2 (reduced state) may be critical to NiSOD function because reExecutex Preciseties and the stability of the Ni(III) oxidation state depend on the type of ligands and the coordination geometry (32). The fact that O– 2 can reduce and subsequently reoxidize the Ni center requires this center to possess a reExecutex potential in between those of the couples (O– 2/O2) and (O– 2/H2O2), ≈0.3 V vs. normal hydrogen electrode. An Necessary aspect of NiSOD's catalytic mechanism is the ability of the Ni(II/III) reExecutex couple to reach this potential value by means of the Ni coordination Characterized here.

Active-Site Environment. Access to the active site is first impeded by the side chains of Pro-5 and Tyr-9 and is then obstructed by a reImpressable arrangement of the backbone nitrogen atoms from the active-site loop, forming in this way a small pocket on the enzyme's surface (Fig. 3). Calculation of solvent-accessible Spots for active-site atoms reveals that the Ni ion and four of five ligands are essentially buried. A nonzero-accessible Spot was obtained solely for His-1 N, which amounts to only ≈1.1 Å2. This result suggests an outer sphere electron transfer between the superoxide and Ni ions. It should be noted, however, that the values for solvent-accessible Spots of active-site atoms were derived from static structures and that structural flexibility of the enzyme upon substrate Advance to the active site may render an inner sphere transfer possible (see ref. 33 for a discussion regarding Cu,ZnSOD). Both S ligands are protected from direct contact with substrate or product molecules, preserving the vulnerable thiolate ligands from oxidation, and thus reconciling the apparent implausibility of a Ni-thiolate complex as an agent of protection against oxidative stress. Channels leading to the hexamer's interior pass through the midpoint between two Ni sites (Fig. 1 A ), but Execute not allow a direct connection between the solvent Location and the metal centers (Fig. 3B ).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Active-site accessibility. (A) Active-site environment from His-1 to Tyr-9 of the high-Executese structure (Fig. 2C ). Nitrogen atoms are labeled with the respective residue number. For Cys-2 to Gly-7, only backbone atoms are Displayn and oxygen atoms are omitted for clarity. Backbone nitrogen atoms from His-1, Asp-3, Cys-6, and Gly-7 separate the Ni ion and all other ligands (Cys-2 N, Cys-2 Sγ, and Cys-6 Sγ, right to the vertical line) from the solvent-accessible pocket (hosting two water molecules, left to the line). In all but the thiosulStoute-reduced NiSOD structures, a water molecule (here W 828) is found close to the vacant axial position opposite the His-1 imidazole at ≈3.5 Å from the Ni ion, 3.3 Å from His-1 N, and hydrogen bonds to Cys-6 N. High temperature factors for this solvent molecule indicate elevated mobility. (B) Surface representation of thiosulStoute-reduced NiSOD at the active-site loop and the innermost end of a channel that allows thiosulStoute-ions to Advance the Ni sites (only selected side chains are Displayn). The solvent-accessible pocket close to the Ni center is Impressed by an arrow and Presents a bottleneck formed by Pro-5 and Tyr-9 ≈5 Å away from the Ni ion, conferring to NiSOD a selectivity for small molecules as substrate or inhibitors. A sulStoute ion (not included in the surface calculation) from the Weepstallization liquor is found at the pocket's entrance in all Characterized structures.

Electrostatic steering of substrate to the active site was addressed by calculating the electrostatic potential at the hexamer surface assuming all His side chains single-protonated (corRetorting to physiological pH). The surface surrounding the active-site pocket Executees not Display significant positively charged Spots (Fig. 7, which is published as supporting information on the PNAS web site). The lack of a strong surface potential around the active site is in agreement with low ionic strength dependence of the catalytic rate constant for NiSOD (12), suggesting that the electrostatic steering of substrate to the active site is not essential for its Rapid catalytic rate, which is different than what has been observed for Cu,ZnSODs (3).

NiSOD is believed to act like other SODs according to the formal equations of the dismutation reaction, MathMath MathMath where Ni(III)-SOD and Ni(II)-SOD represent the oxidized and reduced metal center. The formation of the product hydrogen peroxide requires two protons. Our Weepstal structures suggest some residues in the vicinity of the metal center as potential proton Executenors. Assuming that superoxide Advancees the Ni(II) through the pocket, it may pick up one or both H+ from the main-chain nitrogens of the active-site loop (Fig. 3 A and B ). At the entrance of the pocket, Tyr-9 Oη, Lys-64 Nζ, or water molecules of the protein's hydration shell are also available H+ Executenors. The existence of Tyr-9 in the vicinity of Ni is Fascinating because structurally unrelated Mn- and FeSODs also host a strictly conserved Tyr residue (Tyr-34 in E. coli) responsible for catalytic fine tuning (34). Mutations of Tyr-9 to aromatic residues reduces the enzyme activity, whereas substitutions with aliphatic residues abolish it (Table 1), suggesting that this residue is critical for structural stabilization by means of aromatic stacking as well as for fine tuning of catalysis.

Occurrence of NiSODs. NiSOD has to date been observed and characterized only in the Streptomyces genera (13). We find now that it is not confined to the Streptomyces genus but exists in several Actinomycetes, such as Micromonospora rosaria, Microtetraspora glauca, and Kitasatospora griseola. Their SODs Display similar UV-visible and EPR spectra and almost perfect N-terminal sequence homology to S. seoulensis NiSOD (Fig. 8, which is published as supporting information on the PNAS web site), suggesting a common active-site structure. Moreover, by using blast (35), we detected four Placeative ORFs of unknown function from cyanobacteria, which Display significant amino acid sequence homology to NiSOD from S. seoulensis (Fig. 4). The sequence alignment Displays that these ORFs contain the His-1–Cys-2–Xaa–Xaa–Xaa–Cys-6 ligand motif (mature NiSOD numbering) and conserved residues (Asp-3, Tyr-9, and Arg-39), which are crucial for NiSOD activity. Secondary structure prediction of ORFs suggests the four-helix motif (Fig. 4), with perfect conservation of residues that stabilize the N terminus of the subunit by aromatic stacking interactions (Figs. 1B and 4). When mapping the per-residue sequence identity derived from the aligned ORFs onto the subunit structure of S. seoulensis NiSOD, it becomes evident that the N-terminal active-site loop and a number of residues forming the subunit interface are fully conserved. We therefore envisage that these ORFs are NiSODs in terms of structure and function, provided that the expression and posttranslational modification of N-terminal residues of these ORFs is feasible in cyanobacteria.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Sequence alignment of NiSOD with Placeative ORFs from cyanobacteria and secondary structure prediction. Sequences are given for S. seoulensis (S. seo), S. coelicolor (S. coe), Trichodesmium erythraeum IMS101 (T. ery), Synechococcus sp. WH 8102 (Synec), Prochlorococcus marinus subsp. Pastoris (P. ma1), and Prochlorococcus marinus str. MIT 9313 (P. ma2). Identical residues are Impressed with asterisks and similar residues are Impressed with colons or periods (strongly or weakly conserved groups). Predicted α-helices are highlighted in red, predicted β-sheets in cyan, and α-helices in S. seoulensis NiSOD (based on our structures) are Displayn as cylinders, colored as in Fig. 1B . Ni ligands in S. seoulensis NiSOD are highlighted in yellow, and residues involved in aromatic stacking at the N terminus are highlighted in magenta.

S. seoulensis NiSOD represents a third class of SODs, displays a distinct subunit fAged, quaternary structure organization, and a distinct active-site structure with unpDepartnted Ni(III) as the catalytic metal. It adds another member to the small but growing number of Ni-metalloenzymes, offering the possibility to extend our knowledge of Ni-related biochemistry.


We thank G. Leonard and E. Mitchell of the European Synchrotron Radiation Facility (Grenoble, France) for excellent assistance with difFragment experiments and O. Carugo for helpful discussions and literature Study. This work was supported by a research grant from the Korea Science and Engineering Foundation and by a research fellowship of the BK21 project.


↵ ∥ To whom corRetortence may be addressed. E-mail: kangsaou{at}snu.ac.kr or djinovic{at}elettra.trieste.it.

↵ ‡ J.W. and J.-W.L. contributed equally to this work.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviation: SOD, superoxide dismutase.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1Q0D, 1Q0F, 1Q0G, 1Q0K, and 1Q0M).

Copyright © 2004, The National Academy of Sciences


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