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. 2015 Aug 27;24(11):1777–1788. doi: 10.1002/pro.2768

The structure of the Caenorhabditis elegans manganese superoxide dismutase MnSOD-3-azide complex

Gary J Hunter 1, Chi H Trinh 2, Rosalin Bonetta 1, Emma E Stewart 2, Diane E Cabelli 3, Therese Hunter 1,*
PMCID: PMC4622211  PMID: 26257399

Abstract

C. elegans MnSOD-3 has been implicated in the longevity pathway and its mechanism of catalysis is relevant to the aging process and carcinogenesis. The structures of MnSOD-3 provide unique crystallographic evidence of a dynamic region of the tetrameric interface (residues 41–54). We have determined the structure of the MnSOD-3-azide complex to 1.77-Å resolution. Analysis of this complex shows that the substrate analog, azide, binds end-on to the manganese center as a sixth ligand and that it ligates directly to a third and new solvent molecule also positioned within interacting distance to the His30 and Tyr34 residues of the substrate access funnel. This is the first structure of a eukaryotic MnSOD-azide complex that demonstrates the extended, uninterrupted hydrogen-bonded network that forms a proton relay incorporating three outer sphere solvent molecules, the substrate analog, the gateway residues, Gln142, and the solvent ligand. This configuration supports the formation and release of the hydrogen peroxide product in agreement with the 5-6-5 catalytic mechanism for MnSOD. The high product dissociation constant k4 of MnSOD-3 reflects low product inhibition making this enzyme efficient even at high levels of superoxide.

Keywords: superoxide dismutase, conformational variation, MnSOD-3-azide complex, catalytic mechanism, product inhibition

Introduction

During cellular respiration, the molecular oxygen in the cell can readily acquire an electron to form superoxide radicalsInline graphic.1 These are rapidly converted to other reactive oxygen species (ROS) such as the hydroxyl radical. Unless removed, these ROS will damage cellular macromolecules.2 Both prokaryotic and eukaryotic cells produce superoxide dismutases (SODs, EC 1.15.1.1), a family of metalloenzymes that eliminate superoxide radicals by dismutation to oxygen and hydrogen peroxide.3,4 This reaction involves the redox cycling of the active site metal cofactor that may be manganese, iron, nickel, or copper5–7 (Scheme 1).

Scheme 1.

Scheme 1

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Dismutation by SOD.

The free-living nematode, Caenorhabditis elegans, has five distinct sod genes, which express two cytosolic Cu/ZnSODs (SOD-1 and SOD-5), an alternatively spliced extracellular Cu/ZnSOD (SOD-4) and two mitochondrial MnSODs (SOD-2 and SOD-3).8–12 The MnSODs are both synthesized as precursor molecules with a N-terminal mitochondrial-targeting sequence and are assembled into homotetramers containing one manganese ion per subunit, a process that requires interaction with hsp60/10.13–15 Sod-2 is constitutively expressed while the sod-3 gene is inducible. The latter is expressed only in the dauer diapause stage and is a direct target of the DAF-16/FOXO transcription factor.16,17 Interest in sod-3 grew when its over-expression was observed in the long-lived daf-2 and age-1 mutants. Both these mutants exhibit the Age phenotype16,18–23 and have altered expression of certain components of the insulin/IGF-1 signaling pathway.24–27 The precise role of MnSOD-3 in life extension has been a topic of debate. As superoxide and hydrogen peroxide both act as signaling molecules,28,29 it is likely that SOD-3, possibly together with SOD-2, serves as a modulator of longevity and other cellular pathways,30–36 by fine-tuning the cellular levels of superoxide and hydrogen peroxide.

Catalysis is a cyclic two-stage process, with the manganese ion switching from the oxidized MnIII to the reduced MnII state in a “ping-pong” mechanism.37,38 At high superoxide concentrations, the human MnSOD undergoes an initial burst of catalysis that lasts only a few milliseconds followed by a region of zero order decay of superoxide indicating product inhibition.39 This inhibition is caused by the formation of the peroxy complex of the manganese ion.40,41 The McAdam scheme37 describes the MnSOD catalytic cycle in four reactions that show the dismutation of superoxide occurring through two simultaneous pathways (Scheme 2).

Scheme 2.

Scheme 2

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Mechanism of catalysis of MnSOD.

Equations (1) and (2) represent the outer-sphere pathway whereby the reduction of superoxide to hydrogen peroxide is instantaneous. The second or inner-sphere pathway shown in Eqs. (3) and (4) involves the formation and dissociation of the product-inhibited complex.7 The degree of product inhibition is determined by the gating ratio (k2/k3) which gives a measure of the amount of superoxide that is reduced by the outer-sphere pathway relative to the inner-sphere pathway and k4, the measure of dissociation of the product inhibited complex.39,42

Of the known structures of native homotetrameric SODs, ten are MnSODs and six are FeSODs.13,43–52 In the active sites of each MnSOD, the manganese cofactor is coordinated to three histidinyl, one aspartyl, and either a hydroxyl or water molecule, forming a trigonal bipyramidal geometry that is highly conserved. A hydrogen bond network, involving the second sphere residues of the active site, extends to include residues from a neighboring subunit to form a dimer interface.13,43,47,53 This network permits the proton transfer needed to reduce superoxide to hydrogen peroxide. To have a clearer understanding of how this enzyme functions, we have determined the first molecular structure of a eukaryotic MnSOD in complex with its substrate analog. The structure of the MnSOD-3-azide complex suggests how the superoxide is positioned in the active site and helps elucidate the catalytic mechanism. This structure is a good model for MnSOD structure/function studies as the only other structural data of the MnSOD-azide complex is that of the T. thermophilus which has a different tetrameric assembly to the known eukaryotic MnSODs.54

Results

Structural data of 293 K MnSOD-3 and MnSOD-3-azide complex

Structural data of the 293 K MnSOD-3 was determined at a resolution of 1.77 as was the 100 K MnSOD-3-azide complex. Both formed pink crystals belonging to the space group P41212, with unit cell parameters of a = b = 81.5, c = 138.0 Å (293 K MnSOD-3)13 and a = b = 82.1, c = 138.0 (MnSOD-3-azide complex). The asymmetric unit-cell consists of two subunits designated A and C. Overall the tetrameric structure is very similar to that of the human MnSOD forming a dimer of dimers and shares the same monomeric fold of mononuclear SODs, having a predominantly α-helical hairpin N-domain and an α/β C-domain. Superimposition of the CA atoms of MnSOD-3 with human MnSOD gives a RMSD value of 0.8 Å.55 The data collection and refinement statistics for the 293 K MnSOD-3 and 100 K MnSOD-3-azide complex are presented in Table1.

Table 1.

Data Collection, Processing, and Refinement Statistics for MnSOD-3 293 K and MnSOD-3-azide Complex

MnSOD-3 293 K MnSOD-3-azide 100 K
Source Rigaku RUH3R rotating anode Daresbury SRS 10.1
Wavelength (Å) 1.541 0.97
Resolution range (Å)a 70.19–1.77 (1.87–1.77) 32.4–1.77 (1.86–1.77)
Space group P41212 P41212
Unit-cell parameters (Å) a = b = 81.5, c = 138.0 a = b = 82.1, c = 138.0
No. of observed reflections 190,572 654,522
No. of unique reflections 45,989 46,922
Redundancy 4.1 (4.0) 13.9 (4.0)
Completeness (%)a 99.9 (99.9) 99.9 (99.9)
< I/σ(I) >a 15.6 (2.3) 16.7 (13.3)
Rmerge (%)a, b 6.2 (33.8) 9.0 (51.7)
Rpim (%)a, c 3.4 (19.4) 2.5 (14.5)
Resolution range for refinement (Å) 70.19–1.77 30.6–1.77
R factor (%) 18.1 18.7
Rfree (%)d 21.4 21.6
No. of protein non-H atoms 3427 3178
No. of water molecules 296 384
No. of manganese ions 2 2
No. of sulfate ions 1 1
No. of malonate-ion 1 1
No. of acetate ion — 1
No. of azide — 2
R.m.s.d bond lengths (Å)e 0.013 0.011
R.m.s.d bond angles (˚)e 1.5 1.4
Average overall B factor (Å2)
Protein 26 31
Water 30 36
Manganese ions 16 19
Sulfate ions 74 48
Malonate-ion 32 48
Acetate ion — 49
Azide — 16
Ramachandran analysis, the percentage of residues in the regions of plot (%)f
Favored region 97.4 96.9
Outliers 0 0
PDB code 4X9Q 4X9Q and 5AG2
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a

Values given in parentheses correspond to those in the outermost shell of the resolution range.

b

Rmerge = ∑hkl ∑i|Ii(hkl)-[I(hkl)]|/∑hkl ∑ Ii(hkl).

c

Rpim = ∑hkl {1/[N(hkl)−1]}421/2 ∑i|Ii(hkl) – [I(hkl)]|/∑hkl ∑i Ii(hkl).

d

Rfree was calculated with 5% of the reflections set aside randomly.

e

Based on the ideal geometry values of Engh and Huber.51

f

Ramachandran analysis using the program MolProbity.52

Structural mobility of the N-domain α-hairpins

The interaction between the four N-domain α-hairpins forms the tetrameric quaternary structure consisting of 2 four-helix bundles as seen in other eukaryotic MnSODs [Fig. 3(A)]. Interestingly, structural mobility is visible in the electron densities of both the 100 K and 293 K MnSOD-3 structures as well as the MnSOD-3-azide complex. There are two conformations (designated A and B) of the N-domain α-helical region composed of the 14 residues from Ile41 to Leu54, in a region that forms symmetrically apposed helices of the tetrameric α-hairpin interface [Fig. 3(B)]. For clarity however, we have modeled only one conformer for each structure submitted to PDB. The 100 K MnSOD-3 and the MnSOD-3-azide structures were modeled with conformer A and the 293 K structure with conformer B.

Figure 3.

Figure 3

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Visible absorbance spectra of MnSOD-3 in potassium phosphate buffer (pH 7.8) in oxidized form (top, dashed line), as isolated (dotted line) or reduced (bottom, solid black line). Also shown is the blue shift observed on binding the inhibitory molecule, azide (grey).

Figure 1.

Figure 1

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The structure of MnSOD-3 from C. elegans. A. The tetrameric assembly of MnSOD-3 with subunits labeled A (blue), B (yellow), C (green), and D (orange). Manganese atoms are shown as purple spheres and indicate the positions of the active sites. Subunits A and B form a biologically active dimer as do C and D. The tetrameric interface forms mostly between the hairpin helical segments seen in the center of the figure. Subunits A and C illustrate one of the two four-helix bundles in this interface (blue and green subunits). Red side chains shown on subunit A highlight the position of the alternate conformer B. B, the tetrameric interface between subunits A (blue) and C (green) illustrating the position of conformer B (red). Side chains indicative of those amino acids where interactions may be enhanced due to the position of conformer B are labeled. C. The salt bridges unique to MnSOD-3 formed between subunits A and D (illustrated) and B and C. All figures were drawn using PyMOL.86 Protein illustrations were prepared from PDB entry 3DC5 with conformer B from PDB entry 4X9Q.

Of significance is the increase in the number of possible hydrophobic interactions between subunits A and C (which form the tetramer) of conformer B [Fig. 3(B)]. 19F-tyrosine NMR and amide H/D exchange studies of the human MnSOD have been reported showing a high degree of structural dynamic mobility in the same region; between residues Val40 and Ile58.56 This suggests that the conformational variability observed in all MnSOD-3 structures is not an artifact of crystal packing or the temperature at the time of data collection. The two 14-residue conformers (A and B) were resolved with a RMSD value of 2.0 Å when superimposed, diverging by more than 3.5 Å at Lys51CA [Fig. 3(B)]. Superimposition with human MnSOD indicates that the position this region adopts in the crystal structure of the human MnSOD approaches that of conformer B. In yeast, the tetrameric interface has been shown to have a direct influence on the dimer interface that forms the catalytically active site.57 Unique to MnSOD-3 are salt bridges that form at the cross chain interface A/D (and B/C) between Arg109 in the first β-sheet of one subunit and Asp131 in the sixth α-helix of the opposite subunit of the tetramer (2.9–3.1 Å) [Fig. 3(C)]. Human MnSOD has a unique A/D and B/C bond between Arg132NH1 and Leu135O where this arginine residue is in the equivalent position to Asp131 of MnSOD-3.

Active site geometry of MnSOD-3

The inner sphere arrangement of MnSOD-3 is, as expected, the characteristic five coordinate trigonal bipyramidal configuration. In the active site, the manganese ion is coordinated to five ligands that include residues from both N and C-domains of the subunit: His26 and His74 from the N-terminal hairpin helices, Asp155 and His159 from the C-domain, and a solvent molecule (designated WAT1). The latter may be either a water or hydroxyl molecule.

Overall the active site residues including the gateway residues Tyr34 and His30 positioned at the top of the solvent access funnel of MnSOD-3 and human MnSOD superimpose well with only negligible deviation, with a Tyr34OH to His30ND1 distance of 5.4 Å (Fig. 4). Despite this similarity however, a noteworthy observation is the presence of two water molecules in both 100 K and 293 K MnSOD-3 (designated WAT2 and WAT3) that participate in the hydrogen bonding between Tyr34 and His30 (Fig. 2). The bond lengths of Tyr34OH-WAT2-WAT3-His30NE2 are 2.7 Å, 2.9 Å, and 2.8 Å, respectively. No hydrogen-bonded network between Tyr34 and His30 involving only one of the water molecules is feasible. This two-water arrangement is present in both the A and C subunits of the asymmetric unit of the 100 K and the 293 K MnSOD-3 structures (3DC5 and 4X9Q) but has not been observed in any other native MnSOD structures.

Figure 4.

Figure 4

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Stereo figure of the active site of native MnSOD-3 (A) and MnSOD-3-azide complex (B). The four amino acid ligands to the manganese atom are shown (His26, His74, Asp155 and His159). The Mn metal (purple) and water molecules (red) are represented by spheres and the azide in (B) is blue; Glu158 and Tyr162 (grey) are contributed by the dimeric partner, subunit B. Bonding is shown as black.

Figure 2.

Figure 2

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Stereo representation of the structural alignment between MnSOD-3 (green) and human MnSOD (pink). Metal ligands are His26, His74, His159, and Asp155 (MnSOD-3 numbering). Major components of the hydrogen-bonded network are shown, although the structure of Tyr162 has been omitted for brevity. WAT2 and WAT3 (red) in MnSOD-3 replace a single molecule (pink) in human MnSOD and bridge the gap between the gateway residues His30 and Tyr34. The position of WAT1 (the solvent ligand to the metal) is slightly closer to the gateway residues in MnSOD-3 (red sphere). The metal ion is illustrated as a sphere (purple, MnSOD-3, red, human MnSOD). Bonding is illustrated by black dashes (MnSOD-3, 3DC5; human MnSOD, 1LUV).

Visible absorption spectra and azide inhibition

The as-isolated MnSOD-3 was predominantly in the Mn3+ oxidized state giving the characteristic 480-nm peak in the visible spectrum (Fig. 3). Incubation with potassium permanganate showed a small increase in absorbance at this wavelength with an accompanying increase at lower wavelengths (400 nm), while the presence of DTT abolished the 480-nm peak as the manganese was reduced to the Mn2+ state. The formation of the MnSOD-3-azide complex caused a shift in absorption from 480 to 410 nm that is indicative of a six-coordinate complex.58 The occurrence of a six-coordinate configuration was verified by the structural data of the MnSOD-3-azide complex. The effectiveness of azide as a competitive inhibitor was demonstrated by the loss of 45 and 61% SOD activity when the enzyme was assayed in the presence of 4 and 10 mM sodium azide, respectively. Without azide the enzyme activity was 3261 ± 26 U/mg/Mn.

Structure of MnSOD-3-azide complex

The crystal structure of the MnSOD-3-azide complex (PDB entry 4X9Q and 5AG2) shows that in both chain A and chain C the azide binds directly to the manganese to form a six-coordinate distorted octahedral active site. This is supported by previous spectroscopic work on other SODs.58–61 In this protein, the azide binds to the manganese ion in an end-on manner [Fig. 4(B)], in an arrangement that is similar to that observed in the E. coli and P. haloplanktis FeSOD azide complexes (PDB entries 1ISC and 3LJ9, respectively)54,62 and supported by theoretical studies.63,64 In MnSOD-3, azide is accommodated into the active site with relatively small perturbations of surrounding residues and has an average B factor value of 16 Å2 in both chain A and chain B active sites. The azide lies directly opposite the Asp155OD1 ligand and in line with the phenolic ring of Tyr34 in the cavity formed by the metal ion, the imidazole ring of His74, the backbone of His30, and the NE2 of Gln142. In subunit A, N1 of the azide is positioned end-on 2.8 Å from the manganese ion at an angle of 94° (Mn-N1-N3) in comparison to the 117° and 123° found in the dimeric E. coli and P. haloplanktis FeSODs, respectively.54,62

In the MnSOD-3-azide complex the manganese ion and solvent ligand WAT1 are displaced slightly toward azide N1. This places the solvent ligand 3.5-Å away from the azide N1. The distance however, between the solvent ligand and the manganese ion remains unchanged at 2.3 Å. The His74-Mn-His159 angle increases by 21° to 145° and both His74NE2 and His159NE2 are within bonding distances to the azide N3 (3.0 Å) and azide N1 (2.8 Å), respectively. The most perturbed residues are the outer sphere Tyr34 and His30. The phenolic group of Tyr34 that lies adjacent to N1 of the azide moves away by 0.5 Å and is 5.7 Å from the manganese ion. Positioned at 3.7-Å away from N1, the Tyr34OH is too far from the azide to interact effectively. Twisting of the imidazole ring of His30 creates a difference in the torsion angle of 5°. Consequently the Tyr34OH-Mn-His30ND1 angle widens from 56° to 61°. The hydrogen bonds between the two water molecules and Tyr34 and His30 at the top of the solvent access funnel change only slightly, with the Tyr34-WAT2 and WAT2-WAT3 remaining unchanged and the bond between WAT3 and His30 stretching slightly to 3.0 Å (from 2.8 Å). The major difference in the arrangement of the water molecules at the solvent funnel access is the appearance of a third and new water molecule (designated WAT4) identified in a position where it can hydrogen bond directly with Tyr34 (2.9 Å), His30 (2.9 Å), WAT3 (2.3 Å), and azide N1 (2.5 Å) (Fig. 4). Tyr34 and His30 are therefore connected to each other by two water molecules WAT2 and WAT3 as seen in the MnSOD-3 structure (PDB entry 3DC5 and 4X9Q) and to the azide (and therefore the peroxy adduct) via WAT4. An identical arrangement is also observed in chain C. This configuration has not been previously reported in the literature and lends support to a 5-6-5 catalytic mechanism.47,54,65 The 30% occupancy of the azide shown by the electron density map compares well with the occupancy achieved by Lah et al. for T. thermophilus MnSOD, the only other known MnSOD-azide structure.54 Low azide occupancy was also observed by EPR in E. coli and R. capsulatus MnSODs.58 Azide soaking gave better diffraction data compared to co-crystallization of MnSOD-3 with azide and was therefore the preferred method.

Kinetic studies by pulse radiolysis

A solution of 32.6 μM MnSOD-3 (100% metallated), pH 7.5, was pulse irradiated such that 2 or 4 μM of superoxide was formed and the change in absorbance with time was followed at 350–650 nm. The formation of the initial spectrum formed within 100 µs [Fig. 5(A) closed circles]. This was followed by the first order conversion of the initial spectrum into the final species [Fig. 5(A) open circles] at a rate of 300 s−1. This final spectrum is that of the oxidized Mn3+SOD. Additionally, the decay of 50 μM superoxide using 6:1 and 13:1 ratios ofInline graphic:Mn3+SOD was followed at 260 nm to determine how the enzyme behaves at relatively low and high levels of superoxide [Fig. 5(B)].

Figure 5.

Figure 5

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A. The Mn3+ SOD-3 spectrum obtained from pulse radiolysis. The sample contained 32.6 μM manganese (protein concentration 0.75 mg mL−1) in potassium phosphate buffer, pH 7.5. The closed circles represent the spectrum formed at the rate constant of 1.2 × 109 M−1 s−1 when 32.6 μM Mn2+SOD reacts with either 2 or 4 μM superoxide. The open circles show the spectrum formed at a rate of 300 s−1 and is the spectrum of Mn3+SOD. B. Observed disappearance of 50 μM superoxide in the presence of 8.15 or 3.26 μM MnSOD (black and blue traces respectively). Both traces were fitted to the McAdam scheme to derive the rate constants k1 to k4.

The disappearance of superoxide by MnSOD-3 as a function of time (1.2 × 109 M−1 s−1) is comparable to that observed for human MnSOD (human MnSOD, 1.5 × 109 M−1 s−1). The gating ratio k2/k3 of 1 (both k2 and k3 are 5 × 108 M−1 s−1) for MnSOD-3 is again similar to that of human MnSOD, meaning that the amount of superoxide reduced by the outer-sphere pathway is equivalent to that reduced by the inner-sphere pathway. The two enzymes however, differ in the rate of dissociation of the Mn-peroxy complex. In MnSOD-3 this protonation off occurs more rapidly (k4 of 300 s−1) compared to that observed for the product inhibited human MnSOD (k4 of 120 s−1).42 As the catalytic rates of superoxide dismutation are close to the limit of the rate of diffusion, a 2.5-fold increase in a kinetic constant has a considerable effect on the catalysis of the enzyme.

Discussion

Evidence is emerging that mitochondrial MnSOD is not just an antioxidant but also a tumor suppressor protein as it affects the levels of superoxide and hydrogen peroxide in the cell.66 We show three structural features that have not been previously observed in native MnSODs. The first is evidence in the crystal structure of conformational variation (designated as conformer A and conformer B, Fig. 3) of the α-helical segment of the N-terminal hairpin that forms the major component of the tetrameric interface and leads to the substrate access funnel. This was observed in the 100 K MnSOD-3, the 293 K MnSOD-3, and in the MnSOD-3-azide complex showing that this protein is a dynamic, breathing molecule and may serve as a model for the study of molecular dynamics of MnSODs in general. Hydrophobic interactions between chain A and chain C are increased in conformer B [Fig. 3(B)] probably contributing to an increase in the stability of the tetrameric interface. Conformer A, however, is more likely to influence and increase the flux of superoxide toward the active site as this region is involved in steering superoxide anions. It is possible, therefore, that the flexibility of this segment of the α-hairpin may support both stability and catalysis, in this and similar SODs.

The second feature is the presence of two and not one, second sphere water molecules involved in the vital network between Tyr34 and His30, designated Tyr34OH-WAT2-WAT3-His30NE2. This arrangement occurs in the two subunits of the asymmetrical unit in both 100 K and 293 K MnSOD-3 structures. Furthermore, hydrogen bonding between Tyr34 and His30 is not feasible using a single water molecule. As the catalytic reaction relies on protonation, the number and arrangement of the water molecules in the proton relay pathway leading to the manganese center is of significance. All other native MnSODs described to date have only one water molecule linking these gateway residues.

And finally we present the structure of the MnSOD-3-azide complex, where the azide is end-on to the manganese center. Despite a low occupancy of 0.3, the azide is observed in the same arrangement in both subunits of the asymmetrical unit and is not bound non-specifically to other parts of the structure. In addition an average azide B factor of 16 indicates that the azide molecules present are firmly bound in the active site. In T. thermophilus, azide was shown to be bound side-on to the manganese and within bonding distance to Tyr34 (PDB entry 1MNG). Because of the absence of structural data of eukaryotic MnSOD-azide complexes, previous studies have relied on superimposition of eukaryotic MnSODs with the T. thermophilus MnSOD-azide complex and postulated hypotheses regarding the mechanism of catalysis derived from the position of the substrate complex and the arrangement of the active site solvent molecules.64,67 As the quaternary assembly of the T. thermophilus protein is different from that of the known eukaryotic MnSODs, the MnSOD-3-azide structure may serve as a better model for the study of substrate binding to eukaryotic MnSOD.

In an arrangement observed for the first time in MnSOD, the azide in MnSOD-3 is linked directly to the Tyr34 hydrogen-bond network through an additional water molecule (WAT4) introduced at this strategic point in the proton relay system [Fig. 3(B)]. This creates an original bonding network directly between the gateway residues, the substrate analog, and the metal center of the enzyme. Although it is not certain whether all three solvent funnel access water molecules (WAT2, WAT3, WAT4) occur in the structure simultaneously, it is likely that the network involving the gateway residues and the three waters does in fact form during catalysis. We propose that this provides a constant and rapid source of protons directly to the peroxy-adduct intermediate thereby facilitating the release of hydrogen peroxide and may contribute to the high product dissociation constant k4 observed in this enzyme. The kinetic information obtained by pulse radiolysis shows that MnSOD-3 is both very similar and yet different from the human MnSOD. The k1, k2, k3 rate constants and therefore the k2/k3 gating ratio are similar. The significant difference between these enzymes is the value of k4. In human MnSOD, this protonation off is slow (120 s−1) and causes the observed product inhibition. On the other hand, product dissociation in MnSOD-3 is faster (300 s−1) indicating lower product inhibition and therefore a more efficient enzyme at high superoxide levels. Because the initial rate constant for interaction of the MnSOD with superoxide is similar, this implies that the peroxy adduct forms at the same rate in both proteins, however, they do differ in how rapidly the peroxy product dissociates from the manganese. A similarly fast product dissociation has been reported for human MnSOD[H30N] and this has been associated with the in vivo antitumorigenic properties of this mutant.68 Curiously, and in support of our conclusions, in the human MnSOD[H30N] with a correspondingly high k4, the N30 side chain is positioned to make room for more solvent molecules in the gap between Tyr34 and Asn30.

Mutations of Tyr34 have been the subject of many studies to investigate whether the phenolic OH is the donor of the hydrogen needed for protonation off to release the peroxy adduct from the active site during product turnover.64,65,69–71 In human MnSOD mutations Y34A, Y34N, and Y34V (k4 values of 330, 200, and 1000 s−1 respectively), the rate of hydrogen product dissociation was faster than in the wild type human MnSOD.42 In these cases, a water molecule in a similar position to that observed for WAT4 can be seen in one subunit of each asymmetrical dimer (PDB entry 1ZSP, 2P4K, and 1ZUQ, respectively). As only few structures of SOD-azide complexes are currently available, the coordination and position of the substrate has been a point of contention for many years whereby even the crystal structure itself has been questioned. Consequently, MnSOD-3 is an elegant model to study the mechanism of catalysis and product inhibition, particularly in view of the tumor suppressor property of MnSOD.

Materials and Methods

Protein expression and purification

The E. coli strain OX326A (ΔsodA, ΔsodB)72 was used throughout for protein expression. The coding region of the mature MnSOD-3 was expressed using the pTrc99A expression system and MnSOD-3 was purified as previously described.13,73 Protein concentrations were determined by optical absorption at 280 nm using an extinction coefficient of 43340 M−1 cm−1 and the subunit molecular weight of MnSOD-3. Measurement of metal content by inductively coupled plasma-mass spectrometry (ICP-MS) was performed by ALS Analytica AB, Sweden.

Protein characterization

Optical absorption spectra of MnSOD-3 (3 mg mL−1) were collected using a Beckman DU7500 diode array spectrophotometer equipped with 50 µL micro cells. The as-isolated MnSOD-3 was oxidized by incubation with potassium permanganate (0.1 mM, 0.66:1 ratio with MnSOD-3) and reduced by 50 mM DTT. The MnSOD-3-azide complex was formed using 100 mM azide.

Superoxide dismutase assays were performed essentially as described by McCord and Fridovich3 and Ysebaert-Vanneste and Vanneste74 using cytochrome c as detector and xanthine oxidase as superoxide generator. The inhibitory effect of azide on SOD activity was determined by adding 4 or 10 mM sodium azide in 10 mM potassium phosphate buffer pH 7.8 to the SOD assay mixtures before the addition of xanthine oxidase.69

Crystallization, data collection, structure solution, and refinements

Crystallization, data collection, structure solution, and refinements for MnSOD-3 at 100 K (PDB accession code 3DC5) have been described previously.13 Although some preliminary data for the 293 K MnSOD-3 was previously published,13 the structure was not deposited in the Protein Data Bank (PDB). Further rounds of refinement were performed to 293 K MnSOD-3 prior to depositing in the PDB. Crystals for the MnSOD-3-azide complex were grown using the hanging-drop vapor diffusion method with drops consisting of 2 µL of the MnSOD-3 (100% metallated, confirmed by ICP-MS) and 2 µL of the reservoir liquor. Optimal protein crystallization was achieved using 0.1M bicine pH 9.2 and 2.7M ammonium sulfate for MnSOD-3 (8 mg mL−1). The MnSOD-3-azide complex was obtained by soaking a crystal in 100 mM sodium azide overnight. The crystal was flash-frozen in 50% (v/v) 3.4M sodium malonate/100 mM azide solution and a data set was recorded at 100 K at Daresbury SRS (Synchroton Radiation Source) station 10.1 on an ADSC Quantum charge-coupled device (CCD) detector).75 Data reduction and subsequent calculations were carried out using the CCP4 program suite. The diffraction data was integrated using MOSFLM76 and scaled with SCALA.77 Five percent of the data (the same set used for both MnSOD-3 at 100 K and 293 K) was excluded from the refinement to constitute the Rfree set using FREERFLAG.78 The MnSOD-3-azide complex was determined using the MnSOD-3 structure as a starting model for the refinement in REFMAC5.79 Ten cycles of rigid body refinement (resolution range 12.0–3.0 Å) were followed by ten cycles of restrained refinement (whole resolution range). Reiterated model building was performed using the program Coot80 and water molecules were added to the Fo-Fc map peaks >3.0 RMSD. The azide ions for chains A and C were added after placement of all the water molecules of the active site using 2Fo-Fc and Fo-Fc maps. TLS and restrained refinement along with occupancy refinement of the azide ions were carried out using REFMAC5.79 The geometry of the final model was checked using MOLPROBITY.81 The final structure of the MnSOD-3-azide complex was refined to R = 18.7% and Rfree = 21.6%.

Pulse radiolysis

The pulse radiolysis experiments were performed using a 2 MeV Van de Graaff accelerator at Brookhaven National Laboratory as described.82 In brief, superoxide anions were produced in situ in an air-saturated aqueous solution of 30 mM sodium formate (as a hydroxyl radical scavenger). All UV/visible spectra were recorded on a Cary 210 spectrophotometer with a path length of 2.0 or 6.1 cm, at a constant temperature of 25°C. The catalytic rates were measured by two methods.4 In the first method, hydrogen peroxide (2:1 [H2O2]:[MnSOD-3])44 was used to reduce the enzyme to the Mn2+ state. This reduced form of the enzyme was then oxidized (32.6 μM in potassium phosphate pH 7.5, 30 mM sodium formate, and 10 μL EDTA) with substoichiometric amounts of superoxide. The respective appearance/disappearance of Mn3+SOD was followed by recording the change in absorbance in the visible range (350–650 nm) of the spectrum. Mn3+SOD exhibits an absorbance band at 480 nm. In the second method, the decay of 50 mM superoxide using 6:1 and a 13:1 ratio ofInline graphic:Mn3+SOD was followed at 260 nm. The rate constants were calculated by fitting the data obtained to the mechanism given in Scheme 2 using the Chemical Kinetics program in PRWIN83 as described previously,82,84 taking into consideration the manganese concentration measured by ICP-MS (rather than the protein concentration) assuming that all the manganese is specifically bound at the active site.

Atomic coordinates

The atomic coordinates and structural factors for 293 K MnSOD-3, and MnSOD-3-azide complex have been deposited in the Protein Data Bank under the accession code 4X9Q and 5AG2, respectively.

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