BNL-108388-2015-JA The structure of the Caenorhabditis elegans Manganese Superoxide Dismutase MnSOD-3-Azide complex Gary J. Hunter1, Chi H. Trinh2, Rosalin Bonetta1, Emma E. Stewart2, Diane E. Cabelli3, Therese Hunter1* 1 Department of Physiology and Biochemistry, Faculty of Medicine and Surgery, University of Malta, Malta 2 Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom 3 Chemistry Department, Brookhaven National Laboratory, Upton, New York, United States *Correspondence to : Therese Hunter, Department of Physiology and Biochemistry, Faculty of Medicine and Surgery, University of Malta, Msida, MSD 2080, Malta. Tel: +356 2340 2916. Email: [email protected] Running title: C.elegans MnSOD azide complex 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 structure of MnSOD-3 determined at room temperature provides crystallographic evidence for the first time of a dynamic region of helix (residues 41 to 54) that plays roles in structural integrity through the tetrameric interface and in substrate steering. We have also 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 1 electron to form superoxide radicals O2- . These are rapidly converted to other reactive oxygen species (ROS) such as the hydroxyl radical. Unless removed, these ROS will damage cellular macromolecules2. 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 peroxide3,4. 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. Dismutation by SOD 3+ - 2+ M + O2 ⟶ M + O2 2+ - + 3+ M + O2 + 2H ⟶ M + H2O2 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 hsp60/1013-15. 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 factor16,17. 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 pathway24-27. The precise role of MnSOD-3 in life extension has been a topic of debate. As superoxide and hydrogen peroxide both act as signaling molecules28,29, it is likely that SOD-3, possibly together with SOD-2, serves as a modulator of longevity and other cellular pathways30-36, 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 oxidised MnIII to the reduced MnII state in a ‘ping-pong’ mechanism37,38. 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 inhibition39. This inhibition is caused by the formation of the peroxy complex of the manganese ion40,41. The McAdam scheme37 describes the MnSOD catalytic cycle in four reactions that show the dismutation of superoxide occurring through two simultaneous pathways (Scheme 2). Equation 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 equations 3 and 4 involves the formation and dissociation of the product-inhibited complex7. 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 complex39,42. Scheme 2. Mechanism of catalysis of MnSOD + k1 H 3+ - - 2+ Mn SOD(OH ) + O2 Mn SOD(H2O) + O2 (Eq.1) + k2 H 2+ - 3+ - Mn SOD(H2O)+ O2 Mn SOD(OH ) + H2O2 (Eq.2) k 3 2+ - 3+ 2- Mn SOD(H2O)+ O2 Mn SOD(H2O)(OO ) (Eq.3) + k4 H 3+ 2- 3+ - Mn SOD(H2O)(OO ) Mn SOD(OH ) + H2O2 (Eq 4) To date there are ten known structures of native homotetrameric MnSOD13,43-47. In the active sites of each, 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 interface13,43,47,48. This network permits the proton transfer needed to reduce superoxide to hydrogen peroxide. In order 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 MnSODs49. 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 Å50. The data collection and refinement statistics for the 293 K MnSOD-3 and 100 K MnSOD-3-azide complex are presented in Table 1. 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 Daresbury SRS 10.1 rotating anode Wavelength (Å) 1.541 0.97 Resolution range (Å) * 70.19–1.77 (1.87– 32.4–1.77 (1.86– 1.77) 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 190572 654522 No. of unique reflections 45989 46922 Redundancy 4.1 (4.0) 13.9 (4.0) Completeness (%) * 99.9 (99.9) 99.9 (99.9) < I/σ(I) >* 15.6 (2.3) 16.7 (13.3) Rmerge (%)§* 6.2 (33.8) 9.0 (51.7) Rpim (%)¥* 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 (%)† 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) ξ 0.013 0.011 R.m.s.d bond angles (˚) ξ 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 (%) ‡ Favored region 97.4 96.9 Outliers 0 0 PDB code 4X9Q 5AG2 *Values given in parentheses correspond to those in the outermost shell of the resolution range. § Rmerge = ∑hkl ∑i|Ii(hkl)-[I(hkl)]|/ ∑hkl ∑ Ii(hkl) 421/2 ¥ Rpim = ∑hkl {1/[N(hkl)-1]} ∑i|Ii(hkl) – [I(hkl)]|/ ∑hkl ∑i Ii(hkl) † Rfree was calculated with 5% of the reflections set aside randomly.