The Role of Mixed Amine/Amide Ligation in Nickel Superoxide Dismutase
Superoxide dismutases (SODs) utilize a ping-pong mechanism in which a redox-active metal cycles between oxidized and reduced forms that differ by one electron in order to catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. Nickel-dependent SOD (NiSOD) is a unique biological solution for controlling superoxide levels. This enzyme relies on the use of cysteinate ligands to bring the Ni(III/II) redox couple into the range required for catalysis (~300 mV). The use of cysteine thiolates, which are not found in any other SOD, is a curious choice because of their well-known oxidation by peroxide and dioxygen. The NiSOD active site cysteinate ligands are resistant to oxidation, and prior studies of synthetic and computational models point to the backbone N-donors in the active site (the N-terminal amine and the amide N atom of Cys2) as being involved in stabilizing the cysteines to oxidation. To test the role of the backbone N-donors, we have constructed a variant of NiSOD wherein an alanine residue is added to the N-terminus (Ala0-NiSOD), effectively altering the amine ligand to an amide. X-ray absorption, electronic absorption, and magnetic circular dichroism (MCD) spectroscopic analyses of as-isolated Ala0-NiSOD coupled with DFT geometry optimized models that were evaluated on the basis of the spectroscopic data within the framework of DFT and TD-DFT computations, are consistent with a diamagnetic Ni(II) site with two cysteinate, one His1 amide and one Cys2 amidate ligands. The variant protein is catalytically inactive, has an altered electronic absorption spectrum associated with the nickel site, and is sensitive to oxidation. Mass spectrometric analysis of the protein exposed to air shows the presence of a mixture of oxidation products, the principal ones being a disulfide, a bis-sulfenate, and a bis-sulfinate derived from the active site cysteine ligands. Details of the electronic structure of the Ni(III) site available from the DFT calculations point to subtle changes in the unpaired spin density on the S-donors as being responsible for the altered sensitivity of Ala0-NiSOD to O2.
Introduction<!>Materials and methods<!>Expression, Purification, Processing, and Reconstitution of Variant Protein.<!>Mass spectrometry.<!>Metal Analysis.<!>Quaternary Structure and Stability.<!>Kinetics.<!>Electronic Spectroscopy.<!>X-Ray Absorption Spectroscopy.<!>Computational Methods.<!>Protein Characterization.<!>Kinetics.<!>XAS.<!>Computational models.<!>Spectroscopy.<!>DFT computations.<!>Bonding Description.<!>Electronic Structure of oxidized Ala0-NiSOD.<!>Discussion<!>Conclusions
<p>Superoxide dismutases (SODs) are a family of metalloenzymes that protect aerobic organisms from oxidative stress caused by the superoxide radical anion (O2•−) and reactive oxygen species (ROS) that are derived from O2•−. Specifically, SODs catalyze the disproportionation of superoxide to molecular oxygen (O2) and hydrogen peroxide (H2O2) at rates that are near the diffusion limit. Therefore, SODs are regarded as the first line of defense against this harmful reactive oxygen species.1 During catalysis, the redox-active metal cofactor cycles between oxidized and reduced states that differ by one electron (Equations 1–3).
(1)M(n+1)+O2•−→Mn++O2
(2)Mn++O2•−+2 H+→M(n+1)+H2O2
(3)2 O2•−+2 H+→O2+H2O2</p><p>SODs can be categorized into three distinct classes based on amino acid sequence homology: CuZnSOD, FeSOD and MnSOD, and NiSOD.1 Much like the Fe- and Mn-SODs, the more recently discovered NiSOD enzyme catalyzes the disproportionation of O2•− by cycling between M(III) and M(II) oxidation states. However, NiSOD has a unique active site structure that is necessary to support redox catalysis by nickel. The first two residues of the NiSOD protein are responsible for donating four of the five ligands involved in coordination of the nickel cofactor. In the resting reduced state of NiSOD, the Ni(II) cofactor adopts a four-coordinate, planar NiN2S2 geometry, involving coordination by the N-terminal amine, a deprotonated amide from the Cys2 backbone, and the side-chains of Cys2 and Cys6. In the oxidized state of NiSOD, the Ni(III) ion additionally binds the imidazole side chain of His1, which results in a five-coordinate pyramidal geometry around the Ni(III) ion (Figure 1).2–4</p><p>Ligation by an N-terminal amine is rare in metalloproteins and to date has only been observed in NiSOD, HypA, E.coli HypB, RcnR, and albumins, of which NiSOD is the only enzyme.5–9 Based on results obtained from computational and synthetic models, mixed amine/amide ligation in NiSOD has been suggested to stabilize the Ni(III) state, making the oxidation more metal-centered and thus also protecting the S-donors from oxidation by substrate, O2•−, and products, H2O2 and O2.10–14 It has been demonstrated for several NiN2S2 complexes featuring bisamine ligation that the thiolate donors are sensitive to oxidation, while bisamide complexes tend to be more stable to sulfur oxidation.10, 15 However, recent studies employing synthetic peptide models reached the opposite conclusion—that "all NiSOD mimics are highly unstable showing sulfur oxidation through the formation of disulfide-bridges between monomeric peptide units and sulfur oxygenation".16 Further, it was proposed that "the ability to degrade superoxide by the model peptides is coupled to an increased sensitivity for oxidation, which results from the asymmetric amine/amide Ni(II) coordination." These trends have not been studied directly in the NiSOD enzyme. In this study, we have produced an insertion mutation involving addition of an alanine residue at the N-terminus adjacent to the His1 residue (Ala0-NiSOD), which extends the N-terminus by one residue. This variant provides an opportunity for a direct comparison between the enzyme and model complexes with altered amine/amide ligation. Our results suggest that extension of the N-terminus causes changes in the structure of the reduced enzyme consistent with coordination of the His1 amide linkage. This small structural change has a large effect on the electronic structure and reactivity, and results in dramatic changes in the redox properties, catalytic activity, and oxygen sensitivity of the active site. These changes are interpreted in the framework of DFT calculations that were validated on the basis of spectroscopic data.</p><!><p>The single Ala0-NiSOD variant was prepared by PCR of a WT-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5'- and 3'-primers using the QuikChange® XL Site-Directed Mutagenesis Kit (supporting information Table S1). NovaBlue (Novagen™) competent cells were transformed with the PCR product and transformants were selected by antibiotic resistance. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit from Qiagen, and DNA sequencing was performed to confirm the expected base sequence for the mutant (W. M. Keck DNA facility at Yale University, New Haven, CT). BL21 (DE3) pLysS (Novagen™) competent cells were transformed with the plasmid containing the desired insertion, transformants were selected by antibiotic resistance, the mutant gene was expressed, and the variant protein purified as previously described.17–19</p><!><p>Single colonies were grown overnight at 37 °C with shaking in 10 mL Luria-Bertani broth, supplemented with chloramphenicol (cam) and kanamycin (kan) for selection. These cultures were added to 1 L of pre-warmed fresh media and grown to an OD600 of 0.6 and then induced with 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3–5 hours. Cells were harvested by centrifugation, resuspended in 40 mL Ni-NTA binding buffer (10 mM imidazole, 50 mM Tris, 300 mM sodium chloride pH 8.0), and then frozen at −80 °C to lyse the cells. The cell harvests were thawed and treated with 100 μL of DNAse I solution (10 mg/ml DNAse I, 10 mM magnesium chloride, 20 mM Tris pH 7.5, 40% glycerol) at 37 °C until the viscosity of the solution was significantly reduced. The cell lysate was centrifuged for 5 min at 8,000 rpm and the supernatant was used for chromatographic protein purification.</p><p>All chromatographic purifications employed an AKTA-FPLC (Amersham Biosciences). The cell lysate supernatant was loaded onto a column (Pharmacia HR10) containing Ni-NTA HisBind Superflow™ resin (Novagen™) at 3 ml/min with Ni-NTA binding buffer. Once the absorbance at 280 nm returned to the baseline, the buffer was changed to 12% elute buffer (250 mM imidazole, 50 mM Tris, 300 mM sodium chloride pH 8.0) in one step, and the column was washed with 7 column volumes of 12% elute buffer. The fusion protein was then eluted from the column using 70% elute buffer. Electrospray ionization mass spectrometry (ESI-MS) was used to confirm the molecular weight (MW) of the expected fusion protein (vide infra).</p><p>To remove imidazole from the protein sample, the fusion protein was loaded onto a column (Pharmicia HR10) containing Q-Sepharose resin at 3 mL/min with bind buffer (50 mM Tris pH 8.50). Once the absorbance at 280 nm returned to the baseline, the buffer was changed to 40% elute buffer (50 mM Tris, 500 mM sodium chloride pH 8.5) in one step, and the fusion protein was then eluted from the column and collected. The eluted sample buffer was then adjusted to the conditions used for N-terminal processing using factor Xa (5 mM CaCl2, 50mM Tris, 100mM NaCl, pH 8.0). The purified fusion protein was cleaved to yield NiSOD with Ala0 at the N-terminus by using factor Xa. The concentration of fusion protein in factor Xa cleavage buffer was determined using a bicinchoninic acid (BCA) assay. The assay was performed using the Enhanced Test Tube Protocol outlined in Pierce's BCA™ Protein Assay Kit instruction manual. Factor Xa was then added (30 μL 2.1 U/μL factor Xa/50 mg fusion protein) and the mixture was incubated at 4 °C. The extent of cleavage was monitored using 16% SDS-PAGE. Processed protein was reduced with a five-fold excess of dithiothreitol (DTT) and reconstituted with a three-fold excess of NiCl2 in an anaerobic glove box (Coy Laboratory Products, Inc.). The hexameric quaternary structure of the holo-protein confirmed by size exclusion chromatography (supporting information Figure S1)</p><!><p>To determine the molecular weight (MW) of the NiSOD expression products, ESI-MS analysis was performed using a Bruker Esquire mass spectrometer equipped with an HP-HPLC. Analysis was performed under protein denaturing conditions, 0.1% formic acid and skimmer voltage set to 120 V, and samples were prepared to a final concentration of 20 μM monomer (Table 1).</p><p>Mass spectrometry was also used to identify the species produced by air-oxidation of Ala0-NiSOD, which resulted in a mixture of Cys-S oxygenates and a disulfide. The presence of a disulfide bond in a portion of the air-oxidized Ala0-NiSOD sample mixture was identified by ESI-MS analysis before and after incubating the sample with 5mM tris(2-carboxyethyl)phosphine (TCEP) for 3 hours. ABI-SCIEX QSTAR-XL equipped with a turbo-spray ESI source in sequence with an Agilent 1100 HPLC system with a BioBasic-8 (Thermo Scientific) column was used, and the protein was eluted with a 0.1% formic acid/5% aqueous acetonitrile solution. As a control, ESI-MS was also used on a similarly treated sample of WT-NiSOD, which showed no evidence of S-oxygenation (see supporting information, Figure S2).</p><p>To identify the position of oxygen modifications, peptide analysis employing a Waters Synapt G2-Si mass spectrometer equipped with a nanoACQUITY UPLC system was used. The sample was run through a 2.1 × 30 mm pepsin immobilized Enymate column (Waters), and the peptides were separated using an analytical HSS T3 column (Waters) before injection into the mass spectrometer. The data were analyzed using Waters ProteinLynx Global Server 3.0.1 (PLGS).</p><!><p>A PerkinElmer Optima 4300 DV inductively coupled plasma-optical emission spectrometer (ICP-OES) was used to quantify the nickel content of the reconstituted variant protein. This instrument is equipped with a 40 MHz free-running generator and a segmented-array charge-coupled device (SCD) detector. The sample introduction system consisted of a concentric nebulizer with a cyclonic spray chamber. The concentration of nickel in the sample was determined at λ = 231.604 nm.</p><!><p>To determine the oligomeric state of the proteins, size-exclusion chromatography was performed using a Superdex 75 15/300 GL (GE lifesciences) column. The column was standardized with albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa). A standard curve was constructed by plotting Ve/V0 vs MW (where Ve = elution volume of the peak, and V0 = elution volume of blue dextran), and the data were fit with a second order polynomial. WT- and Ala0-NiSOD were injected onto the column at a concentration of 500 μM (monomer) in 20 mM Tris, 100 mM NaCl pH 8.0, and the retention volume of the peak(s) (Ve/V0) from the chromatogram was analyzed using the standard curve to determine the molecular weight of the eluted protein (see Figure S1).</p><p>Melting temperature (Tm) was measured with a Microcal VP-DSC with a 0.5 mL sample and reference cells. The sample was concentrated to 100–140 μM monomer in 50 mM Tris buffer. The protein sample and blank (50 mM Tris buffer) were degassed under vacuum for 10 min and syringed into the cells using a pulsing motion to expel air bubbles. The protein sample was run at 30 psi over 25–100 °C at a scan rate of 30 °C/hour. Baseline correction and normalization were performed with the Microcal interface to the Origin graphing program, and the Tm was taken to be the peak maximum of the thermogram.</p><!><p>Enzyme kinetics were studied using pulse radiolytic generation of O2•− and were carried out using a 2 MeV van de Graaff accelerator at Brookhaven National Laboratory. Superoxide radicals were generated upon pulse radiolysis of an aqueous, air/O2 saturated solution containing 10 mM phosphate, 30 mM formate, and 5 μM ethylenediaminetetraacetic acid (EDTA) (Equations 4–8). Catalytic rate constants were obtained by monitoring the disappearance of O2•−
(6)OH⋅+HCO2−→H2O+CO2⋅−
(7)CO2⋅−+O2→CO2+O2⋅−
(8)eaq−+O2→O2⋅−
(9)H⋅+O2→HO2
(10)HO2⇌O2⋅−+H+
at 260 nm in the presence of micromolar concentrations of WT- and Ala0-NiSOD. The path length of the quartz cell used was 2.0 cm and a 200–1900 ns pulse width was chosen, resulting in the generation of ~6–45 μM O2•− per pulse. The disappearance of O2•− was followed in identical pH = 8.5 solutions where one contained 10μM Ala0-NiSOD and one only buffer. The bimolecular rate for the disappearance of O2•− was 3×104 M−1s−1. In the presence of enzyme, there was some evidence for a first order rate of 1s−1, leading to a possible bimolecular rate for catalytic activity of the enzyme of ≤ 1×105 M−1s−1. This bimolecular rate was calculated using the nickel concentration, with the assumption that all nickel ions are specifically bound and equally contributing to SOD activity.</p><!><p>Variable temperature absorption and magnetic circular dichroism (MCD) spectra of Ala0-NiSOD were obtained on a Jasco J-715 spectropolarimeter in conjunction with an Oxford Instruments SM-4000 8T magnetocryostat. To eliminate contributions from the CD background and glass strain to the MCD signal, the difference between MCD data collected with the magnetic field aligned parallel and antiparallel to the light propagation axis was taken. The final protein concentrations used for samples of Ala0-NiSOD prepared in an anaerobic environment was 0.655 mM, while the final concentration of Ala0-NiSOD after long-term exposure to air was 0.720 mM. All samples contained at least 55% (v/v) of the glassing agent glycerol.</p><!><p>X-ray absorption spectroscopy (XAS) data collection and analysis were performed as previously described.20 Nickel K-edge XAS data were collected on beamline X9B at the National Synchrotron Light Source (Brookhaven National Laboratory). Samples of frozen protein solutions (1 mM, based on nickel content, in 20 mM Tris•HCl, pH 8.0) were placed in polycarbonate holders inserted into aluminum blocks and held near 50 K using a He displex cryostat. The ring conditions for data collection were 2.8 GeV and 120–300 mA. A sagittally focusing Si(111) double crystal monochromator and a 13-channel Ge fluorescence detector (Canberra) were used for data collection. X-ray absorption near-edge spectroscopy (XANES) data were collected from ± 200 eV relative to the nickel K-edge. The edge energy reported was taken to be the maximum of the first-derivative of the XANES spectrum. Extended X-ray absorption fine structure (EXAFS) data were collected to 9307 eV (k = 16 Å−1). The X-ray energy for the K-edge of nickel was internally calibrated to 8331.6 eV using transmission data from a nickel foil. The data shown are the average of 6 scans. Sixpack software was used to remove bad detector signals, calibrate the edge energy of Ni foil, and average the data. Athena software was used for data reduction and normalization. The EXAFS123 software package was used for XANES analysis.21</p><p>EXAFS data were analyzed using the Artemis software as previously described.22 Scattering parameters were generated using the FEFF 8 software package23 with the starting point for refinement derived from the atomic coordinates of the geometry-optimized computational model (vide infra). The k3-weighted data were fitted by setting the FT window to 2.0−12.5 Å and fitting from r = 1.0 −4.0 Å in r-space with an S0 value of 0.9. Scattering from atoms in the first, second and third (where noted) coordination shells was accounted for in the fits, including multiple-scattering pathways that contribute more than 15% to the EXAFS. Imidazole ligands were fit as rigid rings with one adjustable Ni-N distance and one value of σ2 for all the atoms in the ring. In order to compare different models to the same data set, ifeffit was used to obtain three goodness of fit parameters, χ2 (Equation 9), reduced χ2, and the R-factor (Equation 10), where Nidp is the number of independent data points, Nε2 is the number of uncertainties to minimize, Re(fi) is the real part of the EXAFS fitting function, and Im(fi) is the imaginary part of the EXAFS fitting function. Reduced χ2 = χ2/(Nind - Nvarys) (where Nvarys is the number of refining parameters) and represents the degrees of freedom in the fit.
(11)χ2=NidpNε2∑i=1N{[Re(fi)]2+[Im(fi)]2}
Ifeffit also calculates the R-factor for the fit, which is given by Equation 10 and is scaled to the magnitude of the data, making it proportional to χ2. To compare different models (fits) the R-factor and reduced χ2 parameters can be evaluated to determine which model provides the best fit, in which case both parameters should be minimized. Although the R-factor will always improve with an increasing number of adjustable parameters, reduced χ2 will go through a minimum and then increase, indicating that the model is over fitting the data.
(12)R=∑i=1N{[Re(fi)]2+[Im(fi)]2}∑i=1N{[Re(χ˜datai)]2+[Im(χ˜datai)]2}</p><!><p>All DFT geometry optimizations and single-point calculations were carried out using the ORCA 3.0 program developed by Dr. Frank Neese.24 X-ray crystallographic data of the active site from subunit A of WT-NiSOD (PDB code 1T6U)2, 3 served as the starting point for the generation of the different computational models investigated. In each case, a constrained geometry optimization was carried out whereby all atoms were allowed to move except for those indicated in boldface type below. All initial structures included the primary coordination sphere residues (His1, Cys2, and Cys6) of NiSOD. The carbonyl group of the amide of Cys2 was transformed to a methyl (-CH3) and Cys6 was truncated at the alpha carbon and modeled as CH3CH2S−. These atoms made up the active site models of reduced and oxidized WT-NiSOD (WTred and WTox, respectively). For models of the Ala0-NiSOD nickel site, alanine was built as a new fragment using the PyMOL program and was incorporated via a peptide bond to the His1 terminal amine to account for possible amide coordination to the Ni(II) center (Ala0red and Ala0ox). The side chain was first truncated and modeled as a glycine to determine whether the terminal amine could also coordinate to the nickel and form a six-coordinate complex. Geometry optimizations of this model led to the dissociation of the terminal amine from the nickel. For this reason, alanine was truncated at the α-carbon and methylated (–COCH2CH3) in all subsequent geometry optimizations of Ala0-NiSOD active site models.</p><p>To explore the protonation state of the His1 backbone amide, an additional model, denoted as Ala0-depred, was constructed using the same initial coordinates as for the other models except that the proton was removed from the His1 backbone N atom.</p><p>Six-coordinate, high-spin Ni(II) models were also generated to evaluate possible Cys thiolate oxidation products. In these models, Ala0 was modeled as a glycine by removing the – CH3 side chain and capping the α-carbon with a hydrogen atom. The terminal amine was also retained. The thiolates from Cys2 and Cys6 were modified to represent sulfinates by the addition of two oxygen atoms to each sulfur. Two models were considered, one in which one of the sulfinate oxygens (6C-O) and another in which the sulfinate sulfurs (6C-S) are coordinated to the Ni(II)-ion. For comparison, a DFT geometry optimization was also performed using previously published coordinates of an active site model of a Cys → Ser NiSOD variant (C2S/C6S6-c)25 using the same functional and basis sets as listed below.</p><p>All DFT geometry optimizations, as well as single point and time-dependent (TD-DFT) calculations, were performed using Ahlrich's polarized split and auxiliary basis sets26 (SV(P) and SV/C, respectively) for all atoms except for nickel and the atoms directly coordinating to it, for which Ahlrich's valence triple-ζ basis set, TZV(P) was employed.26 Becke's three-parameter hybrid functional27 in conjunction with the Lee-Yang-Parr functional28 (B3LYP) were employed in all computations. Spin-restricted and spin-unrestricted formalisms were used for calculations involving diamagnetic, S = 0 Ni(II) (i.e., WTred, Ala0red and Ala0-depred) and paramagnetic, S = 1 Ni(II) (i.e., 6C-O and 6C-S) or S = 1/2 Ni(III) (i.e., WTox and Ala0ox) complexes, respectively. Isosurface plots of molecular orbitals (MOs), electron density difference maps, and total spin density plots were generated using Pymol29 with isodensity values of 0.05, 0.005, and 0.005 a.u, respectively. All MO assignments were made by visual inspection.</p><!><p>The purified Ala0-NiSOD fusion protein had the MW expected for the single alanine insertion (Table 1). Upon N-terminal processing with Factor Xa to remove the fusion peptide and nickelation, the Ala0-NiSOD variant bound nickel with a stoichiometric ratio of ~1:1 (Table 1). Unlike the native enzyme, which is golden-brown in color, the Ala0-NiSOD variant was a deep reddish-brown. This color change corresponds to an apparent red-shift of the predominantly S → Ni ligand-to-metal charge transfer (LMCT) transitions from the UV in the reduced WT enzyme to 457 nm in Ala0-NiSOD (Figure 2). The variant was unstable during storage in air; over a period of about two weeks, the 457 nm peak intensity slowly decreased, ultimately resulting in a light tan-colored sample (Figure 2), suggesting oxidation of the Ni site. This change in the electronic absorption spectrum was accompanied by a change in the ESI mass spectrum that is consistent with the formation of a mixture of protein products that feature oxidation of the two cysteinate ligands to form a disulfide as well as the addition of oxygen atoms to form oxygenates (Figure 3a and supporting information Figure S3). The mass associated with the major peak of the air exposed Ala0-NiSOD mass spectrum is 13269.9 Da, which was 2 Da less than the expected unmodified mass. When 5 mM TCEP was added to the solution, the mass associated with the major peak increased to 13271.6 Da, indicating that the mass difference was caused by the presence of a disulfide bond (Figure 3b). Two other major peaks in the deconvoluted spectrum correspond to Ala0-NiSOD oxygenates with + 2 O and + 4 O atoms. Features that might correspond to trace amounts of other products (e.g., + 1 O and + 3 O) are comparable to the noise level of the data. There is no evidence for unmodified Ala0-NiSOD, and thus the air-exposed sample is 100% oxidized, but features a mixture of several protein species.</p><p>Based on the chemistry of model Ni thiolate complexes, the sulfoxygenates likely represent sulfenate and/or sulfinate complexes.30–32 Pepsin digestion shows that the oxygen incorporation is associated with peptides that contain Cys2 and Cys6 (Figures S2). MS/MS analyses of N-terminal peptides containing the Cys residues show modifications of both Cys residues by one O atom in the + 2O product, consistent with a bis-sulfenate, and modification of both Cys residues by 2 O atoms in the + 4 O product, consistent with a bis-sulfinate. Other oxidation products were not present in amounts sufficient for MS/MS analysis.</p><p>Size-exclusion chromatography of as-isolated Ala0-NiSOD gives rise to an elution peak with a retention volume corresponding to the molecular weight appropriate for a homohexamer (supporting information Figure S1). The protein thermal stability was minimally altered by the Ala0 N-terminal extension, as revealed by the DSC data (Figure 4), melting temperatures (Tm, Table 1), and stability of the hexamer. The DSC thermogram associated with Ala0-NiSOD is similar in general appearance to that of WT-NiSOD, but reveals a lower melting temperature (Figure 4, Table 1). The DSC thermograms of Ala0- and WT-NiSOD both exhibit an asymmetric curve with a tail to the low temperature side that suggests a complex thermal denaturation of these proteins, perhaps reflecting dissociation of the hexamer followed by unfolding of monomers.19</p><!><p>The rate of superoxide dismutation in the presence of Ala0-NiSOD was determined by monitoring the disappearance of the optical absorbance of pulse radiolytically generated O2•− at 260 nm in the presence and absence of enzyme. The catalytic rate observed for WT-NiSOD at pH 7.5 is 0.71×109 M−1s−1.18 In contrast, in the presence of Ala0-NiSOD, virtually no increase in the rate of disappearance of superoxide above the spontaneous rate of superoxide dismutation under these conditions (~2×105 M−1s−1) is observed (Table 1). At pH 8.5, there is only a trace of catalytic activity of ≤ 1×105 M−1s−1; Figure 5. Thus, the activity of NiSOD is essentially abolished by the Ala N-terminal extension.</p><!><p>Ni K-edge XAS was used to examine the structure of the Ni site in Ala0-NiSOD prepared anaerobically. Features in the X-ray absorption near-edge structure (XANES) region of the nickel K-edge XAS spectrum provide information about the nickel center coordination number and geometry.33 The XANES spectrum obtained for Ala0-NiSOD (Figure 6) has a peak near 8331 eV that is associated with a 1s → 3d electronic transition, the area of which (Table 2) is most consistent with the presence of a five-coordinate nickel site. Although a highly distorted four-coordinate structure cannot be completely ruled out, such as a C2v idealized geometry with dihedral angles greater than 0° (the value of a planar complex),34 this is not supported by our computational model (vide infra). The presence of a shoulder near 8338 eV, rather than a resolved maximum, in the Ala0-NiSOD XANES spectrum is associated with the Ni 1s → 4pz electronic transition, and is typical of Ni(II) complexes with a five-coordinate pyramidal geometry.33 This is distinct from dithionite-reduced WT-NiSOD (Figure 6), where the XANES spectrum exhibits a much more resolved maximum near 8338 eV that provides unambiguous evidence for a Ni(II) site with four-coordinate planar geometry,33 which has been confirmed by crystallographic analysis.2, 3 The comparison of the XANES spectra for Ala0-NiSOD and reduced WT-NiSOD also reveals a more intense maximum (white line) for Ala0-NiSOD, indicating an increase in the N:S donor ratio relative to reduced WT-NiSOD.33</p><p>The EXAFS region of the XAS spectrum provides information regarding the metal ligands, including the donor-atom types, numbers, and bond distances. Analysis of the EXAFS (Table 3 and Figure 7) was performed by first modeling the primary coordination sphere provided by ligands other than the His1 imidazole. This gave a best fit for N2S2 coordination with two resolved Ni-N distances and a single average Ni-S distance (Table 2). Attempts to split the S-scattering atoms into separate shells gave Ni-S distance differences that were below the resolution of the data (~0.15 Å) (Table S2). Next, C atoms from chelate rings formed by coordination of backbone N atoms (His1 amide N and Cys2 amide N atoms and by coordination of the Cys2 amide N and thiolate S atoms) were added to the model and the distances refined, improving the fit (Table 2). Addition of two third-coordination sphere O atoms to model the amide carbonyl O atoms that should be ordered by the five-membered chelate rings also improved the fit. This four-coordinate fit is compared with the computational model structure in Table 3.</p><p>To test whether the His1 imidazole is coordinated, an imidazole ring was added to the planar model. This gave a very modest improvement to the fit over the four-coordinate model and gave a Ni-N(Im) distance (Ni-N = 2.61(8) Å) that is in reasonable agreement with a crystallographically determined Ni-N(Im) distance (2.70 Å) in oxidized WT-NiSOD.3 This result is also in agreement with the XANES analysis (vide supra), and raises the possibility that under the conditions used for XAS data collection the complex is a low-spin five-coordinate Ni(II) complex with an apical imidazole ligand. However, a five-coordinate Ni(II) site in Ala0-NiSOD is at odds with the computational structures, all of which are consistent with a four-coordinate planar site and indicate that the imidazole sidechain of His1 is not a ligand (vide infra). This finding suggests that while the Ni(II) center favors a four-coordinate, square planar coordination environment, outer-sphere interactions not accounted for in our computational models may prevent complete dissociation of the His1 side chain by limiting the Ni-N(Im) distance to ~2.6 Å (note that this weak axial Ni-N interaction is not expected to affect the electronic structure of the Ni(II) site of Ala0-NiSOD). In any event, the XAS data are consistent with coordination of the two amide N-donors and two Cys S-donors predicted by the computational models.</p><!><p>To elucidate the Ni coordination environment of Ala0-NiSOD under anaerobic conditions, active site models were generated starting from the published crystallographic coordinates of the oxidized active site of WT NiSOD2 and optimized using DFT. Because our MCD data indicate that Ala0-NiSOD possesses a diamagnetic ground state (vide infra), only S = 0, Ni(II) models were considered. The optimized structures of these models are compared to the analogous active site model of reduced WT-NiSOD (WTred) in Figure 8 and Table 3.</p><p>From the optimized bond distances for Ala0red and Ala0-depred (Table 3), these active site models are not predicted to maintain the five-coordinate geometry initially imposed (chosen on the basis of XAS data, vide supra). Instead, the optimized bond distances for the geometry optimized WTred model closely match those determined by X-ray crystallography and EXAFS analysis for the reduced WT-NiSOD enzyme (Table 3) and Ala0-NiSOD (note, however, that a weak axial Ni-N interaction as suggested by our XAS data cannot be ruled out on the basis of these computations, as the WTred model did not include outer-sphere interactions that may limit the conformational freedom of the His1 side chain). The Ni(II) ion in all reduced models resides in a four-coordinate, distorted planar ligand environment with coordination from the His1 backbone NHis1,eq, the Cys2 backbone amidate, and the Cys2 and Cys6 thiolates. Below, the novel electronic absorption features exhibited by Ala0-NiSOD will be used to determine which of the two Ala0-NiSOD computational models more faithfully reproduces the Ni(II) coordination environment in this variant.</p><!><p>The Ala0 insertion dramatically affects the electronic properties of the nickel center. X-band EPR spectroscopy was used to characterize the redox state of the nickel center in the as-isolated protein. Resting (as-isolated) native and recombinant WT-NiSOD exhibit a rhombic EPR spectrum that arises from a five-coordinate, low-spin (S = 1/2) Ni(III) center, with the unpaired electron residing in the Ni dz2-based molecular orbital.18 Anaerobic Ala0-NiSOD and air-oxidized Ala0-NiSODox samples lack this EPR signal; both were EPR-silent (data not shown), indicating that only Ni(II) was present.</p><p>The low temperature electronic absorption spectrum of Ala0-NiSOD under anaerobic conditions (Figure 9, top) exhibits an intense feature centered around 22,300 cm−1 along with several higher energy bands at ~29,000 and 31,300 cm−1 and a lower energy shoulder at ~19,000 cm−1. This spectrum is vastly different from that obtained for reduced WT-NiSOD,10 most notably in terms of the appearance of the intense band at 22,300 cm−1, indicating that Ala0-NiSOD possesses a unique Ni(II) site. The corresponding MCD spectrum is essentially featureless (Figure 9, bottom). The lack of temperature dependent features coinciding with the dominant absorption bands provides compelling evidence that Ala0-NiSOD contains a low-spin, diamagnetic (S = 0) Ni(II) center like reduced WT-NiSOD.10</p><p>After long-term exposure of Ala0-NiSOD to air, the absorption feature at 22,300 cm−1 is no longer present (Figures 1, 10), indicating a change in the electronic structure of the active site, possibly due to thiolate oxidation. Consistent with this hypothesis, the corresponding MCD spectrum (Figure 10, bottom) is distinctly different from that of the oxidized form of WTNiSOD.10 Most notable is the appearance of temperature-dependent, negatively signed features located at ~16,800 cm−1 and ~28,000 cm−1, indicating the presence of a paramagnetic species in the mixture of protein oxidation products. The Ala0-NiSODox MCD spectrum closely resembles the MCD spectrum obtained for active site Cys → Ser NiSOD variants, which have no thiolate ligands and feature high-spin, S = 1, Ni(II) centers. This result is consistent with oxidation of Cys2 and Cys6 in Ala0-NiSODox.19, 25 Because Ala0-NiSODox was shown to be a mixture by mass spectrometry (vide supra), the spectra were not analyzed in further detail.</p><!><p>To assess which geometry-optimized Ala0-NiSOD model is most likely representative of the coordination environment of Ala0-NiSOD, TD-DFT was used to compute absorption spectra for Ala0red, Ala0-depred, and WTred. The appearance of an intense feature at 22,300 cm−1 in the experimental Ala0-NiSOD absorption spectrum that is absent in the absorption spectrum of WT-NiSOD served as marker for evaluating the computational models.</p><p>The TD-DFT computed absorption spectra for all models are shown in Figure 11. Interestingly, even though all models possess similar ligand environments, the TD-DFT computed absorption spectrum for Ala0red exhibits striking differences from those predicted for WTred and Ala0-depred. The presence of an intense feature for this model, absent in the calculated spectra for WTred and Ala0-depred, is consistent with the experimental absorption spectrum of Ala0-NiSOD prepared under anaerobic conditions (Figure 9). The energy of this feature is overestimated compared to the experimental data, which is common for TD-DFT calculated spectra.35 To facilitate a direct comparison with the experimental data, the TD-DFT computed absorption spectra of all models were red-shifted by 10,000 cm−1.</p><p>The predicted absorption spectrum for Ala0red is dominated by an intense feature at ~23,400 cm−1 and two higher energy bands at ~26,600 and 29,700 cm−1, which have no counterparts in the computed WTred and Ala0-depred spectra. An electron density difference map reveals the LMCT character of the transition associated with the ~23,400 cm−1 feature (Figure 11). Specifically, this transition involves electronic excitation from the deprotonated amide (N/O) of the Cys2 backbone to the Ni(II) ion. These results indicate that replacing a coordinating amine with an amide results in dramatic changes to the interaction between Ni(II) and the Cys2 amidate ligand. Note that for WT-NiSOD, a similar amidate → Ni(II) LMCT transition is predicted at ~31,100 cm−1. The slightly decreased S 3p orbital contribution from Cys6 (which is trans to the Cys2 backbone amide) to the LUMO of the variant likely contributes to the red-shift of this transition from WT-NiSOD to Ala0-NiSOD.</p><p>Alternatively, the computed absorption spectrum for Ala0-depred only exhibits slight differences from the WTred spectrum (Figure 11). Two features, located at ~21,500 and 23,100 cm−1, have intensities that are reasonably similar to that of the prominent band at 22,300 cm−1 in the experimental Ala0-NiSOD absorption spectrum (Figure 9). However, nearly identical features are present in the computed absorption spectrum for WTred, which is inconsistent with the drastic changes to the experimental absorption spectrum in response to the Ala0 substitution. Thus, we conclude that the Ala0red model more faithfully reproduces the active site environment of the Ala0-NiSOD variant under anaerobic conditions.</p><!><p>Our results suggest that amide coordination to the Ni(II) center by the His1 backbone dramatically changes the electronic structure of the active site, the effects of which are most evident in the appearance of an intense feature in the absorption spectrum of Ala0red that has no counterpart in the WTred spectrum. The results from a DFT single point calculation for Ala0red reveal extensive mixing between the Ni and ligand frontier orbitals (Figure 12). The lowest unoccupied molecular orbital (LUMO) is Ni dx2-y2 based (45.9% 3d-character), consistent with the LUMO composition of WTred and the d-orbital splitting of a four coordinate, square planar Ni(II) complex.10 The highest occupied molecular orbital (HOMO) is ligand based with large contributions from the S 3p-orbitals of both Cys2 and Cys6 (64.6%) that engage in π-antibonding interactions with the Ni dxz-based orbital (26.2% character). The HOMO-2 is predominantly Ni dz2-based (79.4% 3d-character), while the remaining Ni 3d-based MOs have large ligand contributions and are difficult to unambiguously identify.</p><!><p>To further examine the consequences of the Ala0 modification of NiSOD, the one-electron oxidized derivative of the Ala0red model (Ala0ox) was generated computationally and compared with WTox (Figure 13 and Table 3). In agreement with X-ray crystal structures of the active site of oxidized Ni(III)SOD,2, 3 both the WTox and Ala0ox models retain a five-coordinate, distorted square pyramidal geometry with additional ligation from the imidazole ring of His1. The most dramatic difference between the Ala0ox and WTox models involves the Ni-NHis1,eq bond distance, consistent with the difference in amine versus amide coordination.</p><p>The DFT-computed MO diagram for Ala0ox is shown in Figure 14. Due to spin-polarization and the consequent mixing between Ni and ligand-based orbitals, only the spin-down (β)-MOs are shown. Plots of the β-spin MOs and the unpaired spin density for Ala0ox (Figure 14, inset) reveal that the formally singly occupied MO has significant orbital contributions from Ni (69.5% 3d-orbital character) and SCys2 (12.9% 3p-orbital character), which participate in a π-antibonding interaction. This sizable percentage of sulfur orbital character (along with other factors, such as an altered ability of the His1 side chain to coordinate to the Ni(III) ion) could be partially responsible for the instability of Ala0-NiSOD in air, as evidenced by the changes in the experimental absorption spectrum and mass spectrometric characterization (Figures 2 and 3). To higher energy, the β-LUMO+1 has significant contributions from the S ligand atoms (23.9% 3p orbital character) as well as Ni (51.3% 3d-orbital character), which engage in σ-antibonding interactions. The remaining Ni 3d-based MOs can be identified at lower energies. Ligand orbitals produce the dominant contributions to the MOs in the energy window between the β-LUMO and the filled Ni 3d-based β-MOs.</p><!><p>Nickel binding in NiSOD is achieved utilizing only three invariant amino acid residues, (His1, Cys2, and Cys6),36 where, in addition to the sidechains, the N-terminal amine of His1 and the backbone amidate of Cys2 provide ligands to the metal binding site (Figure 1).2, 3 A remarkable characteristic of the NiSOD active site is the presence of metal-thiolate bonds that remain intact during the catalytic cycle despite the propensity of thiolates to oxidize in the presence of reactive oxygen species (e.g., peroxide37, 38) or even O2.30–32 This remarkable stability, as well as the Ni-centered rather than S-centered oxidation have, in part, been attributed to the presence of mixed amine/amidate N-donors in the active site.10–15 In fact, a structural survey of redox active Ni-containing enzymes, with the exception of methyl coenzyme M reductase, reveals a trend: Ni-enzymes featuring amidate ligands, such as acetyl-coenzyme synthase39 and Ni superoxide dismutase,2, 3 are oxygen tolerant, whereas, redox active Nienzymes that do not have any amidate ligation, such as NiFe-hydrogenase40 and carbon monoxide dehydrogenase,39 are oxygen-sensitive.41</p><p>As suggested by the above observation, the tendency toward S-centered redox reactions in Ni(II) thiolate complexes with an N2S2 ligand donor atom set is modulated by the nature of the N-donors. Various NiN2S2 complexes have been synthesized in which the N-donor ligands were varied from bis-amine to amine/amide to bis-amidate and their redox and air-stability examined.11, 15, 42–44 In the bis-amine complexes (e.g., Ni(bmmp-dmed), the redox chemistry is irreversible and sulfur centered, but the complexes are stable to air oxidation.12 Bis-amide N-donor ligand environments (e.g., Ni(emi))2–) result in reversible Ni-centered redox chemistry; however, the sulfur ligands are susceptible to air-oxidation.12 Model complexes with a mixed amine/amide N-donor ligand environment feature Ni(II) centers that have reversible redox properties and are also stable in air. One example is (Ni(BEAAM))−, which is a planar N2S2 nickel complex with ligand distances similar to those observed for reduced WT-NiSOD. In MeCN, this complex features a diamagnetic nickel center and the S-donors are stable toward oxygen for weeks.12</p><p>DFT studies have suggested an explanation for this trend: The S-orbital character in the "redox-active" MO (RAMO) increases as the N-donors are systematically changed from amide to amine: A bis-amide has the lowest, amine-amide (e.g.,Ni(BEAAM)−) has intermediate, and bis-amine (e.g., Ni(bmmp-dmed)) has the highest S-orbital character in the RAMO.12,15 Thus, in the bis-amine complexes, the irreversible redox properties are associated with increased sulfur character in the RAMO, leading to S-centered rather than Ni-centered oxidation.12 The presence of mixed amine/amide N-donors in NiSOD was proposed to promote metal-centered oxidation without activating the S-donors toward oxidation.10</p><p>Conversion of the Cys2 amidate to a secondary amine was explored in a semi-synthetic variant of NiSOD, and resulted a Ni active site that was minimally structurally perturbed (XAS), could access the Ni(III) state (EPR), but only had only ~1% of WT-NiSOD catalytic activity.45 Here we report that alteration of the His1 N-terminal amine by the addition of Ala to the N-terminus also results in a loss of SOD activity, as well as the loss of a stable Ni(III) species in the resting enzyme, clearly establishing the role of mixed amine/amidate ligation in producing enzyme activity.</p><p>In principle, the addition of Ala to the N-terminus could result in the formation of a Ni center with either amide/amidate or bis-amidate coordination. XAS was used to show that the Ni center in Ala0-NiSOD is in the correct locus to bind to both Cys residues in the Ni site and that the remaining ligands are N/O-donors, consistent with ligation of two backbone N-donors. However, EXAFS data are not suitable to characterize the types of N-donor ligands that are present. To assess whether Ala0-NiSOD features amide/amidate or bis-amidate coordination, DFT geometry optimizations were performed to generate models for each alternative and the resulting models were evaluated on the basis of our spectroscopic data within the framework of DFT and TD-DFT computations. The prominent absorption feature at ~22,300 cm−1 displayed by Ala0-NiSOD under anaerobic conditions is nicely reproduced in the TD-DFT computed absorption spectrum of Ala0red, leading to the conclusion that this species most likely features an amide/amidate N-donor set, rather than a bis-amidate ligand environment.</p><p>In addition to the loss of SOD activity and altered spectroscopic features, Ala0-NiSOD proved to be unstable in air, leading to oxidation of the protein to a mixture of species containing disulfide and S-oxygenated complexes as revealed by ESI-MS. Similar oxidation products to all of these have been observed for a set of peptide models of the WT active site (albeit using KO2 as an oxidant),16 whereas the WT enzyme does not undergo oxidation in air, suggesting a role for the protein in protecting the thiolates from oxidation. There are also precedents in synthetic model chemistry involving oxidation by O2 of planar, low-spin Ni(II) dithiolates leading to disulfide complexes16, 46 and to sulfoxygenates including sulfenate and sulfinate complexes.30, 32 The observation that the sulfoxygenate NiSOD products feature only even numbers of O atoms is consistent with a dioxygenase-like mechanism incorporating both oxygen atoms from a single O2 unit,31 and the observation of a bis-sulfenate suggests addition of O2 across the cis-S donors followed by intramolecular O-O bond cleavage, as has been observed in a model system that features cis-thiolate coordination.47</p><p>Oxidation of the thiolate S-donor ligands to a disulfide ligand or to sulfoxygenates would be expected to decrease the σ-donor ability of the S-donor ligand and favor a high-spin Ni electronic configuration. Indeed, the MCD spectrum obtained from air-oxidized Ala0-NiSOD shows negatively-signed, temperature dependent features at ~16,800 cm−1 and ~28,000 cm− that are remarkably similar to those displayed by the active site Cys → Ser NiSOD single and double variants.25 These Cys variants contain a high-spin (S = 1) Ni(II) ion in a six-coordinate ligand environment, with likely coordination from the Ser oxygen(s) and solvent molecule(s) in addition to ligation from the terminal His1 amine, a nitrogen from the His1 imidazole ring, and the deprotonated backbone amide of Cys2.25</p><p>The EPR spectra of anaerobic Ala0-NiSOD and the mixture of oxidation products also lack the signature rhombic spectrum of low-spin d7 Ni(III). A comparison of the HOMO of Ala0red and of the β-LUMO of the hypothetical Ni(III)-bound Ala0ox model, which is the formally singly occupied orbital, to the corresponding MOs of WTred and WTox provides further insight into the origin of the unique properties of Ala0 NiSOD (Figure 15). Substitution of an amine with a weakly coordinating amide in the reduced state (Ala0red) causes a relatively insignificant decrease in the amount of Ni 3d-orbital character in the HOMO from ~28% to 26%. Similarly, the total S 3p orbital contribution to this orbital remains essentially constant, though the individual contribution from SCys2 that coordinates trans to Neq,His1 increases by 5.9% and that from SCys6 decreases by 5.6% (Figure 15a). A comparison of the β-LUMOs of the oxidized counterparts shows that the amount of Ni 3d-orbital character in this orbital decreases slightly from ~73% in WTox to ~70% in Ala0ox. While the β-LUMO of WTox contains predominantly Ni dz2 orbital character, this MO has contributions from several Ni 3d orbitals in the case of Ala0ox (Figure 15b). Moreover, the sulfur 3p orbital contribution to the β-LUMO increases from ~8% in WTox to ~13% in Ala0ox, and in the latter model this "hole character" (and thus the unpaired spin density in the occupied spin-up counterpart) is localized entirely on SCys2. This increase in SCys2 3p orbital contribution to the β-LUMO likely explains why Ala0-NiSOD is unstable in air despite the similarities in the MO compositions of WTred and Ala0red. This conclusion is supported by a previous spectroscopic and computational study of a one-electron oxidized derivative of a NiN2S2 complex possessing a bis-amidate N-donor set.10</p><p>On the basis of DFT calculations for model systems of NiSOD, it has been proposed that protonation of the metal bound cysteine thiolates48 and the electronic preference for metal-centered redox chemistry10 protects the S-donors from oxidation by the O2•− substrate or the O2/H2O2 products. The results obtained in the present study provide compelling evidence that the mixed amine/amide ligation in NiSOD plays an important role by adjusting the redox properties of the active site to optimize Ni center reduction/oxidation while protecting the S-donors from oxidation by the O2•− substrate or the O2/H2O2 products. The role of the His imidazole ligand in Ala0-NiSOD is less clear. The DFT studies support an unligated, His-off, imidazole, whereas the best EXAFS fits are obtained when it is included as a ligand, though the His-off model also provided a good fit. Five-coordinate Ni(II) complexes can adopt either square pyramidal or trigonal bipyramidal geometries and the nickel ion can be either high-spin (S = 1) or low-spin (S = 0).49 It has been demonstrated that low-spin complexes are typically formed utilizing low electronegative donor ligands such as P, As, and C, and high-spin complexes are formed utilizing high electronegative donor ligands such as O and N, with S-donor ligands found in both low- and high-spin complexes.50 A similar five-coordinate His-on complex was observed as an intermediate in an inner-sphere reaction pathway for NiSOD addressed using DFT to explore the potential energy surface.51 However, in this case the complex was calculated to be high-spin and the dismutation reaction was proposed to involve protonation of one Cys ligand.</p><!><p>From an analysis of our spectroscopic data within the framework of DFT and TD-DFT computations, we propose that the Ala0 insertion to the N-terminus of NiSOD results in a change from the amine/amidate coordination of the Ni(II) ion to an amide/amidate N-donor environment. Substitution of the N-terminal amine in the WT enzyme with an amide in the Ala0-NiSOD variant promotes Cys-thiolate oxidation, highlighting the necessity of the mixed amine/amidate ligand environment in the WT enzyme to promote Ni-based and reversible oxidation. The DFT-computed MO compositions for WT-NiSOD and Ala0red reveal small but significant differences in the composition of the HOMO, and lead us to propose that the reason why Ala0-NiSOD is unstable in air is the altered electronic structure of its one-electron oxidized form, most importantly the composition of the β-LUMO. In the case of Ala0ox, this orbital contains significantly more sulfur 3p character than its counterpart of WTox, which renders the Ni-S bond more readily susceptible to S-based oxidation.</p>