Inorg. Chem. 2006, 45, 10552−10566
The Influence of Amine/Amide versus Bisamide Coordination in Nickel Superoxide Dismutase Kosh P. Neupane and Jason Shearer* Department of Chemistry, UniVersity of NeVada, Reno, NeVada 89557 Received June 25, 2006
Nickel superoxide dismutase (NiSOD) is a mononuclear nickel-containing metalloenzyme that catalyzes the disproportionation of superoxide by cycling between NiII and NiIII oxidation states. In the reduced NiII oxidation state, the metal center is ligated by two cysteinate sulfurs, one amide nitrogen, and one amine nitrogen (from the N-terminus), while in the oxidized NiIII state, an imidazole nitrogen coordinates to the metal center. Herein, we expand on a previous report in which we described a functional metallopeptide-based NiSOD model compound [NiII(SODM1)] (SODM1 ) H2N-HCDLPCGVYDPA-COOH) by exploring how acylation of the N-terminus (producing [NiII(SODM1-Ac)]) influences the properties of the metallopeptide. Titration results, GPC data, and mass-spectrometry data demonstrate that NiII coordinates to SODM1-Ac in a 1:1 ratio, while variable pH studies show that NiII coordination is strong at a pH of 7.5 and above but not observed below a pH of 6.2. This is higher than [NiII(SODM1)] by ∼1.0 pH unit consistent with bisamide ligation. Ni K-edge XAS demonstrates that the NiII center is coordinated in a square-planar NiN2S2 coordination environment with Ni−N distances of 1.846(4) Å and Ni−S distances of 2.174(3) Å. Comparison of the electronic absorption and CD spectrum of [NiII(SODM1)] versus [NiII(SODM1-Ac)] in conjunction with time-dependent DFT calculations suggests a decrease in Ni covalency in the acylated versus unacylated metallopeptide. This decrease in covalency was also supported by DFT calculations and Ni L-edge XAS. [NiII(SODM1Ac)] has a quasireversible NiII/NiIII redox couple of 0.49(1) V vs Ag/AgCl, which represents a −0.2 V shift compared with [NiII(SODM1)], while the peak separation suggests a change in the coordination environment upon oxidation (i.e., axial imidazole ligation). Using the xanthine/xanthine oxidase assay, we determine that [NiII(SODM1-Ac)] is less active than [NiII(SODM1)] by over 2 orders of magnitude (IC50 ) 3(1) × 10-5 vs 2(1) × 10-7 M). Possible reasons for the decrease in activity are discussed.
Introduction Superoxide (O2•-) is a toxic byproduct of aerobic respiration.1-4 In multicellular organisms O2•- can lead to a number of diseases and disorders. For example in humans O2•- has been linked to amyotrophic lateral sclerosis (Lou Gehrig’s disease),5-7 Parkinson’s disease,8,9 myocardial and cerebral ischemia-reperfusion injury,10,11 complications from
diabetes,12 rheumatoid arthritis,13 certain cancers,14,15 and aging in general.16 For single cellular organisms, the rapid and efficient response to O2•- is more imperative since overexposure to O2•- could lead to the rapid death of the
* To whom correspondence should be addressed. E-mail: shearer@ chem.unr.edu. (1) Imlay, J. A. Annu. ReV. Microbiol. 2003, 57, 395-418. (2) Valentine, J. S.; Wertz, D. L.; Lyons, T. J.; Liou, L. L.; Goto, J. J.; Gralla, E. B. Curr. Opin. Chem. Biol. 1998, 2, 253-262. (3) Miller, A.-F.; Sorkin, D. L. Comments Mol. Cell. Biophys. 1997, 9, 1-48. (4) Fridovich, I. J. Biol. Chem. 1989, 264, 7761-7764. (5) Potter, S. Z.; Valentine, J. S. J. Biol. Inorg. Chem. 2003, 8, 373380. (6) Valentine, J. S.; Hart, P. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3617-3622. (7) Valentine, J. S. Free Radical Biol. Med. 2002, 33, 1314-1320.
(8) Mendez-Alvarez, E.; Soto-Otero, R.; Hermida-Ameijeiras, A.; LopezReal, A. M.; Labandeira-Garcia, J. L. Biochim. Biophys. Acta 2002, 1586, 155-168. (9) Jenner, P.; Olanow, C. W. Ann. Neurol. 1998, 44, S72-S84. (10) Collino, M.; Aragno, M.; Mastrocola, R.; Gallicchio, M.; Rosa, A. C.; Dianzani, C.; Danni, O.; Thiemermann, C.; Fantozzi, R. Euro. J. Pharm. 2006, 530, 70-80. (11) Kim, G. W.; Kondo, T.; Noshita, N.; Chan, P. H. Stroke 2002, 33, 809-815. (12) (a) Ferdinandy, P.; Schulz, R. Br. J. Pharmacol. 2003, 138, 532543. (b) Niedowicz, D. M.; Daleke, D. L. Cell Biochem. Biophys. 2005, 43, 289-330. (13) Okamoto, T. Oxid. Stress Dis. 2005, 18, 245-270. (14) Chatterjee, S.; Fisher, A. B. Phytopharm. Cancer ChemopreV. 2005, 171-186. (15) Akman, S. A. Crit. Rev. Oxid. Stress Aging 2003, 2, 925-954. (16) Passi, S.; Ricci, R.; Aleo, E.; Cocchi, M. Prog. Nutr. 2005, 7, 3-22.
10552 Inorganic Chemistry, Vol. 45, No. 26, 2006
10.1021/ic061156o CCC: $33.50
© 2006 American Chemical Society Published on Web 11/30/2006
Amine Ws Amide Coordination in NiSOD
organism.17 To destroy O2•- before it can cause extensive cellular damage aerobic organisms use metalloenzymes capable of facilitating its rapid degradation.2,18,19 The most common O2•- detoxification pathways involve metalloenzymes called superoxide dismutases (SODs).2,19,20 SODs catalyze the disproportionation of O2•- by cycling between reduced and oxidized states
Figure 1. NiSOD structures of the reduced NiII (NiSOD-red) and oxidized NiIII (NiSOD-ox) forms highlighting the six-residue binding loop (H2NHCDLPC).25,26
These metalloenzymes fall into one of four classes based on the transition metal cofactor(s) contained in their active site. The three best known SODs are Cu/Zn-, Mn-, and FeSOD.19-21 The fourth and most recently discovered class of SOD contains a mononuclear nickel at its active site (NiSOD) and is found in several Streptomyces species and cyanobacteria.22-24 NiSOD is a homohexamer in solution and contains one nickel center per monomer.25,26 The nickel center is found at the N-terminus of each monomer, is ligated within a “Nibinding hook” composed of the first six residues from the N-terminus, and cycles between the NiII and NiIII oxidation state during catalysis. X-ray crystallographic studies25,26 have demonstrated that the reduced NiII center is contained in a square planar coordination geometry with ligands derived from two cis-cysteinate sulfurs (C2 and C6), an amide nitrogen from the peptide backbone (derived from C2), and the terminal amine nitrogen (from H1). Upon oxidation to NiIII, the imidazole moiety from H1 coordinates to the apical position affording Ni with a square-pyramidal coordination geometry (see Figure 1). Despite the fact that a number of structural,25-27 biochemical,22,23,27,28 spectroscopic,27,29,30 and computational studies30-32 have been reported concerning (17) Demple, B.; Ding, H.; Jorgensen, M. Methods Enzymol. 2002, 348, 355-364. (18) Kurtz, D. M., Jr. Acc. Chem. Res. 2004, 37, 902-908. (19) Fridovich, I. Annu. ReV. Biochem. 1995, 64, 97-112. (20) Miller, A.-F. Curr. Opin. Chem. Biol. 2004, 8, 162-168. (21) Fridovich, I. Encycl. Biol. Chem. 2004, 4, 135-138. (22) Youn, H.-D.; Kim, E.-J.; Roe, J.-H.; Hah, Y. C.; Kang, S.-O. Biochem. J. 1996, 318, 889-896. (23) Youn, H.-D.; Youn, H.; Lee, J.-W.; Yim, Y.-I.; Lee, J. K.; Hah, Y. C.; Kang, S.-O. Arch. Biochem. Biophys. 1996, 334, 341-348. (24) Palenik, B.; Brahamsha, B.; Larimer, F. W.; Land, M.; Hauser, L.; Chain, P.; Lamerdin, J.; Regala, W.; Allen, E. E.; McCarren, J.; Paulsen, I.; Dufresne, A.; Partensky, F.; Webb, E. A.; Waterbury, J. Nature (London) 2003, 424, 1037-1042. (25) Wuerges, J.; Lee, J.-W.; Yim, Y.-I.; Yim, H.-S.; Kang, S.-O.; Carugo, K. D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 8569-8574. (26) Barondeau, D. P.; Kassmann, C. J.; Bruns, C. K.; Tainer, J. A.; Getzoff, E. D. Biochemistry 2004, 43, 8038-8047. (27) Choudhury, S. B.; Lee, J.-W.; Davidson, G.; Yim, Y.-I.; Bose, K.; Sharma, M. L.; Kang, S.-O.; Cabelli, D. E.; Maroney, M. J. Biochemistry 1999, 38, 3744-3752. (28) Bryngelson, P. A.; Arobo, S. E.; Pinkham, J. L.; Cabelli, D. E.; Maroney, M. J. J. Am. Chem. Soc. 2004, 126, 460-461. (29) Szilagyi, R. K.; Bryngelson, P. A.; Maroney, M. J.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 30183019. (30) Fiedler, A. T.; Bryngelson, P. A.; Maroney, M. J.; Brunold, T. C. J. Am. Chem. Soc. 2005, 127, 5449-5462. (31) Pelmenschikov, V.; Siegbahn, P. E. M. J. Am. Chem. Soc. 2006, 128, 7466-7475.
NiSOD, there are a number of questions concerning both the mechanism of O2•- disproportionation and how the unusual structure of NiSOD contributes to its reactivity. One feature of the NiSOD active site that is very unusual is the nitrogenous ligands that coordinate to Ni in the equatorial positions. The metal-Namide coordination motif is only found in a handful of metalloenzymes,33,34 while the utilization of an N-terminal amine as a ligand is even less well represented in biology. To our knowledge, the Nterminal amine ligand is found in one other metalloenzyme, CooA,35 where the amine from an N-terminal proline residue is bound in the axial position of an Fe-porphyrin. In a recent spectroscopic and computational study, Brunold and coworkers have suggested that anionic amide coordination in NiSOD lowers the degree of sulfur character in the redoxactive orbitals, thus protecting the cysteinate sulfurs from oxidative damage.30 Such a finding might seem to suggest that a bisamide NiN2S2 motif would enhance the protection of the cysteinate-sulfurs from oxidative damage36 compared to the amine/amide motif since the oxidant would be more likely to extract a Ni-centered electron over a S-centered electron because of decreased S-character in the redox active orbital. Grapperhaus has recently readdressed this notion using DFT calculations probing the influence of amide coordination in NiN2S2 complexes.32 In that study it was suggested that amine/amide coordination may provide the S-based ligands protection from oxidative damage by lowering the inherent reactivity of the Ni/S-based HOMO toward dioxygen compared with the bisamide coordination motif.37,38 Thus the bisamide motif should afford a less, not more, stable NiN2S2 Ni center than the amide/amine motif. These DFT results are in line with synthetic studies examining NiN2S2 reactivity toward dioxygen, which show that bisamine-ligated complexes are more stable toward dioxygen than bisamideligated complexes. (32) Mullins, C. S.; Grapperhaus, C. A.; Kozlowski, P. M. J. Biol. Inorg. Chem. 2006, 11, 617-625. (33) Kovacs, J. A. Chem. ReV. 2005, 104, 825-848. (34) Doukov, T. I.; Iverson, T. M.; Seravalli, J.; Ragsdale, S. W.; Drennan, C. L. Science 2002, 298, 567-572. (35) Lanzilotta, W. N.; Schuller, D. J.; Thorsteinsson, M. V.; Kerby, R. L.; Roberts, G. P.; Poulos, T. L. Nat. Struc. Biol. 2000, 7, 876-880. (36) Grapperhaus, C. A.; Darensbourg, M. Y. Acc. Chem. Res. 1998, 31, 451-459. (37) Grapperhaus, C. A.; Mullins, C. S.; Kozlowski, P. M.; Mashuta, M. S. Inorg. Chem. 2004, 43, 2859-2866. (38) Hatlevik, O.; Blanksma, M. C.; Mathrubootham, V.; Arif, A. M.; Hegg, E. L. J. Biol. Inorg. Chem. 2004, 9, 238-246.
Inorganic Chemistry, Vol. 45, No. 26, 2006
10553
Neupane and Shearer This study probes how amine/amide versus bisamide coordination to Ni in functional NiSOD model compounds influences their SOD activities. Recently, we communicated the preparation and spectroscopic and reactivity properties of a metallopeptide-based NiSOD mimic, [NiII(SODM1)].39 SODM1 is a peptide composed of the first 12-residues of NiSOD (H2N-HCDLPCGVYDPA-COOH) from Streptomyces coelicolor, and will coordinate 1 equiv of NiII in a manner nearly identical to that of NiSOD. [NiII(SODM1)] possess spectroscopic properties similar to NiSOD and will catalytically disproportionate O2•-. Herein, we examine how the physical and reactive properties of the metallopeptide are altered upon acylation of the N-terminus (producing [NiII(SODM1-Ac)]; SODM1-Ac ) AcN-HCDLPCGVYDPACOOH). It will be demonstrated that this modification lowers the degree of sulfur character in the d-manifold, optimizes the NiII/III redox potential for SOD catalysis, and yet, gives a poorer SOD. Experimental Section General. All manipulations were performed under an atmosphere of dinitrogen using glovebox and Schlenk line techniques unless otherwise noted. Diethyl ether was purified by distilling it from LiAlH4 under dinitrogen, while water was purified by a Barnstead E-pure water purification system to a resistance of at least 17.8 MΩ. All other reagents were of the highest quality available and were used as received. Chemicals were purchased from Acros Chemical Company, except Fmoc-protected amino acids and coupling reagents (Advanced ChemTech), xanthine and xanthine oxidase (Sigma), and nitro blue tetrazolium (MP Biochemical). SODM1-Ac Synthesis and Purification. SODM1-Ac (AcNHCDLPCGVYDPA-COOH) was prepared by both manual (in solid-phase peptide reaction vessels) and automated synthesis (on a Protein Technologies PS3 automated peptide synthesizer). We used standard Fmoc/tBu-based protection strategies on Wang resin with HBTU/HOBt/DIEPA coupling methods.40 After the deprotection of the final Fmoc group, the free amine was capped with a solution containing 5% Ac2O, 89% DMF, and 6% diisopropylethylamine. The peptide was then cleaved from the resin using a mixture of 95% TFA, 2.5% ethanedithiol, and 2.5% triisopropyl silane. Once cleaved, the crude cleavage solution was filtered, and the filtrate evaporated under vacuum to a glassy film. The film was washed several times with freshly distilled diethyl ether, and the resulting tan crude peptides were subsequently purified by reverse-phase HPLC on a Waters DeltPrep 60 equipped with Atlantis C-18 reverse-phase column (5 µm; 50 × 100 mm). The fractions containing the desired product were then lyophilized yielding white powders. The purity of each peptide was assessed by analytical reverse-phase HPLC (Atlantis C-18 reverse phase column 5 µm; 4.6 × 100 mm) and mass spectrometry on a Waters Micromass 20 ESI mass spectrometer (positive ion mode). The following gradients, retention times, and parent ion peaks were obtained for AcN-HCDLPCGVYDPA-COOH (SODM1-Ac): gradient 10-65% MeCN (0.1% TFA) in H2O (0.1% TFA) over 45 min (preparative 40 mL/min ) 11.3 min; analytical 1 mL/min Rt )15.3 min.). Yield ) 38%. (M)+ Calcd: 1331.4928. Found: 1331.5443. Preparation of [NiII(SODM1-Ac)]. Metalated [NiII(SODM1-Ac)] was prepared by the addition of 1 equiv of NiCl2 to 1 equiv of (39) Shearer, J.; Long, L. M. Inorg. Chem. 2006, 45, 2358-2360. (40) HOBt, 1-hydroxybenotriazole; HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexaflurophosphate N-oxide; DIPEA, N,N-diisopropylethylamine.
10554 Inorganic Chemistry, Vol. 45, No. 26, 2006
in 50 mM N-ethylmorpholine (NEM) buffer (pH ) 7.5), which produced a brownish-red solution. Gel permeation chromatography was performed on a Waters DeltPrep 60 equipped with a Protein-Pak GPC column (7.8 × 300 mm, 60 Å pore size) using an aqueous solution of NaHCO3 (sat.) under a positive pressure of He. Calibrations were made using a Waters poly(ethylene glycol) standard kit. Electronic absorption spectrum λmax/nm (/M-1 cm-1)): 694 (110), 535 (512), 418 (1320), 304 (8760). (M)+ Calcd: 1389.4282. Found: 1386.9413. (M)2+ Calcd: 694.7141. Found: 692.8316. Electronic Absorption and CD Spectroscopic Studies. All electronic absorption measurements were carried out on either a Varian Cary 50 or an OLIS-CARY 14 using quartz cuvettes with 1 cm path lengths. Experiments were performed in 50 mM NEM solutions with the pH adjusted by the addition of dilute NaOH and HCl. Peptide concentrations were quantified by the procedure of Ellman.41 NiII was added to solution as NiCl2 in neutral water (pH ) 7.0). The concentration of the NiCl2 solution was high relative to that of the peptide so that addition of NiII to the peptide did not significantly alter the pH of the peptide solution. SOD activity assays were performed at pH 7.5 using the modified xanthine/ xanthine oxidase assay of Tabbi.42 In this assay the inhibition of nitro blue tetrazolium (NBT) reduction by the SOD mimic is monitored. CD spectroscopic measurements were performed on an OLIS DSM 17 CD spectropolarimeter. In the visible region, cuvettes with a 1 cm path length were used, while in the UV region, we used peptide concentration of 1 mg mL-1 and 1 mm path length cuvettes. In the UV region, CD spectra were recorded in bufferless solutions with the pH carefully adjusted with dilute NaOH and HCl. All spectroscopic measurements were performed at pH 7.5 unless otherwise noted. The simultaneous deconvolutions of the CD and electronic absorption spectra were performed using an in-housewritten procedure for Igor Pro 5.0 (Wavemetrics, Lake Oswego, OR). Electrochemistry. Electrochemical measurements were performed on a Princeton Applied Research PARSTAT 2273 potentiostat in a Faraday cage to minimize noise in the cyclic voltammograms. A standard three-electrode cell (glassy carbon working electrode, Pt disk auxiliary electrode, Ag/AgCl (aq) reference electrode) was used. The cell solutions were sparged with Ar prior to measurement and consisted of 1.0 mM [Ni(SODM1-Ac)] and 100 mM NaCl buffered to a pH of 7.5 (50 mM N-ethylmorpholine). Ni K-Edge X-ray Absorption Spectroscopy. A 1.8 mM solution of [Ni(SODM1-Ac)] (prepared by adding 0.8 equiv of NiCl2 to 1 equiv of SODM1-Ac at a pH 7.5, 50 mM NEM) was injected into an aluminum sample holder between windows made from Kapton tape (3M, Minneapolis, MN, catalog no. 1205) and quickly frozen in liquid nitrogen. Data were collected at the National Synchrotron Light Source, Brookhaven National Laboratories (Upton, NY) on a Beamline X9b. A focused Si(111) double monochromator was used for energy selection and a low-angle (4.5 mrad) polished Ni mirror for harmonic rejection. The sample was maintained at 20 K throughout data collection with a He Displex cryostat. All spectra are reported as fluorescence data and recorded on a Canberra 13element solid-state Ge fluorescence detector. Total count rates were maintained under 30 kHz per channel. For the edge spectrum, the primary hutch aperture was set to 0.4 mm to obtain the maximum spectral resolution, and data were collected in 10 eV steps in the pre-edge region (8131-8310 eV), 0.3 eV steps in the edge region (8310-8350 eV), and 2.0 eV steps in the near-edge region. EXAFS
SODM1-Ac
(41) Ellman, G. L. Arch. Biochem. Biophys. 1958, 74, 443-450. (42) Tabbi, G.; Driessen, W. L.; Reedijk, J.; Bonomo, R. P.; Veldman, N.; Spek, A. L. Inorg. Chem. 1997, 36, 1168-1175.
Amine Ws Amide Coordination in NiSOD data collection used a primary hutch aperture height of 0.8 mm, and data were collected in 10 eV steps in the pre-edge region (8131-8310 eV), 0.5 eV steps in the edge region (8310-8350 eV), 2.0 eV steps in the near-edge region (8352-8630 eV), and 5.0 eV steps in the far-edge region (8631 eV to 15.5 keV). The spectra represent the average of four data sets for the edge spectrum and eight data sets for the EXAFS spectrum. A blank recorded prior to data collection revealed that there was no observable signal above the baseline noise from the mirror, sample holder windows, or frozen solutions. Data analysis were performed using the XAS refinement and graphing package EXAFS123 provided by Prof. R. C. Scarrow (Haverford College, Haverford, PA).43 We present refinements based on unfiltered k3-weighted data performed in a manner analogous to that previously described,39 except we note that glitches had to be removed from the XAS prior to analysis. Although the EXAFS data were collected to 15.5 keV, we analyzed the data out to only 14.3 keV because of noise at higher energies. The goodness of fit was judged using 2 ) [nidp/(nidp - np)] × average[(ydata - ymodel)/σ]
(2)
where nidp is the number of independent data points, np is the number of refined parameters, σ is the estimated uncertainty in the data, ydata is the experimental k3 EXAFS spectrum, and ymodel is the simulated k3 EXAFS spectrum. Ni L-Edge X-ray Absorption Spectroscopy. Ni L-edge X-ray absorption spectra were collected at the National Synchrotron Light Source, Brookhaven National Laboratories (Upton, NY) on Beamline U4b.44 Thin metallopeptide films were prepared by applying 10 µL of a freshly prepared solution of either [Ni(SODM1)] or [Ni(SODM1-Ac)] to a 7.0 × 5.0 mm silicon wafer (SPI Supplies, West Chester, PA). The solutions were then allowed to slowly evaporate in an evaporation chamber under Ar. Silicon wafers were then applied to the sample paddle using silver paste. Spectra were recorded in total electron-yield mode at ambient temperature under a vacuum of less than 5 × 10-9 mbar. The incident flux was measured from the total electron yield of a gold-coated grid placed between the monochromator and focusing mirror. Data were recorded in 0.4 eV steps prior to the L3-edge (830-850 eV), 0.1 eV steps between the L3 and L2 edges (850-876 eV), and 0.4 eV steps beyond the L2-edge (876-980 eV). Each spectrum reported represents the average of at least 10 data sets. Individual data sets were calibrated against a simultaneously recorded spectrum of NiO (absorption maximum at 853.2 eV) and were inspected to ensure that sample decomposition was not occurring. The background was removed from each spectrum by first fitting the region before the L3-edge and after the L2-edge to a polynomial function. We then subtracted this contribution to the background from the intrinsic background resulting from the promotion of the 2p electrons to the vacuum using the function
[ [
Ibckg ) I3 + I2
( (
(3)
)] )]
I3 ) H3
E - P3 1 1 + arctan 2 π Γ/2
(4)
I2 ) H2
E - P2 1 1 + arctan 2 π Γ/2
(5)
where Γ is the width of the step, E is energy, H is the height of the step, and P2 and P3 are the inflection points for the L2- and L3(43) Scarrow, R. C. EXAFS123; Haverford College: Haverford, PA, 2005. (44) Chen, C. T.; Sette, F. ReV. Sci. Instrum. 1989, 60, 1616-1621.
edges, respectively. We chose the peak maxima as inflection points. The calculated spectra were generated using the TT-Multiplets suite of programs.45-47 The atomic Hamiltonian for NiII was extended to include perturbations resulting from both crystal-field48 and charge-transfer effects.46,49 Transition to two different configurations were examined: Ni2p3d8 f Ni2p3d9 and Ni2p3d9L f Ni2p3d10L, where 2p represents a hole in a Ni 2p orbital and L represents a hole in a previously filled ligand-based orbital. For the simulated spectra we utilized a D4h symmetry and refined the spectra by varying the parameters 10Dq, Ds, Dt, the energy difference between the two ground and excited-state configurations, and the strengths of the transfer integrals to the four sets of d-orbitals under D4h symmetry. Spectral simulations were then produced by applying a Lorentzian line shape, followed by a Gaussian line shape, to each of the calculated transitions to approximate broadening caused by lifetime and instrumental effects. Electronic Structure Calculations. Geometry optimizations (GOs) were performed using the Amsterdam Density Functional (ADF) software package version 2005.01.50 For the minimized NiSOD active-site ((NiSODmin)1-), all non-hydrogen atoms were first positioned according to the crystal structure of reduced NiSOD, followed by the addition of the appropriate number of hydrogen atoms. The structure was first optimized with the positions of all non-hydrogen atoms restrained to obtain a starting structure. To simulate the constraints that may exist in the peptide, we then performed a second GO with only with only two geometry restraints: the Ni-S-C bond angle (from Ni-Cys6, 110.8°) and C-S-Ni-S dihedral angle (Cys6-Ni-Cys2, 30.6°). This second GO used the conductorlike screening model to simulate solvation effects ( ) 78.4, r ) 1.3 Å).51,52 The second geometry-optimized structure is the one that is discussed (see Supporting Information for coordinates of the optimized structure). For all calculations, the frozen-core approximation was used for the 1s orbital of all second row elements and the 1s, 2s, and 2p orbitals for Ni and S. The valance orbitals were treated with the ADF TZDP basis set (triple-ξ basis set with double polarization). Calculations were performed using the local density approximation of Vosko, Wilk, and Nussair53 and the nonlocal gradient corrections of Beck54-56 and Perdew.57,58 Atomic orbital contributions to the corresponding molecular orbitals are reported as Mulliken populations. Calculations using acylated (NiSODmin-Ac)2- were performed in an identical manner except, in addition to hydrogen atoms, an N-terminal acetyl group was also added to the structure, and in the first GO, all non-hydrogen atoms except the acetyl group were restrained (see Supporting Information for coordinates of the optimized structure). Time-dependent DFT (TD-DFT) calculations were then performed on the GO-minimized (45) Cowan, T. The Theory of Atomic Structure and Spectra; University of California Press: Berkeley, CA, 1981. (46) deGroot, F. M. F. Chem. ReV. 2001, 101, 1779-1808. (47) deGroot, F. M. F. J. Electron Spectrosc. Relat. Phenom. 1994, 67, 529-622. (48) Butler, P. H. Point Group Symmetry: Applications, Methods, and Tables; Plenum Press: New York, 1981. (49) Okada, K.; Kotani, A.; Thole, B. T. J. Electron Spectrosc. Relat. Phenom. 1992, 58, 325-343. (50) Te Velde, G.; Bickelhaupt, F. M.; Baerends, E. J.; Guerra, C. F.; Van Gisbergen, S. J. A.; Snijders, J. G.; Ziegler, T. J. Comput. Chem. 2001, 22, 931-967. (51) Klamt, A. J. Chem. Phys. 1995, 99, 2224-2235. (52) Klamt, A.; Jones, V. J. Chem. Phys. 1996, 105, 9972-9981. (53) Vosko, S. J.; Wilk, M.; Nussair, M. Can. J. Phys. 1980, 58, 12001211. (54) Becke, A. J. Chem. Phys. 1986, 84, 4524-4529. (55) Becke, A. J. Chem. Phys. 1988, 88, 1053-1062. (56) Becke, A. Phys. ReV. A 1988, 38, 3098-3100. (57) Perdew, J. P. Phys. ReV. B 1986, 34, 7406. (58) Perdew, J. P. Phys. ReV. B 1986, 33, 8822-8824.
Inorganic Chemistry, Vol. 45, No. 26, 2006
10555
Neupane and Shearer structures using the TD-DFT implementation found in the ADF 2005.01 software package using the iterative Davidson method for matrix diagonalization (as opposed to direct diagonalization of the matrix to extract excitation energies and oscillator strengths).59 For the TD-DFT calculations, we used identical computational methods, as noted above, except the exchange and correlation functional of Van Leeuwen and Baerends was used,60 and we did not apply a solvation model.
Results and Discussion Peptide Synthesis and NiII Binding. The peptide SODM1Ac (AcN-HCDLPCGVYDPA-COOH) was prepared by standard solid-phase synthetic techniques and obtained in moderate yields following purification and lyophilization. The anaerobic addition of 1 equiv of NiCl2 to slightly basic (pH 7.5) solutions of SODM1-Ac produces reddish-brown solutions, which are in contrast to the light pinkish-beige solutions of [NiII(SODM1)]. Similar to [NiII(SODM1)], solutions of metalated SODM1-Ac do not change color when exposed to air, indicating that NiII is not being oxidized to NiIII by dioxygen or immediately decomposing. The NiII oxidation state is also indicated by the fact that the addition of the reductant sodium dithionite to aerobic solutions of metalated SODM1-Ac produces no change in the corresponding electronic absorption spectrum of the metallopeptide. We next sought to determine the stoichiometry of NiII binding to SODM1-Ac and the influence that pH has on nickel coordination. Upon addition of NiCl2 to SODM1-Ac, a ligandfield band centered at 535 nm is produced in the electronic absorption spectrum (535 ) 512 M-1 cm-1, Vide infra). Spectrophotometric titrations were therefore used to determine the NiII/SODM1-Ac Kd. It was observed that the addition of ∼1.3 equiv of NiCl2 affords complete coordination of NiII to the peptide suggesting a 1:1 SODM1-Ac/NiII stoichiometry. From the plots of [SODM1-Ac] versus [NiCl2] added to solution, an approximate Kd ) 34(1) µM (Figure 2) for [NiII(SODM1-Ac)] was obtained. This indicates weaker NiII coordination to SODM1-Ac than SODM1, which has a NiII Kd value of
