Metallopeptide based mimics with substituted histidines approximate a key hydrogen bonding network in the metalloenzyme Nickel superoxide dismutase

Article abstract of DOI:10.1021/ic9010407

Nickel Superoxide dismutase (NiSOD) is a recently discovered superoxide dismutase that utilizes the Ni^III/Ni^II couple to facilitate the disproportionate of O₂^.- into H ₂O₂ and O₂

Full text of DOI:10.1021/ic9010407

1
0560 Inorg. Chem. 2009, 48, 10560–10571
DOI: 10.1021/ic9010407
Metallopeptide Based Mimics with Substituted Histidines Approximate a Key
Hydrogen Bonding Network in the Metalloenzyme Nickel Superoxide Dismutase
Jason Shearer,* Kosh P. Neupane, and Paige E. Callan
Department of Chemistry, University of Nevada, Reno, Nevada 89557
Received June 1, 2009
III II
Nickel superoxide dismutase (NiSOD) is a recently discovered superoxide dismutase that utilizes the Ni /Ni couple to
•
-
facilitate the disproportionation of O into H O and O . A key structural component of NiSOD is an elongated axial
2
2
2
2
III
˚
His-imidazole Ni bond (2.3-2.6 A) that is the result of a H-bonding network between His(1), Glu(17), and Arg(47).
Herein we utilize metallopeptide based mimics of NiSOD with His(1) ε-nitrogen substituted imidazoles to approximate
the electronic influence of this H-bonding network ({Ni (SOD -Im-X)} X = Me, H, DNP, and Tos;
SOD -Im-X = H CDLPCGVYDPA where H is an N-substituted His). All reduced {Ni (SOD -Im-X)} are
similar to one another as assessed by electronic absorption spectroscopy, circular dichroism (CD) spectroscopy, and
Ni K-edge x-ray absorption (XAS). This indicates that the change in His(1) is having little influence on the square-
planar Ni N S center. In contrast, changes to the axial His(1) ligand impart differential spectroscopic properties on the
2
oxidized {Ni (SOD -Im-X)} metallopeptides. Resonance Raman spectroscopy (405 nm excitation) in conjunc-
tion with a normal coordinate analysis indicates that as the axial His imidazole is made less Lewis basic there is an
III/II
M1
M1
0
0
II
M1
II
2
III
M1
III
increase in Ni -S bond strength in the equatorial plane, with force constants for the Ni-S bond trans to the amine
ranging from 1.54 to 1.70 mdyn A . The rhombic electron paramagnetic resonance (EPR) spectra of the four oxidized
-
1
˚
III
metallopeptides are all consistent with low-spin Ni contained in a square pyramidal coordination environment, but
1
show changes in the hyperfine coupling to N along g . This is attributable to a reorientation of the g vector in the more
4
z
z
III
imidazole
III
amine
(
along the Ni -N
bond) versus less (along the S-Ni -N
bond) Lewis basic imidazole bases. This
reorientation of g along the xy plane translates into a decrease in A by ∼20 MHz. A decrease in Lewis-basicity of the
z
zz
axial imidazole also translates into a 2 orders of magnitude increase in SOD catalysis across the metallopeptide series,
6
-1 -1
8
with k ranging from 6(1) ꢀ 10 M
s
for the metallopeptide with the least basic imidazole. This likely results from a fine-tuning of the electron
for the metallopeptide with the most Lewis basic imidazole to 6(2) ꢀ 10
cat
1 -1
-
M
transfer properties of the Ni-center, which optimize it for SOD catalysis.
s
5
6,7
Introduction
and fall into one of four classes (Cu/ZnSOD, FeSOD,
6,7
8
•
-
MnSOD, and NiSOD ) based on the metal cofactor(s)
found at their active-site. Nickel containing superoxide dis-
mutase (NiSOD) is the most recently discovered SOD, and
Superoxide (O2 ) is a toxic byproduct of anaerobic
respiration, and its cellular degradation is typically catalyzed
by redox-active metalloenzymes called superoxide dismu-
tases (SODs).
by catalyzing its disproportionation into H O and O :
1-4
•-
was first found in several Streptomyces species and cy-
SODs facilitate the degradation of O2
9-12
anobacteria.
A more recent bioinformatics study has
2
2
2
SOD þ O2• - þ 2Hþ f SOD þ H
red
ox
O
2
ð1Þ
ð2Þ
(5) Fridovich, I. Encycl. Biol. Chem. 2004, 4, 135–138.
6) Grove, L. E.; Brunold, T. C. Comments Inorg. Chem. 2008, 29, 134–
68.
2
(
1
4
(
7) Miller, A.-F. Acc. Chem. Res. 2008, 41, 501–510.
8) Bryngelson, P. A.; Maroney, M. J. Met. Ions Life Sci. 2007, 2, 417–
SOD þ O2• - f SOD þ O
ox
red
(
2
43.
9) Youn, H.-D.; Kim, E.-J.; Roe, J.-H.; Hah, Y. C.; Kang, S.-O.
Biochem. J. 1996, 318, 889–896.
10) Lee, J.-W.; Roe, J.-H.; Kang, S.-O. Methods Enzymol. 2002, 349, 90–
01.
11) 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.
12) Palenik, B.; Brahamsha, B.; Larimer, F. W.; Land, M.; Hauser, L.;
(
(
*
To whom correspondence should be addressed. E-mail: shearer@
1
unr.edu.
(
(
(
1) Bai, K. V. K.; Azeez, S. Adv. Plant Physiol. 2006, 9, 87–100.
2) Cabelli, D. E.; Riley, D.; Rodriguez, J. A.; Valentine, J. S.; Zhu, H.
(
Biomimetic Oxid. Catal. Transition Met. Complexes 2000, 461–508.
(
(
3) Fridovich, I. Annu. Rev. Biochem. 1995, 64, 97–112.
Chain, P.; Lamerdin, J.; Regala, W.; Allen, E. E.; McCarren, J.; Paulsen, I.;
Dufresne, A.; Partensky, F.; Webb, E. A.; Waterbury, J. Nature 2003, 424,
1037–1042.
4) Stallings, W. C.; Metzger, A. L.; Pattridge, K. A.; Fee, J. A.; Ludwig,
M. L. Free Radical Res. Commun. 1991, 12-13, 259–68.
pubs.acs.org/IC
Published on Web 10/15/2009
r 2009 American Chemical Society

Article
Inorganic Chemistry, Vol. 48, No. 22, 2009 10561
Scheme 1
bond length will have an influence on the physical and
reactivity properties of the metalloenzyme when compared
to similar Ni-centers where this Ni-N bond would be at a
typical length.
We and others have successfully modeled many of the
structural, spectroscopic, and functional properties of
NiSOD using small metallopeptide based biomimetic com-
pounds containing the first 7 to 12 residues from the
18,23-26
N-terminal sequence of S. coelicolor NiSOD.
In one
of these studies we found that the inclusion of an amino acid
residue that has the potential to coordinate Ni in the axial
position greatly enhances the SOD activity of the resulting
demonstrated that the gene encoding for NiSOD is found in
several aquatic bacteria and invertebrates.
18
metallopeptide. If the N-terminal histidine was replaced
13
with alanine, the activity of the resulting metallopeptide
dropped by over an order of magnitude. In contrast, the
replacement of the N-terminal histidine with glutamic acid
afforded a metallopeptide with only a modest reduction in
SOD activity. This is in line with mutation studies performed
by Maroney and co-workers who have shown that mutation
of His(1) to a residue with a non-coordinating side-chain all
NiSOD exists as a homohexamer in solution with each
monomer containing one mononuclear Ni-ion. The nickel
center is ligated within a loop composed of residues 1-6 from
1
4-16
the N-terminus (the “nickel binding hook”).
In the
II
reduced Ni oxidation state the nickel center is found in a
square planar N2S2 coordination environment with two
cis-cysteinate ligands from Cys(2) and Cys(6), the N-terminal
27
but eliminated SOD activity in the metalloenzyme.
amine from His(1), and the amidate nitrogen from Cys(2)
Incorporation of intricate outer-sphere interactions found
in metalloproteins is difficult to accomplish using metallo-
peptide based mimics. Furthermore, changing these interac-
tions in a systematic and predictable manner in metall-
opeptide based mimics is nearly impossible. Therefore, to
III
(
Scheme 1). Upon oxidation to Ni , the imidazole δ-nitro-
gen from His(1) will coordinate to the nickel ion in an axial
position forming a square-pyramidal geometry about nickel
(Scheme 1).
X-ray crystallographic analysis of oxidized NiSOD shows
III
more finely probe the role that the axial ligand to Ni has in
that the axial Ni-N bond is substantially elongated com-
controlling the activity of the metalloenzyme NiSOD, we
III
pared to “typical” Ni -N bonds with neutral N-donors; in
M1
have prepared a series of metallopeptides ({Ni(SOD
-
˚
NiSOD this bond is greater than 2.3 A long, compared
M1
0
Im-X)}; SOD -Im-X=H N-H CDLPCGVYDPA-COOH
and H is an N-substituted imidazole) that have variable
III
17-21
2
˚
to ∼2.0 A long for a typical Ni -N bond.
Brunold
0
and co-workers have suggested that this elongated Ni-N
bond is due to a hydrogen bonding network between the ε
N-H group of His(1) and Glu(17) and Arg(47) on an adjacent
N-substitution of the His(1) imidazole ε-nitrogen (Chart 1).
The resulting N-substituted imidazoles will impart changes to
the electron donating ability of the imidazole δ-nitrogen,
1
7
protein monomer. Density functional theory (DFT) and
hybrid-DFT calculations of computational models of the
NiSOD active site show that when the hydrogen bonding
network is omitted from the model a normal Ni-imidazole
III
making it a more or less Lewis-basic donor to the Ni center.
Although changing the electronics of the imidazole donor
cannot account for all of the fine-tuning provided by the
protein environment, it will be shown that this one subtle
change is capable of reproducing a number of the key aspects
of the electronic and reactivity properties of the metalloen-
zyme. In fact, we will demonstrate that weakening the axial
1
7,18,22
˚
bond length of ∼2.0 A is obtained.
In contrast, inclu-
sion of the outersphere hydrogen bonding interaction to the ε
17
˚
N-H group elongates the Ni-imidazole bond to 2.17 A. This
strongly suggests that the hydrogen bonding network is at
least partially responsible for the increase in Ni-imidazole
bond length; the H-bonding network is structurally con-
straining the imidazole in a position relatively far-removed
from the Ni-center. Thus, from the standpoint of the Ni-
center, it is being presented with a less Lewis-basic donor. It
III
N-Ni bond through these subtle electronic changes to the
axial imidazole ligand enhances SOD activity and makes the
resulting metallopeptide-based mimic more NiSOD-like.
Possible reasons for these findings will be discussed.
Experimental Section
II
III
seems highly likely that this alteration of the axial Ni -N
M1
M1
Preparation of {Ni SOD H(1)X}. SOD -Im-X (H
2
N-
0
0
Me
H CDLPCGVYDPA-COOH; H = H, H , H , H
Tos
DNP
) were
prepared by manual solid phase peptide synthesis on Wang resin
(
13) Dupont, C. L.; Neupane, K.; Shearer, J.; Palenik, B. Environ.
t
using Fmoc/ Bu protecting groups in a manner similar to what
Microbiol. 2008, 10, 1831–1843.
(
(
14) Kurtz, D. M., Jr. Chemtracts 2004, 17, 212–217.
24
M1
has been previously described, except for SOD -Im-DNP,
which was synthesized using HMPB-BHA resin. Fmoc-pro-
15) Barondeau, D. P.; Kassmann, C. J.; Bruns, C. K.; Tainer, J. A.;
Getzoff, E. D. Biochemistry 2004, 43, 8038–8047.
16) 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.
17) Fiedler, A. T.; Bryngelson, P. A.; Maroney, M. J.; Brunold, T. C. J.
Am. Chem. Soc. 2005, 127, 5449–5462.
18) Neupane, K. P.; Gearty, K.; Francis, A.; Shearer, J. J. Am. Chem.
Soc. 2007, 129, 14605–14618.
ε
Me
tected N-methyl histidine (H ) was obtained from Bachem
(
ε
Tos
(Torrance, CA), while N-tosyl histidine (H ) and N-2,
ε
DNP
(
4
-dinitrophenyl histidine (H
) were purchased from
(
(23) Neupane, K. P.; Shearer, J. Inorg. Chem. 2006, 45, 10552–10566.
(24) Shearer, J.; Long, L. M. Inorg. Chem. 2006, 45, 2358–2360.
(25) Schmidt, M.; Zahn, S.; Carella, M.; Ohlenschlaeger, O.; Goerlach,
(
19) Kruger, H. J.; Holm, R. H. J. Am. Chem. Soc. 1990, 112, 2955–1963.
(
20) Machida, R.; Kimura, E.; Kushi, Y. Inorg. Chem. 1986, 25, 3461–
3
466.
21) Orpen, A. G.; Brammer, L.; Allen, F. H.; Kennard, O.; Watson, D.
G.; Taylor, R. J. Chem. Soc., Dalton Trans. 1989, S1–S83.
22) Pelmenschikov, V.; Siegbahn, P. E. M. J. Am. Chem. Soc. 2006, 128,
466–7475.
M.; Kothe, E.; Weston, J. ChemBioChem 2008, 9, 2135–2146.
(26) Tietze, D.; Breitzke, H.; Imhof, D.; Kothe, E.; Weston, J.; Bunt-
kowsky, G. Chem.;Eur. J 2009, 15, 517–523.
(27) Bryngelson, P. A.; Arobo, S. E.; Pinkham, J. L.; Cabelli, D. E.;
Maroney, M. J. J. Am. Chem. Soc. 2004, 126, 460–461.
(
(
7

1
0562 Inorganic Chemistry, Vol. 48, No. 22, 2009
Shearer et al.
Chart 1
Advanced ChemTech (Louisville, KY). The peptides were
cleaved from the resin using 95% TFA/2.5% ethanedithiol/
substitution of 0.51 equiv of I
2
followed by dialysis of the
solution against 50 mM NEM buffer (pH 7.4) to remove all
Mn compounds from solution. All GPC data for oxidized
2
.5% triisopropyl silane (95% TFA/5% triisopropyl silane for
M1
III
M1
SOD -Im-DNP), and the cleavage solution was evaporated
under vacuum to dryness. The glassy crude product was washed
with peroxide-free diethyl ether, and the peptides were purified
by reverse phase HPLC on a Waters X-terra C-18 column (semi-
preparative: 5 μm 50 ꢀ 100 mm; analytical: 5 μm 4.6 ꢀ 100 mm)
{Ni (SOD -Im-Me)} were collected using a Waters Pro-
TM
˚
tein-Pak GPC column (7.8 ꢀ 300 mm; 60 A pore size) using a
M1
3
saturated NaHCO mobile phase (pH 8.3). GPC traces were
monitored at 350 nm where the free SOD -Im-Me peptide
-
1
)
does not absorb. Electronic absorption spectrum λmax (cm
M1
-1
-1
and lyophilized yielding white powders (yellow for SOD H-
(ε (M cm )): 28,575 (3,260);25,575 (sh);18,210 (740). Electron
paramagnetic resonance (EPR) (77 K 1:1 NEM buffer/glycerol):
DNP
(
1)H
).
M1
SOD -Im-Me. R
MeCN/Water 0.1% TFA over 45 min), R = 3.4 min (semi-
t
= 15.6 min (analytical: 10-65%
g = 2.33; g = 2.24; g = 2.00 (Azz = 84 MHz). ESI-MS
x y z
+
(positive ion mode) M m/z: calcd 1359.1; found 1359.2.
Stopped-Flow Kinetic Studies. A HI-TECH SF-61 stopped
flow kinetics instrument equipped with double mixing syringes
t
preparative: 10-29% MeCN/Water 0.1% TFA over 15 min).
ESI-MS (positive ion mode) M m/z: calcd 1303.4; found
+
•
-
1
303.3. Yield: 132 mg (56%).
M1
was utilized for the measurement of the catalytic O
2
dispro-
M1
SOD -Im-Tos. R
t
= 22.4 min (analytical: 10-65%
portionation reactions facilitated by {Ni(SOD -Im-X)}.
One of the 2.5 mL syringes was filled with buffer (50 mM
NEM pH 8.0). The second 2.5 mL syringe was filled with a
MeCN/Water 0.1% TFA over 45 min), R = 3.8 min (semi-
preparative: 10-29% MeCN/Water 0.1% TFA over 15 min).
ESI-MS (positive ion mode) M m/z: calcd 1443.4; found
t
+
II/III
M1
solution of {Ni
tions ranging from 1.0-60 μM). A third 0.5 mL (or 2.5 mL)
syringe was filled with dry DMSO, 18-crown-6, and KO (final
(SOD -Im-X)} (final mixing concentra-
1
443.8. Yield: 35 mg (14%).
M1
SOD -Im-DNP. R = 19.3 min (analytical: 10-65%
t
2
concentrations ranging between 2.5-50 mM). Thus, mixing of
the three solutions results in a 10:1 ratio (or 2:1) of buffer/
DMSO, which substantially minimizes any optical disturbances
from the mixing of the water and DMSO at the beginning of the
MeCN/Water 0.1% TFA over 45 min), R = 18.0 min (semi-
preparative: 10-30% MeOH/Water 0.1% TFA over 25 min).
ESI-MS (positive ion mode) M m/z: calcd 1455.4; found
t
+
1
455.3. Yield: 60 mg (21%).
Under a dinitrogen atmosphere in a glovebag (or a 3:97 H2/
2
9,30
kinetics run
treatment because of such a disturbance). The disappearance of
KO was subsequently monitored by measuring the change in
(the first 5 ms were disregarded in the kinetic
N
2
mixed atmosphere in a COY anaerobic chamber) solutions
of the peptides were then prepared in 50 mM NEM buffer
pH = 7.4). The peptide concentrations of these solutions were
2
-1
absorbance at 245 nm as a function of time (ε ∼ 2250 M
(
-
1 29
cm ). All kinetics studies were performed at 24.5(5) °C. Prior
to all kinetic experiments, several shots of buffer and 18-crown-
2
8
determined by the methods of Ellman. To these peptide
solutions 1 equiv of NiCl dissolved in water (pH = 7.4) was
added affording the reduced Ni metallopeptides.
2
II
M1
II
6
DMSO) were recorded and averaged together to use as the
/DMSO (or buffer, {Ni (SOD -Im-X)}, and 18-crown-6/
II
M1
{
Ni (SOD -Im-Me)}. Electronic absorption spectrum
•
-
-1 -1 -1
baseline. As a test reaction the catalytic O2 disproportionation
afforded by Cu/Zn SOD (from bovine erythrocytes; Sigma Cat.
No. S2515) was recorded indicating that we can observe the tail-
λmax (cm ) (ε (M cm )): 27,400 (940); 21,690 (360); 18,050
+
160, sh). ESI-MS (positive ion mode) M m/z: calcd 1359.1;
(
found 1358.3.
•
-
II
M1
end of catalytic O
2
9
disproportion reactions with a rate con-
.
{
Ni (SOD -Im-Tos)}. Electronic absorption spectrum
-1 -1 31
-1 -1 -1
stant of ∼2 ꢀ 10 M
s
All data workup and the sub-
λ
max (cm ) (ε (M cm )): 21,730 (405, sh); 18,110 (180, sh).
+
ESI-MS (positive ion mode) M m/z: calcd 1499.1; found
sequent rate constants were then extracted from the data using
in-house designed procedures for the software package Igor Pro
6.02 (Wavemetrics; Lake Oswego, OR).
1
498.9.
II
M1
{
Ni (SOD -Im-DNP)}. Electronic absorption spectrum
-1 -1 -1
Physical Methods. Electronic absorption spectra were ob-
tained on a CARY 50 UV-vis-NIR spectrophotometer in
quartz cells with 1 cm pathlengths. Circular dichroism (CD)
spectra were also recorded in quartz cells with 1 cm pathlengths,
and were obtained on an OLIS-DCM 17 spectropolarimeter
using photomultiplier detectors in the visible region (300-
λmax (cm ) (ε (M cm )): 38,127 (25,700), 29,130 (5,590),
2
M
1,560 (410, sh), 18,110 (150, sh). ESI-MS (positive ion mode)
+
m/z: calcd 1511.1; found 1510.8.
M1
III
Preparation of {Ni (SOD -Im-Me)}. In a COY anaero-
II
M1
bic chamber a ∼1-3 mM solution of {Ni (SOD -Im-Me)}
was prepared in 50 mM NEM buffer (pH = 7.4) and placed on
700 nm) and InGaAs detectors in the NIR region (600-1000 nm).
2
an ice bath. To this 0.51 equiv of I in ethanol was added slowly
dropwise. Over the course of the addition, the solution changed
from a light pink/beige to dark orange/brown. This solution was
(
29) Riley, D. P.; Rivers, W. J.; Weiss, R. H. Anal. Biochem. 1991, 196,
III
M1
344–349.
then used as prepared. Alternatively, {Ni (SOD -Im-Me)}
could be prepared from the addition of 1.0 equiv of KMnO in
(30) Weiss, R. H.; Flickinger, A. G.; Rivers, W. J.; Hardy, M. M.; Aston,
4
K. W.; Ryan, U. S.; Riley, D. P. J. Biol. Chem. 1993, 268, 23049–23054.
(31) Goldstein, S.; Fridovich, I.; Czapski, G. Free Radical Biol. Med.
(
28) Ellman, G. L. Arch. Biochem. Biophys. 1958, 74, 443–450.
2006, 41, 937–941.

Article
Inorganic Chemistry, Vol. 48, No. 22, 2009 10563
3
All CD data sets were averaged together in the overlapping
spectral regions.
Electrochemical data were obtained on a Princeton Applied
Research PARSTAT 2273 potentiostat using a standard three-
electrode cell (pyrolytic graphite edge working electrode, Pt disk
auxiliary electrode, SCE reference electrode). Concentrations of
refinements are based on Fourier Filtered k (χ) data over the
-
1
˚
energy range of k = 2.0-12.5 A and back-transformed from
0
˚
r = 1.0-2.5 A.
Electronic Structure, Excited State, and EPR Calculations. All
electronic structure calculations were performed using Neese’s
3
6
software package ORCA version 2.6.35. Geometry optimized
(GO) structures were generated from minimized NiSOD active-
sites on oxidized forms, and used convergence criteria (in au) of
root-mean-square (rms) and maximum forces of 0.0003 and
0.0001, respectively, and rms and maximum gradients of 0.002
and 0.001, respectively. The group attached to the N(ε) of the
II
M1
{
Ni (SOD -Im-X)} in 50 mM NEM buffer were ∼0.7-1.0
mM, and NaCl was used as the supporting electrolyte (100 mM).
The solutions were sparged with Ar prior to recording the data.
Square wave voltammograms were recorded with a pulse height
of 15 mV at a frequency of 5 Hz. The oxidation and reduction
waves were then extracted from the background capacitance by
applying a cubic spline function before and after the oxidation/
reduction peaks. Peak positions were then determined by taking
the first derivatives of these data.
III
Me
His(1) imidazole ring was varied between Me (Ni (SOD )), H
)), and -SO H
III
(Ni (SOD )), 2,4 dinitrophenyl (Ni (SOD
H
III
DNP
2
III Tos
(Ni (SOD )). GO calculation on all species utilized the local
3
density approximation of Vosko, Wilk, and Nusair, the
7
3
8-40
41,42
EPR spectra were recorded using ∼0.05 mM solutions of
gradient correction of Becke
VWN), the TZVP
aug-TZVPP
and Perdew
for all non-ligating atoms, and the
basis set for Ni, S, and all ligating N atoms.
(BP86/
III
M1
43-45
{
of I in EtOH) as 1:1 mixtures of buffer/glycerol (buffer =
Ni (SOD -Im-X)} (generated by the addition of 0.5 equiv
4
3-47
2
5
0 mM NEM pH 7.4) in quartz EPR tubes. The spectra were
Frequency calculations on all GO structures were performed
using the Becke’s three parameter hybrid functional for ex-
change with the Lee-Yang-Parr functional for correlation
recorded at 77 K in a quartz EPR finger dewar on a Varian E4
EPR spectrometer interfaced to a PC through a Scientific Soft-
ware Services EWWIN system. Data were averaged over 40
scans. The following experimental parameters were used: micro-
wave frequency = 9.07-9.12 GHz; microwave power =
4
8,49
(the B3LYP hybrid functional),
two sided displacements,
4
3-45
43-47
the TZVP for all non-ligating atoms, the aug-TZVPP
basis set for Ni, S, and all ligating N atoms, a 434 point Lebedev
-
7
angular mesh, and convergence criteria of 10 E in energy. In
2
1
.5-5.0 mW; modulation amplitude = 10 G; time constant =
0 ms; conversion time = 10 ms; gain = 2 ꢀ 10 .
h
4
addition, vibrational frequencies were also obtained using
5
0
III
Me
III
Gaussian 03 for Ni SOD , Ni SOD
DNP
III
, Ni SOD
Tos
Resonance Raman data were obtained using a modified Jobin
to
provide input-files for Lehnert’s program for performing the
Yvon U1000 high resolution double monochromator employ-
ing 1800 groves/mm gratings and a PMT detector. The gratings
were driven by a microstepper motor (50 nm/step resolution)
attached to the cosecant bar, and the output from the PMT was
recorded using a DATAQ DI-158 data acquisition module. The
microstepper motor and data acquisition module were con-
trolled by a PC using in-house written software in C++. The
monochromator was calibrated using the Hg emission lines
from a low-pressure Hg(Ar) lamp. Excitations were provided
with a 405 nm solid state diode laser (bandwidth ∼5 nm)
provided by Opto Engine LLC with ∼30 mW of laser power
at the sample. The light from the laser was passed through a
monochromator containing a 2400 grooves/mm holographic
grating (for selecting λex = 405.0 nm) and then focused onto the
sample. Data were collected at room temperature in quartz
fluorescence cuvettes or in NMR tubes in a quartz EPR finger
dewar at 77 K using a 135° backscattering geometry. Samples
were recorded in 1:1 mixtures of buffer/glycerol with concentra-
3
2,51-53
QCA-NCA.
As these calculations were to provide for
an initial set of force constants as input for the QCA-NCA
3
8-42
programs, we utilized the BP86/VWN functional
TZVP basis set on all atoms.
and
4
3-45
EPR g-values and superhyperfine coupling constants were
calculated by solving the coupled-perturbed SCF (CP-SCF)
5
4,55
equations.
tional,
These calculations utilized the B3LYP func-
basis-set for all non-ligating atoms,
basis set for Ni and S, and Kutzelnigg’s
4
8,49
43-45
the TZVP
4
3-47
the aug-TZVPP
NMR/EPR basis set (IGLO-III) for all ligating N atoms. All
5
6
orbitals from -100 to 100 E of the HOMO/LUMO gap were
h
considered for the EPR calculations, with the center of electro-
nic charge defined as the origin of the g-matrix.
(36) Neese, F. In ORCA, An Ab Initio, Density Functional, and Semi-
empirical Program Package, 2.6.35 ed.; Universitat Bonn: Bonn, Germany,
2008.
III
tions ranging between 1-5 mM. All Ni species were generated
by adding concentrated I dissolved in EtOH to the reduced Ni
2
II
(37) Vosko, S. H.; Wilk, L.; Nusair, M. Can. J. Phys. 1980, 58, 1200–1211.
(
38) Becke, A. D. J. Chem. Phys. 1986, 84, 4524–4529.
metallopeptides. All spectra were independently calibrated by
using an internal standard of acetaminophen. Force constants
were obtained initially from DFT calculations (vide infra) and
then subsequently refined using the Quantum Chemistry As-
sisted Normal Coordinate Analysis (QCA-NCA) procedure of
(39) Becke, A. D. Phys. Rev. A: Gen. Phys. 1988, 38, 3098–3100.
(40) Becke, A. D. J. Chem. Phys. 1988, 88, 1053–1062.
(
41) Perdew, J. P. Phys. Rev. B: Condens. Matter Mater. Phys. 1986, 33,
822–8824.
42) Perdew, J. P. Phys. Rev. B: Condens. Matter Mater. Phys. 1986, 34,
406.
(43) Schaefer, A.; Horn, H.; Ahlrichs, R. J. Chem. Phys. 1992, 97, 2571–
8
7
(
3
2
Lehnert and Tuczek.
Ni K-edge X-ray absorption data were collected on beamline
X3b at the National Synchrotron Light Source (Brookhaven
National Laboratories; Upton, NY) as previously described.
2577.
(
44) Ahlrichs, R.; coworkers unpublished.
2
4
(45) Neese, F. unpublished results.
(
46) Balabanov, N. B.; Peterson, K. A. J. Chem. Phys. 2005, 123, 64107.
III/II
M1
Samples of ∼0.7-3 mM of {Ni
(SOD -Im-X)} (50 mM
(47) Woon, D. E.; Dunning, T. H., Jr. J. Chem. Phys. 1993, 98, 1358–
NEM buffer, pH 7.4) were injected between windows made from
Kapton tape in aluminum sample holders, and quickly frozen in
liquid nitrogen. All spectra represent the average of 5 to 10
data sets. Data were analyzed using the software packages
1371.
(48) Becke, A. D. J. Chem. Phys. 1993, 98, 5648–5652.
49) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B: Condens. Matter Mater.
Phys. 1988, 37, 785–789.
(
(
(
50) Gaussian 03, Revision C.02; Gaussian, Inc.: Wallingford, CT, 2004.
51) Praneeth, V. K. K.; Naether, C.; Peters, G.; Lehnert, N. Inorg. Chem.
33
34,35
EXAFS123 and FEFF 8.2
as previously described. All
2
006, 45, 2795–2811.
52) Allouche, A.; Pourcin, J. Spectrochim. Acta, Part A 1993, 49A, 571–
580.
(53) Peterson, M. R.; McIntosh, D. F. In QCPE 576.
(
(
(
32) Lehnert, N.; Tuczek, F. Inorg. Chem. 1999, 38, 1659–1670.
33) Scarrow, R. C. In EXAFS123; Haverford College: Haverford, PA,
2
005.
34) Ankudinov, A. L.; Ravel, B.; Rehr, J. J.; Conradson, S. D. Phys. Rev.
B: Condens. Matter Mater. Phys. 1998, 58, 7565–7576.
35) Ankudinov, A. L.; Bouldin, C. E.; Rehr, J. J.; Sims, J.; Hung, H.
Phys. Rev. B: Condens. Matter Mater. Phys 2002, 65, 104107/1–104107/11.
(54) Neese, F. J. Chem. Phys. 2001, 115, 11080–11096.
(
(55) Neese, F. Curr. Opin. Chem. Biol. 2003, 7, 125–135.
(56) Kutzelnigg, W.;Fleischer, U.;Schindler, M.The IGLO-Method: Ab-initio
Calculation and Interpretation of NMR Chemical Shifts and Magnetic Suscept-
ibilities; Springer Verlag: Berlin/Heidelberg, Germany, 1991; Vol. 213.
(

1
0564 Inorganic Chemistry, Vol. 48, No. 22, 2009
Shearer et al.
Results and Discussion
We have previously reported on the synthesis and pro-
perties of the NiSOD metallopeptide-based mimic
II
M1
M1
{
Ni (SOD -Im-H)} (SOD : H N-HCDLPCGVYD-
2
24 M1
PA-COOH). Briefly, the peptide SOD was prepared by
standard Fmoc based solid-phase peptide synthesis.
Upon the addition of 1 equiv of NiCl to a slightly basic
2
(
pH 7.4; 50 mM in N-ethylmorpholine (NEM) or phos-
M1
phate buffer) solution of SOD
the metallopeptide
Ni (SOD -Im-H)} is produced, as signified by a pink-
ish-beige solution. The resulting spectroscopic and structural
II
M1
{
II
M1
properties of {Ni (SOD -Im-H)} are reminiscent of
reduced NiSOD, and the resulting metallopeptide displays
high SOD activity (vide infra). Considering the utility of
using this and similar mimics to understand some of the
II
properties of NiSOD, it was decided to modify {Ni -
M1
(
SOD -Im-H)} to probe how subtle alterations to the
III
electronics of the imidazole axial ligand to Ni will influence
the reactivity and properties of the NiSOD mimic.
Preparation and Spectroscopic Properties of the Re-
II
M1
Figure 1. Electronic absorption spectra of {Ni (SOD -Im-H)}
II
M1
II
M1
(
blue trace), {Ni (SOD -Im-Me)} (green trace), {Ni (SOD
-
II
M1
Im-Tos)} (red trace), and {Ni (SOD -Im-DNP)} (orange trace).
Inset depicts the expanded spectrum of {Ni (SOD -Im-DNP)}.
II
M1
II
M1
duced Metallopeptide Based Mimics {Ni (SOD
-
M1
Im-X)} (X = Me, Tos, DNP). The other SOD -Im-X
(
prepared by standard solid-phase peptide synthesis. Both
X = Me, DNP, Tos) peptides used in this study were
M1
M1
SOD -Im-Tos and SOD -Im-DNP were prepared
in low overall yields (less than 25%). In both cases a
M1
significant quantity of SOD was recovered because of
the unavoidable cleavage of the tosyl or 2,4-dinitrophenyl
groups from the His(1) imidazole. To avoid the excessive
cleavage of the 2,4-dinitrophenyl group from the N-
M1
DNP
terminal His residue of SOD (H(1)H
1
) we removed
,2-dithiolethane from the cleavage cocktail and replaced
it with an equal volume of triisopropyl silane. Failure to
perform this substitution resulted in extremely low yields
) (less than 1%) because of the
M1
of SOD (H(1)H
DNP
thiol-assisted cleavage of the dinitrophenyl protecting
group.
II
M1
The metallopeptides {Ni (SOD -Im-X)} were pre-
pared in a manner analogous to that previously described
II
M1
24
for {Ni (SOD -Im-H)}. Solutions of the metallo-
peptides were prepared in aqueous 50 mM NEM
buffer (pH 7.4), and 1 equiv of NiCl (in water) was
2
added to solution. Upon the addition of NiCl , both
2
II
M1
II
M1
{
yielded light beige-pink solutions, while {Ni (SOD
Ni (SOD -Im-Me)} and {Ni (SOD -Im-Tos)}
II M1
-
Figure 2. Electronic absorption (bottom) and CD spectrum (top) of
Im-DNP)} remained mostly yellow because of the in-
tense 2,4-dinitrophenyl chromophore.
II
M1
{
Ni (SOD -Im-Me)}.
II
M1
The
electronic
absorption
spectrum
of
spectrum of {Ni (SOD -Im-DNP)} is obscured by
the intense 2,4-dinitrophenyl chromophore (Figure 1,
Table 1).
II
M1
{
Ni (SOD -Im-Me)} is reminiscent of that reported
red
for reduced NiSOD (NiSOD ; Figures 1 and 2; Table 1);
it contains weak transitions in the ligand-field region of
the spectrum at 27,400, 21,690, and 18,050 cm
The Ni K-edge X-ray absorption spectra of the three
Ni metallopeptides are consistent with a square planar
-
1
II
red
-1 17
II
coordination geometry about Ni . The XANES spectra
(
NiSOD : ∼28,000, 22,000, and 18,000 cm ). The
red
II
M1
CD spectrum of {Ni (SOD -Im-Me)} also resembles
that of NiSOD , with positively signed bands at 17,670
(Figure 3, Table 2) all show a prominent pre-edge peak
corresponding to Ni(1s) f Ni(4p ) transitions at ∼8338
57
z
-
1
and 30,300 cm and a negatively signed band at 22,940
II
eV, which is a characteristic feature of square planar Ni
centers. The EXAFS region (Figure 3, Table 2) of the Ni
-
1
II
M1
cm (Figure 2). {Ni (SOD -Im-Tos)} possesses an
electronic absorption spectrum that is also reminiscent of
K-edge spectra are best modeled with Ni contained in an
N S coordination environment with the average Ni-S
red
NiSOD , except that in the high-energy regions the tosyl
2
2
group dominates the spectrum (Figure 1, Table 1). In
contrast to the other metallopeptides investigated in this
study, the ligand-field region of the electronic absorption
(
57) Colpas, G. J.; Maroney, M. J.; Bagyinka, C.; Kumar, M.; Willis, W.
S.; Suib, S. L.; Mascharak, P. K.; Baidya, N. Inorg. Chem. 1991, 30, 920–928.

Article
Inorganic Chemistry, Vol. 48, No. 22, 2009 10565
II
M1
III
M1
Table 1. Electronic Absorption, Circular Dichroism, Electrochemical Data for {Ni (SOD -Im-X)} (X = Me, DNP, and Tos) and {Ni (SOD -Im-Me)}
UV/vis
CD
max (cm 1) (ε (M-1 cm
-
-1
)
λ (cm-1) (Δε (M-1 cm-1
)
+
E (V vs Ag/Ag )
λ
II
M1
{
Ni (SOD -Im-Me)}
18,050 (160, sh)
17,670 (1.60)
22,940 (-2.04)
30,300 (2.31)
18,797 (-2.32)
21,740 (6.00)
26,880 (70.7)
282(4)
2
2
1,690 (360)
7,400 (940)
II
M1
{
Ni (SOD -Im-DNP)}
18,110 (150, sh)
470(10)
598(5)
2
3
1,560 (410, sh)
8,127 (25,700)
29,940 (63.4)
17,185 (1.90)
II
M1
{
Ni (SOD -Im-Tos)}
18,110 (180, sh)
1,730 (405, sh)
2
22,420 (-2.95)
3
0,490 (5.33)
III
M1
a
nd
{
Ni (SOD -Im-Me)}
18,210 (740)
13,950 (4.19)
16,445 (-1.73)
20,660 (-6.59)
2
2
5,575 (sh)
8,575 (3,260)
2
6,738 (-24.5)
a
nd. = not determined.
II
M1
II
M1
II
M1
Figure 3. Ni K-edge X-ray absorption datafor {Ni (SOD -Im-Me)} (A), {Ni (SOD -Im-Tos)} (B), and {Ni (SOD -Im-DNP)} (C). The left
II
) transitionsindicative of square-planarNi , the middle contains the FT
portionof the figure contains the edge spectra, whichdisplayprominent Ni(1sf 4p
z
3
k EXAFS data, and the left displays the FF k EXAFS data. Experimental data are shown in red, simulations to the data are shown in dashed blue, and the
3
II
M1
II
M1
difference spectrum is shown in green. {Ni (SOD -Im-Me)} was obtained at a concentration of ∼3 mM, while {Ni (SOD -Im-Tos)} and
II
M1
{
Ni (SOD -Im-DNP)} were recorded at submillimolar concentrations because of solubility issues.
˚
bond length being between 2.17-2.18 A and the average
was obtained as a thin-film on a microelectrode surface.
Although thin-film electrochemistry has proven useful
in the studies of many redox active materials, it can
sometimes be problematic for gaining insight into solution
electrochemical properties because differences in the
microenvironment provided by the film versus solution
˚
Ni-N bond length being ∼1.92 A. These metric para-
red 58
meters compare well with EXAFS data reported for
NiSOD
,
and are nearly identical to what was pre-
II
M1
viously reported by us for {Ni (SOD -Im-H)} and
1
3,18,24
other NiSOD metallopeptide based mimics. It
therefore appears that all four metallopeptides reported
can dramatically influence the redox potential and elec-
trode kinetics of the electroactive material.
II
in this study display nearly identical Ni structures, and
59-62
This
that imidazole substitution is having a minimal influence
on the structure of the reduced Ni metallopeptides. This
should be especially problematic when applied to small
metallopeptides where the redox center is largely solvent
exposed. We therefore decided to investigate the electro-
chemical behavior of all of these metallopeptides by
solution electrochemistry as opposed to thin film electro-
chemistry.
II
is to be expected as the His(1) imidazole is not directly
involved in coordination to the reduced Ni -center.
II
M1
Solution Electrochemical Properties of {Ni(SOD
-
Im-X)}. In a previous report we obtained a redox
III
potential for the Ni /Ni couple of {Ni(SOD
II
M1
-
+
24
Im-H)} at 0.70(2) V versus Ag/Ag . This potential
(
(
59) Hu, Y.; Hu, N. J. Phys. Chem. B 2008, 112, 9523–9531.
60) Wang, L.; Waldeck, D. H. J. Phys. Chem. C 2008, 112, 1351–1356.
(
58) 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
999, 38, 3744–3752.
(61) Sumner, J. J.; Weber, K. S.; Hockett, L. A.; Creager, S. E. J. Phys.
Chem. B 2000, 104, 7449–7454.
(62) Sumner, J. J.; Creager, S. E. J. Phys. Chem. B 2001, 105, 8739–8745.
1

1
0566 Inorganic Chemistry, Vol. 48, No. 22, 2009
Shearer et al.
II
M1
III
M1
Table 2. Ni K-Edge X-ray Absorption Refinement Parameters for {Ni (SOD -Im-X)} (X = Me, DNP, and Tos) and {Ni (SOD -Im-Me)}
II
M1
II
M1
II
M1
III
M1
{
Ni (SOD -Im-Me)}
{Ni (SOD -Im-Tos)}
{Ni (SOD -Im-DNP)}
{Ni (SOD -Im-Me)}
edge energy (eV)
pre-edge peak 1 (eV)
8340.2(4)
8332.7(2)
0.03
8337.9(2)
0.42
8340.3(2)
8332.4(2)
0.026
8338.0(1)
0.43
8340.2(1)
8332.5(2)
0.028
8338.1(1)
0.46
8342.5(1)
8334.1(1)
0.079
a
intensity
pre-edge peak 2 (eV)
intensity
a
E
o
(eV)
8343.5
8343.2
8343.2
8344.1
N Shell
n
r (A)
σ (A )
2
2
2
3
˚
1.91(1)
0.0026(3)
1.87(2)
0.0029(6)
1.89(1)
0.0028(5)
1.894(3)
0.0037(2)
2
2
˚
S Shell
n
r (A)
σ (A )
2
2
2
2
˚
2.182(1)
0.0022(8)
2.174(2)
0.0034(4)
2.179(2)
0.0027(3)
2.174(1)
0.0029(3)
2
2
˚
b
GOF
0.31
0.68
0.54
0.33
a
b
1/2
Intensity relative to the edge with an uncertainty of ∼ ( 5%. GOF = R[n{idp}/(n{idp} - n{p})] ; R = ave[(data-simulation)/esd(data); n(p)=
number of varied paramters; n(idp) = 2(rmax - rmin)(kmax - kmin)/π.
III
II
III
species. As an initial test to determine if the Ni oxida-
All four metallopeptides yield quasireversible Ni /Ni
redox couples in solution (Supporting Information). So-
tion state could be generated and stabilized we added a
concentrated solution of KMnO to buffered solutions of
II
M1
lutions of {Ni (SOD -Im-H)} (1.0 mM in: 50 mM
NEM; 100 mM NaCl; pH 7.4) display a Ni /Ni redox
4
III
II
II
M1
{Ni (SOD -Im-Me)} (pH 7.4; 50 mM NEM). This
resulted in an intense red/brown colored solution and an
increase in the intensity and number of transitions ob-
served in visible region of the UV/vis spectrum (Figure 4).
+
potential at E = 434(3) mV versus Ag/Ag . This is more
in line with the solution electrochemical measurements
obtained for NiSOD metallopeptide based mimics in our
1
8
II
M1
laboratory.
which possesses a more electron donating imidazole
In contrast, {Ni (SOD -Im-Me)},
After the addition of 1 equiv of KMnO to solutions of
4
II
M1
{Ni (SOD -Im-Me)} no further change to the elec-
tronic absorption spectrum was noted outside of the
III
donor, stabilizes the Ni oxidation state to a greater
extent with a redox couple of E = 282(4) mV versus Ag/
Ag . As expected, the less electron donating ligands
changes due solely to excess KMnO being present in
4
solution. We should note that although MnO4 is a
+
-
provide for more positive redox potentials with E =
4
multielectron oxidation reagent we require 1 equiv
of permanganate to effect the complete oxidation of
the metallopeptide. This is despite the fact that there
is sufficient electrochemical driving force to effect fur-
ther metallopeptide oxidations by some of the reduced
Mn-based species that would be present in solution.
We are currently probing the mechanism of elec-
+ II
70(10) and 598(5) mV versus Ag/Ag for {Ni -
M1
II
M1
(
SOD -Im-DNP)} and {Ni (SOD -Im-Tos)}, re-
spectively. We note that all four metallopeptides display
relatively large peak to peak separations in their voltam-
mograms (ΔE ∼ 250 mV). This is likely due to the
imidazole ligands binding to and dissociating from the
III
II
II
M1
Ni center during the Ni /Ni redox process at these
tron transfer to {Ni (SOD -Im-Me)} to sort this
issue out.
-
1
18
relatively slow (ν = 100 mV s ) scan velocities. Very
recently Maroney and co-workers reported that the Ni /
II
Addition of 0.5 equivalents of ascorbate to the fully
oxidized nickel-containing metallopeptide resulted in
III
Ni couple for NiSOD is 290 mV versus NHE (∼91 mV
+
63
II
M1
II
M1
vs Ag/Ag ). {Ni (SOD -Im-Me)} therefore has
the complete reformation of {Ni (SOD -Im-Me)}
(Figure 4, inset). This process could be cycled through
at least six times without the observable decomposition
III
II
the closest redox potential to the Ni /Ni couple of
NiSOD.
III
M1
III/II
M1
Preparation of {Ni (SOD -Im-Me)} via Chemical
of {Ni
no metallopeptide decomposition following repeated
(SOD -Im-Me)}. Surprisingly, we observe
II
M1
Oxidation. One reason that {Ni (SOD -Im-Me)} was
initially prepared was that we reasoned the additional
Lewis-basicity of the N-methyl imidazole would aid in the
KMnO /ascorbate additions by both ESI-MS and GPC
4
(Supporting Information). As a milder oxidizing agent I2
dissolved in EtOH was also investigated for the formation
III
stabilization of the Ni oxidation state. This was indeed
confirmed by our CV measurements, which showed a
considerably more negative redox potential for
{Ni (SOD -Im-Me)} than the other three metallo-
peptides investigated (vide supra). Thus, attempts were made
III
M1
of the {Ni (SOD -Im-Me)}, which yielded similar
results as KMnO . Similarly, no metallopeptide damage
is noted from ESI-MS, GPC, and HPLC data following
4
II
M1
III
M1
{Ni (SOD -Im-Me)} generation using I as an oxi-
2
III
M1
III
dant. Attempts were made to isolate the pure Ni forms
to generate and isolate t3he stable {Ni (SOD -Im-Me)}
of the other metallopeptides probed in this study, but
these all proved to be too unstable to handle above liquid
nitrogen temperatures for even brief periods of time (vide
infra).
(
63) Herbst, R. W.; Guce, A.; Bryngelson, P. A.; Higgins, K. A.; Ryan, K.
C.; Cabelli, D. E.; Garman, S. C.; Maroney, M. J. Biochemistry 2009, 48,
354–3369.
3

Article
Inorganic Chemistry, Vol. 48, No. 22, 2009 10567
III
M1
Figure 5. EPR spectra (77 K) of {Ni (SOD -Im-Me)} (A),
III
M1
III
M1
{
{
Ni (SOD -Im-H)} (B), {Ni (SOD -Im-DNP)} (C), and
III M1
Ni (SOD -Im-Tos)} (D).
environment. This symmetry change combined with an
increase in the number of final states available will
II
M1
Figure 4. Oxidation of {Ni (SOD -Im-Me)} promoted by the addi-
II
M1
6
4,65
tionof1 equiv ofKMnO topH7.4 solutions of{Ni (SOD -Im-Me)}
in0.1equiv aliquots. The green traceisthe beginning pointofthe titration,
and the red trace is the end point. The inset depicts the tail-end of the
ascorbate effected reduction of oxidized {Ni (SOD -Im-Me)} by
the addition of ascorbate in aliquots of 0.05 reducing equiv at pH 7.4. The
green trace is the “beginning point” of the titration, and the red trace is the
end point.
4
increase the overall Ni 1s f 3d peak intensity.
The EXAFS region of the Ni K-edge X-ray absorption
III
M1
III
M1
spectrum for {Ni (SOD -Im-Me)} is consistent with
Ni contained in a five coordinate N S ligand environ-
ments (Figure 6; Table 2). We can best model the EXAFS
3
2
˚
region with two Ni-S scatterers at 2.19 A and three Ni-N
scatterers at 1.89 A. These data are nearly identical to
˚
III
III
M1
The Ni oxidation state of {Ni (SOD -Im-Me)}
was confirmed by X-band EPR spectroscopy. Figure 5
displays the 77 K EPR spectrum of I generated
2
what was previously reported by Maroney and co-work-
ers for NiSOD , and are also consistent with the change
ox
in oxidation state and coordination number. The negli-
gible change of the average Ni-S would be expected
because of the increase in charge about the Ni-center
being offset by the increase in coordination number about
Ni from 4 to 5.
III
M1
{
Ni (SOD -Im-Me)}, which is consistent with a
III
low-spin S = 1/2 Ni species. The EPR spectrum is
rhombic with g values at g = 2.33, 2.24, and 2.00 (oxidi-
zed NiSOD; NiSOD has g = 2.30, 2.24, 2.01; Table 3).
ox
Furthermore, there is strong superhyperfine coupling in
g (A = 84 MHz), indicative of coupling of the unpaired
The vis-NIR electronic absorption and CD spectra of
III
M1
z
zz
I generated {Ni (SOD -Im-Me)} are displayed in
1
4
electron with an N nucleus. This coupling is also
2
Figure 7 (Table 1). The electronic absorption spectrum is
similar to what has been previously reported for
NiSOD ; it is a relatively broad featureless spectrum
ox
observed in NiSOD , but the superhyperfine coupling
ox
constant is considerably smaller in NiSOD (A = 69.7
ox 17
zz
9
,17,58
MHz), indicating that there is a stronger interaction
between the unpaired spin on the Ni center and a
nitrogen nucleus in {Ni (SOD -Im-Me)} versus Ni-
SOD .
The XANES portion of the {Ni (SOD -Im-Me)}
Ni K-edge X-ray absorption spectrum also shows
changes relative to that recorded for {Ni (SOD -Im-
Me)} that are consistent with a change from the Ni
to Ni oxidation state (Figure 6; Table 2). The energy
of the edge jump shifts by 2.8(6) eV higher in energy
-1
from 35,000-12,000 cm punctuated with a peak at
III
-1
-1
28,410 cm and shoulders at 25,645 and 18,115 cm .
In contrast to the electronic absorption spectrum of
III
M1
ox
III
M1
{
from that observed for NiSOD . The CD spectrum of
Ni (SOD -Im-Me)}, its CD spectrum is distinct
III
M1
ox
III
M1
-1
{
is characterized by a positively signed low-energy transi-
Ni (SOD -Im-Me)} between 35,000-12,000 cm
II
M1
II
-1
tion at 13,775 cm and a series of negatively signed
III
57
-1
higher energy transitions between 16,500-27,000 cm
This can be contrasted with the CD spectrum of
.
III
M1
II
in {Ni (SOD -Im-Me)} compared with {Ni -
ox
NiSOD , which displays a series of positively and nega-
tively signed features over the energy region of
M1
(
SOD -Im-Me)}. Furthermore, the Ni 1s f 4p tran-
z
II M1
sition displayed in {Ni (SOD -Im-Me)} now be-
comes blurred into the edge of the XANES of
-1
17
1
6,500-27,000 cm in the CD spectrum. This is likely
a manifestation of the differential electronic properties of
the Ni-center imparted by weak versus strong axial imida-
III
M1
{
Ni (SOD -Im-Me)} while the Ni 1s f 3d transition
III M1
in {Ni (SOD -Im-Me)} is considerably more intense
III
M1
ox
zole donor in {Ni (SOD -Im-Me)} versus NiSOD .
II
M1
than for {Ni (SOD -Im-Me)}. The increase in inten-
sity of the Ni 1s f 3d transition is consistent with both
a change in symmetry about the Ni center and an
increase in the number of holes in the 3d-manifold. Each
of these would be expected with a change from four-
III
M1
{
Ni (SOD -Im-Me)} was probed further by reso-
nance Raman (rR) spectroscopy. Excitation of
III
M1
{
in a significant enhancement of three Raman active
Ni (SOD -Im-Me)} using 405.0 nm light resulted
-1
vibrational modes at 323, 343, and 388 cm (Figure 8,
Table 4). We note that these are due exclusively to the
II
coordinate square planar Ni to five coordinate square
pyramidal Ni ; the pseudocenter of inversion would be
lost in {Ni (SOD -Im-Me)} versus {Ni (SOD
Im-Me)}, thus the parity forbidden Ni 1s f 3d transi-
tion gains intensity through a dipole mechanism in a
square-pyramidal versus square-planar coordination
III
III
M1
II
M1
-
(
64) Westre, T. E.; Kennepohl, P.; DeWitt, J. G.; Hedman, B.; Hodgson,
K. O.; Solomon, E. I. J. Am. Chem. Soc. 1997, 119, 6297–6314.
65) Roe, A. L.; Schneider, D. J.; Mayer, R. J.; Pyrz, J. W.; Widom, J.;
Que, L., Jr. J. Am. Chem. Soc. 1984, 106, 1676–1681.
(

1
0568 Inorganic Chemistry, Vol. 48, No. 22, 2009
Shearer et al.
III
M1
III
M1
III
M1
III
M1
a
Table 3. EPR Results for {Ni (SOD -Im-Me)}, {Ni (SOD -Im-H)}, {Ni (SOD -Im-Tos)}, {Ni (SOD -Im-DNP)}
g
x
(Axx MHz) (Ayy MHz)
g
y
g
z
(Azz MHz)
exp
calcd.
exp
calcd.
exp
calcd.
III
M1
{
{
{
{
Ni (SOD -Im-Me)}
2.36 (nd)
2.33 (nd)
2.33 (nd)
2.33 (nd)
2.284 (69.2)
2.288 (69.5)
2.282 (59.9)
2.286 (57.2)
2.29 (nd)
2.27 (nd)
2.22 (nd)
2.24 (nd)
2.260 (55.3)
2.263 (55.2)
2.169 (48.2)
2.206 (46.9)
2.02 (83)
2.01 (81)
2.00 (55)
2.00 (67)
2.040 (74.0)
2.032 (73.9)
2.048 (51.3)
2.045 (65.7)
III
M1
Ni (SOD -Im-H)}
III
M1
Ni (SOD -Im-DNP)}
III
M1
Ni (SOD -Im-Tos)}
a
Calculated results were obtained using the B3LYP functional by solving the CP-SCF equations.
III
M1
Figure 6. Ni K-edge X-ray absorption data for I
simulations to the data are shown in dashed blue, and the difference spectrum is shown in green. The left figure shows the XANES region of the x-ray
2
generated {Ni (SOD -Im-Me)} (concentration ∼ 3 mM). Experimental data are shown in red,
3
3
absorption (XAS), the middle figure shows the FT k EXAFS, and the right figure shows the FF k EXAFS. The inset presented in the XANES panel
displays an expansion of the Ni(1s f 3d) transition.
III
M1
Figure 8. Resonance Raman spectra of {Ni (SOD -Im-Me)}
(
{
A and B, recorded at room temperature and 77 K, respectively),
Ni (SOD -Im-DNP)} (C; 77 K), and {Ni (SOD -Im-Tos)}
D; 77 K) following 405 nm excitation. The symmetric N-Ni-S stretch-
III
M1
III
M1
(
ing modes are highlighted in red.
Figure 7. Electronic absorption spectrum (bottom) and CD spectrum
III
M1
(
top) of {Ni (SOD -Im-Me)}.
III
Ni form of the metallopeptide as excitation of identical
It should be noted that the Ni-S vibrational modes
observed in {Ni (SOD (H(1)H ))} occur at lower
II
M1
III
M1
Me
concentrations of {Ni (SOD -Im-Me)} at 405.0 nm
using similar integration times did not lead to a significant
signal in this region of the rR spectrum. These resonance
enhanced Raman modes are similar as those observed for
ox
ox
energy than those observed in NiSOD (NiSOD νNi-S
-
1 17,66
=
349, 365, and 391 cm ).
This reduction in stretch-
ing frequency is reflected in the calculated force cons-
ox
17,66
III
M1
NiSOD , and correspond to N-Ni-S stretches.
tants for the Ni-S bonds in {Ni (SOD -Im-Me)}
A normal coordinate analysis
(NCA) yields force constants of 1.54 and 1.31 mdyn
ox
versus NiSOD .
(
66) Fiedler, A. T.; Brunold, T. C. Inorg. Chem. 2007, 46, 8511–8523.

Article
Inorganic Chemistry, Vol. 48, No. 22, 2009 10569
III
M1
Table 4. Resonance Raman Derived Ni-S Force Constants and Vibrational Modes for {Ni (SOD -Im-X)} (X = Me, DNP and Tos)
exp. ν (cm-
1
)
calcd. ν (cm-1
)
normal-mode description
-1
)
˚
f (mdyn A
III
M1
a
S rNi + NifN
amide
amine
a
{
Ni (SOD -Im-Me)}
323
325
340
386
341
349
345
350
Ni-S : 1.31
b
S rNi + NifN
b
3
3
43
88
Ni-S : 1.54
a
amine
amide
S rNi - NifN
S rNi + NifN
III
M1
a
a
{
{
Ni (SOD -Im-DNP)}
338
49
343
Ni-S : 1.47
b
amine
amide
amine
b
3
S rNi + NifN
Ni-S : 1.69
III
M1
a
S rNi + NifN
a
Ni (SOD -Im-Tos)}
Ni-S : 1.51
b
b
3
52
S rNi + NifN
Ni-S : 1.70
a
b
Cysteinate trans to the amide nitrogen. Cysteinate trans to the amine nitrogen.
-
1
III
M1
˚
A
for the Ni-S bond trans to the amine and trans to the
a decrease in A . For {Ni (SOD -Im-H)} Azz is
zz
amide, respectively. These are significantly reduced when
compared to the corresponding force constants of 1.79
and 1.68 mdyn A found in NiSOD , indicating that
strong axial coordination by the imidazole is significantly
weakening the equatorial Ni-S bonds, as might be
expected.
Low Temperature Generation and Trapping of
{Ni (SOD -Im-X)} (X = H, DNP, and Tos). At-
tempts were made to prepare and isolate the Ni forms of
{Ni (SOD -Im-X)} (X = H, DNP, and Tos) on an
ice-bath for detailed study without success. The oxidation
of {Ni (SOD -Im-X)} (X = H, DNP, and Tos) by I2,
KMnO , KO , and Oxone all yielded intractable poly-
meric materials. It was possible, however, to generate
sufficient quantities of {Ni (SOD -Im-X)} in EPR
and NMR tubes at low temperatures (∼ -15 °C) in 1:1
buffer/glycerol mixtures (buffer = 50 mM NEM pH 7.4)
followed by rapidly freezing the solutions in liquid nitro-
gen for study by EPR and rR spectroscopy.
equal to 80 MHz, while it decreases to 65 and 67 MHz
III
M1
III
M1
for {Ni (SOD -Im-DNP)} and {Ni (SOD
Im-Tos)}, respectively. The EPR spectra of the metallo-
peptides with the weaker imidazole donors ({Ni -
-
-
1
ox 66
˚
III
M1
III
M1
(SOD -Im-DNP)} and {Ni (SOD -Im-Tos)})
therefore appear to better approximate the EPR spectrum
When all of the
ox
of NiSOD (Azz = 69.7 MHz).
9,17,58
III
M1
solutions were warmed to room temperature and the EPR
spectra were re-recorded at 77 K no signal could be
detected above baseline, indicating the decomposition
III
III
M1
III
of the Ni metallopeptides upon warming.
II
M1
III
M1
Curiously, comparing {Ni (SOD -Im-DNP)} ver-
III M1
sus {Ni (SOD -Im-Tos)}, it was noted that the
superhyperfine coupling constant is larger for the metal-
lopeptide with the weaker axial imidazole ligand
4
2
III
M1
III
M1
({Ni (SOD -Im-Tos)}). To gain insight into the
origin of this difference we turned our attention to
electronic structure calculations. The EPR spectra of
the four metallopeptides were simulated using Neese’s
methodology at the B3LYP level.
revealed a surprising aspect of the influence imparted
by changing the nature of the axial ligand. We find that as
the axial ligand is made less Lewis-basic the orientation
III
M1
III
54,55
For both {Ni (SOD -Im-DNP)} and {Ni -
These calculations
M1
(
following excitation at 405.0 nm. As the axial imidazole is
SOD -Im-Tos)} we were able to obtain rR spectra
made less Lewis-basic the resulting N-Ni-S vibrational
III
M1
III
Me
modes observed in {Ni (SOD -Im-Me)} increase in
of the g vector changes. For both Ni (SOD ) and
z
Ni (SOD ) the g vector is oriented along the
z
-
1
III
H
energy by 8-18 cm (Figure 8, Table 4). This corre-
sponds to a strengthening of the calculated force con-
III
imidazole
III
Ni -N
bond, as would be expected. In Ni -
) the g vector becomes more oriented into
molecular xy plane. For Ni (SOD ), which has the
III
M1
DNP
stants for the Ni-S bonds. For {Ni (SOD
-
(SOD
z
III
Tos
Im-DNP)} the NCA derived force constants are 1.69
-
1
˚
and 1.47 mdyn A for the Ni-S bond trans to the amine
and trans to the amide, respectively. The Ni-S bond of
least Lewis-basic imidazole donor, the g vector becomes
z
amine
oriented along the S-Ni-N
bond. Furthermore, the
III
M1
{
Ni (SOD -Im-Tos)} are even stronger at 1.70 and
singly occupied molecular orbital (SOMO) composition
switches from a relatively large contribution from the
-
1
˚
1
bonds found in NiSOD of the oxidized metallopeptides
.51 mdyn A , which are the closest to the strong Ni-S
ox
III
imidazole δ-nitrogen atomic orbital (AO) in Ni -
(SOD ) and Ni (SOD ) to negligible contributions
6
6
Me
III
H
investigated in this study. Warming the solutions to
room temperature for several minutes resulted in both the
decomposition of the Ni metallopeptides and the dis-
III
from this AO in Ni (SOD
DNP
III
) and Ni (SOD
Tos
)
(Supporting Information). Therefore, it appears that in
III
III
III
M1
III
M1
appearance of the bands attributable to the Ni -S
{Ni (SOD -Im-DNP)} and {Ni (SOD -Im-
III
M1
stretches in both {Ni (SOD -Im-DNP)} and
Tos)} the observed superhyperfine coupling in g appears
to be due to the amine nitrogen not the imidazole nitro-
gen.
z
III
M1
{
Ni (SOD -Im-Tos)}. Attempts were made to probe
III
M1
the rR spectrum of {Ni (SOD -Im-H)} without
success as excitation of this metallopeptide at 405 nm
In a study performed by Brunold and co-workers it was
found that variation of the axial imidazole N-Ni bond
length from 2.07-2.16 A (because of outersphere H-
bonding interactions) in NiSOD computational models
effected a change in the g-values, but not a significant
change in superhyperfine coupling. It was reasoned that
(and other visible wavelengths) leads to significant pho-
tobleaching of the sample.
The EPR spectra of {Ni (SOD -Im-X)} (X = H,
DNP, and Tos) are displayed in Figure 5. As can be seen
all three EPR spectra are similar in appearance to that
˚
III
M1
1
7
III
M1
III
obtained for {Ni (SOD -Im-Me)}, including the
the increase in the Ni -N bond length dropped the
superhyperfine coupling in g , which is indicative of
energy of the SOMO, which increased the anisotropy of
the g-tensor via mixing of the SOMO with the Ni-
based MOs through a spin-orbit coupling mechanism.
z
III
low-spin Ni with nitrogen coordination along g . As
the Lewis-basicity of the axial ligand is decreased there is
z

1
0570 Inorganic Chemistry, Vol. 48, No. 22, 2009
Shearer et al.
III
In contrast, the change in the Ni -N bond length did
not have a dramatic influence on the composition of the
SOMO, and thus the superhyperfine coupling constant
remained fairly constant as the bond length was changed.
We observe something quite different in our systems
where the axial ligands themselves are changed, not the
III
axial Ni -N bond lengths. As the imidazole ligand is
changed from a more to a less Lewis-basic ligand we
observe a large change in the composition of the AO, as
noted above, but not a large change in the relative
energetics of the SOMO versus the other filled Ni-based
MOs. Therefore orbital mixing via a spin-orbit coupling
mechanism would be similar for the four metallopeptides
studied, resulting in similar g-values across the series.
However, because of the changes in the composition of
the SOMO we observe larger changes in the computa-
tionally derived superhyperfine coupling constants. This
is what is observed experimentally.
Figure 9. Decay trace for the disproportionation of 50 mM KO
2
(18-c-6
M1
These observations may help explain why superhyper-
fine coupling was observed in the oxidized His(1)Gln
mutant of NiSOD.
salt) {Ni(SOD -Im-X)} (pH 8.0 T = 24.7 °C). The black trace is the
M1
KO
(
2
self-disproportionation, the blue trace is {Ni(SOD -Im-Me)}
1
8,27
III
This Ni center likely lacks
M1
60 μM), the green trace is {Ni(SOD -Im-H)} (12 μM), the red trace is
{Ni(SOD -Im-DNP)} (2.0 μM), and the purple blue trace is
M1
III
axial Ni -N ligation yet still displays the characteristic
M1
{Ni(SOD -Im-Tos)} (1.0 μM).
three line splitting in g observed for native NiSOD. The
above data suggest that the superhyperfine coupling
z
a double mixing device allows us to minimize any optical
disturbances resulting from mixing of the two dissimilar
solvents. However, the first 5 ms of the kinetics run
beyond the instrument’s deadtime still had to be elimi-
nated because of optical disturbances that could not be
compensated for by baseline subtraction.
ox
III
observed in the g of NiSOD , which has a Ni -N
z
˚
distance between 2.3-2.6 A, is possibly due to coupling of
the unpaired spin with the amine nitrogen not the axial
imidazole.
M1
SOD Activity of {Ni(SOD -Im-X)} (X = Me, H,
DNP, and Tos) Investigated By Stopped-Flow Kinetics.
All reactions employed 50 mM NEM buffer at pH =
8.0. The increase in pH from 7.4 to 8.0 was done to slow
M1
We previously reported that {Ni(SOD -Im-H)} is
SOD active based on both a simple NBT assay and the
•-
down the O
self-disproportionation reaction, which
2
2
3,24,67
4
xanthine/xanthine oxidase assay.
xanthine/xanthine oxidase assay we obtained an IC
From the
=
0
under these conditions we measure to be 7(1) ꢀ 10
-
1
-1
M1
M
to solution we observe a change in the decay kinetics of
s
(Figure 9). When {Ni(SOD -Im-H)} is added
5
-
7
M1
23
2
(1) ꢀ 10 M for {Ni(SOD -Im-H)}. This is less
•-
than an order of magnitude poorer than the activity
obtained from naturally occurring SODs; Cu/Zn SOD
O
process (Figure 9). This is due to the second order
2
from a second order process to a pseudo-first order
-
8
-8
•-
M1
displays an IC = 4 ꢀ 10 M, or 8 ꢀ 10 M per
catalytic decomposition of O
H)}, which is first order in both reactant (Table 2, Sup-
porting Information). Although the reaction between
{Ni(SOD -Im-H)} and O
disproportionation reaction (k = 7(3) ꢀ 10 M
is at least one and a half orders of magnitude slower than
2
by {Ni(SOD -Im-
5
0
6
subunit. The NBT assay also indicated high SOD
7
M1
22
activity for {Ni(SOD -Im-H)}.
M1
•-
Both the NBT and the xanthine/xanthine oxidase
assays are indirect methods for monitoring SOD activity;
a competition reaction is set up between the SOD mimic
and a reporter molecule. The drawback with these meth-
ods is that it is impossible to know if the SOD mimic is
2
is faster than the self-
7
-1 -1
s
), it
•
-
the reaction observed between O
and NiSOD at this
s ). These data therefore
2
9
-1 -1 27
pH (kcat ∼ 1 ꢀ 10 M
•
-
•-
M1
reacting with O2 directly (i.e., catalyzing O2 dis-
proportionation) or if it is interfering with the O
demonstrate that {Ni(SOD -Im-H)} is a poorer cat-
alyst than one would have concluded from the xanthine/
xanthine oxidase assay; using the xanthine/xanthine oxi-
•-
2
•
-
production, the indicator molecule, or O
itself in a
2
•
-
68
M1
manner not related to O2 disproportionation. To
circumvent this issue we monitored the direct decomposi-
tion of O2 using stopped-flow kinetics by monitoring
dase assay the activity of {Ni(SOD -Im-H)} was
overestimated by over an order of magnitude.
The O
•
-
•-
disproportionation kinetics of the other three
2
•
-
-1 -1
the disappearance of O2 at 245 nm (ε = 2250 M
s
).
metallopeptides were also investigated using stopped-
flow kinetics. We found that all three of the
N-substituted imidazole substituted metallopeptides
We employed a double mixing stopped flow system to
increase the dilution of anhydrous DMSO (the solvent
used for solubilizing the KO 18-crown-6 salt) into the
are capable of facilitating the catalytic disproportiona-
2
2
9
•-
buffer. This was done for two reasons. One is that by
diluting the DMSO in buffer we can better approximate
the situation observed in pure buffer. The second reason
is that dilution of the DMSO into an excess of buffer using
tion of O . The metallopeptide with the most electron
2
III
III
M1
rich Ni -center, {Ni (SOD -Im-Me)}, affords the
•-
slowest O
degradation kinetics of the four metallopep-
2
6
-1 -1
tides investigated (k = 6(1) ꢀ 10 M s ). In con-
III
M1
III
trast, both {Ni (SOD -Im-DNP)} and {Ni -
M1
•-
(
SOD -Im-Tos)} display O degradation kinetics that
2
III M1 8
(
67) Tabbi, G.; Driessen, W. L.; Reedijk, J.; Bonomo, R. P.; Veldman, N.;
Spek, A. L. Inorg. Chem. 1997, 36, 1168–1175.
68) Riley, D. P. Chem. Rev. 1999, 99, 2573–2587.
are faster than {Ni (SOD -Im-H)} with k=4(2) ꢀ 10
-
1
-1
8
-1 -1
(
M
s
and 6(2) ꢀ 10 M s , respectively.

Article
Inorganic Chemistry, Vol. 48, No. 22, 2009 10571
Summary and Conclusion
electrochemically optimized to perform SOD chemistry un-
der near physiological conditions; neither the oxidation nor
the reduction reaction will slow down the overall dispropor-
tionation reaction. In contrast, the metallopeptide with the
In this study we have utilized unnatural amino acids in
metallopeptide based NiSOD mimics to probe the influence
of a H-bonding network in the metalloenzyme. It was
reasoned that we could approximate the electronic conse-
quence of this structural feature in the metalloenzyme by
altering the ligand based electronics of the axial His(1)
imidazole donor by appending electron donating and with-
drawing groups to the imidazole ε-nitrogen. In essence, it was
reasoned that by utilizing less Lewis-basic imidazole donors
the electronic properties of the nickel center in NiSOD could
be approximated.
M1
most Lewis basic imidazole donor, {Ni(SOD -Im-Me)},
II
III
has a Ni /Ni redox potential that is nearly 300 mV more
negative than the midpoint of the superoxide oxidation/
reduction potentials. Therefore, the reduction of superoxide
II
M1
by {Ni (SOD -Im-Me)} will dramatically retard the
•-
overall
{
rate
of
O
2
disproportionation
by
M1
Ni(SOD -Im-Me)}. There are also significant changes
in Ni-ligand bond strengths as the imidazole ligand’s elec-
tronics are changed. This will likely influence the rate of SOD
kinetics for this metalloenzyme as this will change the inner-
In all cases, we did not observe significant influences on the
structure and properties of the reduced Ni forms of the
II
sphere reorganization energy (λ ) from metallopeptide to
i
metallopeptides as the His(1) imidazole was modified. This
should be expected because the imidazole does not coordi-
metallopeptide. It is highly likely that both of these factors
are combining in a serendipitous manner to allow for efficient
II
nate to the reduced Ni center. In contrast, significant
M1
catalytic SOD activity in {Ni(SOD -Im-Tos)} and
differences in the spectroscopic and reactivity properties of
M1
{
Ni(SOD -Im-DNP)}. NiSOD itself, with the limited
III
the oxidized Ni metallopeptides were observed as the
number of naturally occurring amino-acid residues available,
utilizes the His(1) Glu(17) Arg(47) H-bonding network
to effectively “reduce” the Lewis-basicity of the imidazole
ligand (from the standpoint of the Ni -center). Thus,
NiSOD likely optimizes its electron transfer properties
imidazole ligand electronics were changed. We observe that
as the imidazole ligand is made less Lewis-basic the metallo-
peptide basedmodelbegins tobetter approximate the proper-
ties of the metalloenzyme; the EPR properties, N-Ni-S
stretching frequencies, and the resulting SOD activity all
become similar to the metalloenzyme as the axial imidazole
donor is made less Lewis basic. We note that some stabilizing
interaction to the Ni-center is required as removal of this
ligand (e.g., the His to Ala modification) produces a cataly-
3
3 3
3 3 3
III
18,69-71
through a rack/entatic state type mechanism.
Acknowledgment. Funding for this project was pro-
vided by the NIH (P20 RR-016464) and the NSF (CHE-
0844234). We also thank Professor Martin L. Kirk
(University of New Mexico) for valuable advice and
Professor Nicolai Lehnert (University of Michigan) for
access to the source code of his QCA-NCA software.
III
tically inactive SOD whose Ni state cannot be stabilized for
18
study. Therefore subtle changes to the axial ligand translate
into profound differences in SOD activity effected by these
NiSOD metallopeptide mimics; too strong of an axial ligand
and too weak of an axial ligand will both dramatically hinder
Supporting Information Available: Contains the full author
list for ref 50, computational, chromatographic, ESI-MS, and
kinetic results. This material is available free of charge via the
Internet at http://pubs.acs.org.
(if not out-right shut-down) SOD activity.
The likely explanations for the influence of the axial
imidazole ligand are both rooted in electron transfer to and
from the Ni-center. The metallopeptide with the least Lewis
M1
(69) Malmstrom, B. G. Eur. J. Biochem. 1994, 223, 711–718.
(70) Williams, R. J. P. Eur. J. Biochem. 1995, 234, 363–381.
(71) Vallee, B. L.; Williams, R. J. Proc. Natl. Acad. Sci. U.S.A. 1968, 59,
basic imidazole donor {Ni(SOD -Im-Tos)} has a redox
potential that is nearly identical to the pH 7 midpoint
•-
65
O2
oxidation/reduction potentials.
It is therefore
498–505.