Versatile methodology toward NiN2S2 complexes as nickel superoxide dismutase models: Structure and proton affinity

Article abstract of DOI:10.1021/ic9009042

Structural features of the reduced form of the nickel superoxide dismutase (Ni-SOD) active site have been modeled with asymmetric NiN₂S ₂ complexes (Et₄N)[Ni(nmp)(SR)] (R = C₆H ₄-p-CI (2) and (S^t

Full text of DOI:10.1021/ic9009042

5620 Inorg. Chem. 2009, 48, 5620–5622
DOI: 10.1021/ic9009042
Versatile Methodology Toward NiN2S2 Complexes as Nickel Superoxide Dismutase
Models: Structure and Proton Affinity
Eric M. Gale, Ashis K. Patra, and Todd C. Harrop*
Department of Chemistry, University of Georgia, 1001 Cedar St, Athens, Georgia 30602
Received May 10, 2009
Structural features of the reduced form of the nickel superoxide
dismutase (Ni-SOD) active site have been modeled with asym-
metric NiN2S2 complexes (Et4N)[Ni(nmp)(SR)] (R = C6H4-p-Cl (2)
and (StBu) (3)) obtained via S,S-bridge splitting of the dimeric
metallosynthon, [Ni2(nmp)2] (1). Complexes 2 and 3 are irreversibly
oxidized at potentials within the window needed for SOD activity,
236 and 75 mV versus Ag/AgCl, respectively. The exogenous
thiolato-S in 2 and 3 serves as a proton acceptor, suggesting
potential involvement of Cys6 in Ni-SOD for H+ storage between
SOD half reactions.
of this SOD.7,8 The overall protein fold, spectroscopy,
ligands, and coordination geometry in Ni-SOD are quite
distinct from those of other SODs. For example, Ni-SOD in
its reduced NiII form (Ni-SODred) is ligated in an N2S2
square-planar geometry arising from the primary amine-N
of His1, carboxamido-N from Cys2, and two thiolato-S
donors from Cys2 and Cys6 (Chart 1). The presence of
carboxamido-N and primary amine-N provides an unusual
set of donors and raises questions with regard to the proper-
ties they impart on the Ni center, with few examples known in
biology.9,10 Another striking feature of Ni-SOD is the pre-
sence of two coordinated cysteine thiolates, which are them-
selves subject to oxidation in the presence of ROS.11
Although the highly covalent nature of thiolate ligation is
believed to aid in modulating the NiII/NiIII redox couple to a
value suitable for physiological function,12 the interactions
directing metal-based redox are not yet fully understood.13,14
Additional questions arise as to how superoxide coordinates,
the source of protons in the oxidative half-reaction, and how
these tie into the overall Ni-SOD catalytic mechanism.
The report of Ni-SOD has inspired us and others15 to
answer these questions through small molecule analogues in
The reactive oxygen species (ROS) superoxide (O2•-) is an
inevitable cytotoxic byproduct of aerobic metabolism, which
if not eliminated can lead to a variety of health disorders.1 To
combat this ROS, all aerobic organisms possess metallo-
enzymes known as superoxide dismutases (SODs) that cata-
•-
lyze the disproportionation of O2 into hydrogen peroxide
(H2O2) and O2 through alternate oxidation and reduction of
their catalytic metal centers.2 These SODs have been exten-
sively characterized and employ metal cofactors such as Fe,3,4
Mn,5 or Cu/Zn6 to catalyze the disproportionation reaction.
Recently, a new class of SODs containing Ni (Ni-SOD) has been
characterized; however, less is known regarding the mechanism
Chart 1. Active Site of Ni-SODred and the Model Complexes, [Ni(nmp)
(SR)]-, Used in This Study (R = C6H4-p-Cl (2) or tBu (3))
*To whom correspondence should be adressed. E-mail: tharrop@chem.
uga.edu.
(1) (a) Beyer, W.; Imlay, J.; Fridovich, I. Prog. Nucleic Acid Res. Mol.
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J. Biochem. Mol. Toxicol. 2003, 17, 24. (c) Valentine, J. S.; Wertz, D. L.;
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R. L.; Roberts, G. P.; Poulos, T. L. Nat. Struct. Biol. 2000, 7, 876.
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r
2009 American Chemical Society
pubs.acs.org/IC
Published on Web 06/05/2009

Communication
Inorganic Chemistry, Vol. 48, No. 13, 2009 5621
Scheme 1
Figure 1. ORTEP diagrams of the anions of (Et4N)[Ni(nmp)(SC6H4-p-
Cl)] (2) (left) and (Et4N)[Ni(nmp)(StBu)] (3) (right) showing 50% thermal
˚
ellipsoids for all non-hydrogen atoms. Selected bond distances (A) and
angles (deg) for 2 and 3, values for 3 are shown in brackets: Ni1-N1,
1.8638(14) [1.882(2)]; Ni1-N2, 1.9470(14) [1.9635(19)]; Ni1-S1, 2.1492
(5) [2.1629(7)]; Ni1-S2, 2.2139(4) [2.1938(7)]; N1-Ni1-N2, 83.25(6)
[82.30(8)]; N1-Ni1-S2, 176.73(5) [174.21(6)]; S1-Ni1-S2, 90.45(2)
[98.14(3)].
Dark red X-ray quality crystals of 2 and 3 were obtained
under anaerobic conditions by slow diffusion of Et2O into
THF solutions of the complexes at RT. The structures
revealed four-coordinate square-planar NiII centers arising
from the tridentate nmp2- ligand and monodentate exo-
genous thiolate completing the N2S2 coordination sphere
(Figure 1). The Ni-N/S distances are somewhat variable and
in line with the electronic nature of the donor atom, as the
order to investigate the physical properties of the models
and shed light into the intrinsic properties of the Ni active
site. Models employing both electronically accurate and
asymmetric N2S2 spatial disposition are also quite scarce.15
Herein, we report our initial work in this area, namely, the
synthesis, structural and spectroscopic/electrochemical char-
acterization, and proton binding studies of an unprecedented
set of four-coordinate square-planar NiIIN2S2 complexes as
biomimetic structural analogues of Ni-SOD employing the
tridentate ligand N-(2-mercaptoethyl)picolinamide (nmpH2,
where H represents dissociable protons; Chart 1).
˚
˚
Ni-Namide bond is shorter (1.8638(14) A for 2 and 1.882(2) A
˚
for 3) than the Ni-Npy bond in both 2 (1.9470(14) A) and 3
˚
(1.9635(19) A), which results in a relative lengthening of the
˚
Ni-S bond that is trans to the carboxamido-N (2.2139(4) A
˚
for 2 and 2.1938(7) A for 3). Slight distortions from a perfect
square plane are also evident from the bond angles about Ni,
with the most acute arising from the tight bite of the pyridine-
2-carboxamide unit (Supporting Information for further
structural information). The observed metric parameters
for 2 and 3 are similar to those of other reported NiII
complexes in similar coordination spheres9,11,15 and are also
strikingly similar to those determined for Ni-SODred,8 pre-
sumably arising from the close match between donor type
and spatial disposition provided by our model system
(see Table S3, Supporting Information).
As described above, both 2 and 3 exhibit square-planar
geometries in the solid state that also appear to be the same
in solution. This property is clearly demonstrated in their
1H NMR spectra, which display no paramagnetically shifted
resonances expected for tetrahedral or octahedral geometry.
This diamagnetism is not solvent dependent, as the square-
planar (S=0) state persists in both donor ((CD3)2CO) and
non-donor (CDCl3, THF-d8) solvents (Supporting Informa-
tion). The red-orange color of solutions of 2 and 3 result from
presumed S-to-Ni charge transfer bands in their UV-vis
spectra and are similar to other NiIIN2S2 complexes9,15,17
with bands at 450 nm (ε = 5450 M-1 cm-1) and 464 nm (ε =
4540 M-1 cm-1) for 2 and 3, respectively (Figure 2). These
When a DMF solution of nmp2- is mixed with 1 mol equiv
of [Ni(H2O)6](ClO4)2 under anaerobic conditions, a red-
orange precipitate forms at room-temp (RT) consistent with
the dinuclear S,S-bridged species, [Ni2(nmp)2] (1) (Scheme 1).
Since similar S,S-bridged species have been shown to break in
the presence of strong ligands like CN-,16 we attempted
bridge-splitting reactions with exogenous thiolate ligands to
generate monomeric NiN2S2 complexes with an asymmetric
coordination sphere, as observed in Ni-SOD. The addition of
2 mol equiv of RS- (where R=C6H4-p-Cl or tBu) to 1 resulted
in formation of the corresponding mononuclear NiII com-
plexes (Et4N)[Ni(nmp)(SC6H4-p-Cl)] (2)and(Et4N)[Ni(nmp)-
(StBu)] (3) in 70% yield for both (Scheme 1, Chart 1). The
nmp2- ligand replicates the two five-member chelate rings
formed by His1 and Cys2 in Ni-SOD, while the exogenous
thiolate allows for unconstrained modeling of Cys6.8
Collectively, the bridge-splitting reaction of metallosynthon
1 with electronically different thiolate ligands provides a new,
versatile, and general synthetic methodology for the prepara-
tion of monomeric and asymmetric NiN2S2 complexes that
can be controlled and varied at the fourth coordination
position.
λmax values are similar to the visible band of Ni-SODred
,
which is also centered near 450 nm.12 Complexes 2 and 3 are
stable in the solid state upon exposure to air while solutions
appear moderately stable over the course of hours as
monitored by UV-vis spectroscopy.
Cyclic voltammetric (CV) measurements of 2 and 3 in
MeCN display irreversible oxidation events at 236 and 75 mV
versus Ag/AgCl, respectively. Their differences are largely
(15) (a) Shearer, J.; Zhao, N. Inorg. Chem. 2006, 45, 9637. (b) Neupane,
K. P.; Gearty, K.; Francis, A.; Shearer, J. J. Am. Chem. Soc. 2007, 129,
14605. (c) Ma, H.; Chattopadhyay, S.; Petersen, J. L.; Jensen, M. P. Inorg.
Chem. 2008, 47, 7966.
(16) Grapperhaus, C. A.; Bellefeuille, J. A.; Reibenspies, J. H.; Darensbourg,
M. Y. Inorg. Chem. 1999, 38, 3698.
::
(17) Kruger, H.-J.; Peng, G.; Holm, R. H. Inorg. Chem. 1991, 30, 734.

5622 Inorganic Chemistry, Vol. 48, No. 13, 2009
Gale et al.
the HOMO.13 In this regard, the capabilities of 2 and 3 as
proton acceptors were tested. The addition of 1 mol equiv of
tosic acid to 2 and 3 leads to near quantitative formation of
1 via protonation of the exogenous thiolate (Scheme 1). No
decomposition of the Ni(nmp) unit resulted from this strong
acid, further underscoring the stability of the coordinated
tridentate ligand and suggesting that Cys6 may be the most
likely candidate for protonation in Ni-SOD.20c The exogen-
ous thiolate in these complexes can also be exchanged by the
addition of a more acidic thiol. Treatment of 3 with 1 mol
equiv of HSC6H4-p-Cl (pKa=5.97) afforded 2 in quantitative
yield (Scheme 1). In contrast, the addition of 1 mol equiv of
tBuSH (pKa =17.9) to 2 displayed no reaction (Scheme 1).
Another proposed source of protons for the oxidative half
reaction is Tyr9,21 and thus we tested the proton-accepting
ability of 2 and 3 with 4-chloro-3,5-dimethylphenol (pKa ∼
18). No proton exchange was observed when 1 mol equiv of
this phenol was added to 2, consistent with our observation
that proton exchange is largely governed by the pKa. When
1 mol equiv of phenol was added to 3, however, 33% of dimer
1 was generated. Although it may be expected that an equal
distribution of 1 and phenol/thiol would be present on the
basis of similar pKa values, one must also consider the
thiophilicity of NiII (borderline hard-soft acid), which sup-
ports the observed reaction path and lack of [Ni(nmp)(OPh)]-
complex formation.
In summary, we have prepared two new complexes which
successfully mimic electronically and spatially the primary
coordination sphere of Ni-SODred. In doing so, we introduce
a novel approach toward the preparation of NiN2S2 com-
plexeswith variable exogenous thiolate ligands that opens the
door for the potential preparation of a large library of
Ni-SOD synthetic analogues. Complexes 2 and 3 show
irreversible oxidation events within the range necessary for
SOD chemistry. However, the irreversibility suggests that the
square-planar N2S2 donor set alone is insufficient to support
NiII/NiIII redox cycling, and stabilization is aided via another
interaction like coordination of an axial N ligand. The
reported complexes are the first to demonstrate a propensity
to accept protons at the exogenous thiolato-S site in the
presence of an appropriate proton donor, supporting a role
for the cysteine thiolate(s) in proton storage and transport
during SOD catalysis. The protonation of 3, featuring a
more “cysteine-like” alkyl thiolate, supports the likelihood of
Tyr9 as a possible proton source in Ni-SOD. Recent studies
on Tyr9 mutants by Maroney and co-workers demonstrate
a decrease in activity and H2O2 saturation kinetics not
observed for wild-type enzyme, highlighting its importance
in catalysis and proposed role to aid in the release of H2O2.21
Figure 2. UV-vis spectra of 2 (blue) and 3 (red) in MeCN at 298 K.
Inset: CV of 5 mM solutions of2 (blue) and 3 (red) (vs Ag/AgCl in MeCN,
0.1 M nBu4NPF6 supporting electrolyte, glassy carbon working electrode,
scan rate: 100 mV/s, RT).
attributed to the coordination of the more basic thiolate in
3 versus 2. As expected, these valuesfallinbetweenanalogous
NiIIN2S2 complexes containing diamine-dithiolate18 and
dicarboxamide-dithiolate ligands.17 While the oxidation po-
tentials observed for 2 and 3 are intermediate between the
aforementioned congeners and within the window acceptable
for SOD chemistry,19 the irreversible behavior is more similar
to that of the bis-amine complexes. Bulk oxidation of MeCN
solutions of 2 and 3 with a chemical oxidant like ferrocenium
resulted in the partial formation of 1 and the corresponding
disulfide, suggesting that the observed redox chemistry is in
part ligand-based. It has been proposed that the N2S2 ligand
environment alone in Ni-SOD is insufficient for attaining
NiIII, and stabilization is imparted from other interactions
like coordination of the axial imidazole-N of His1 as ob-
served in the Ni-SODox structure.8,12,15b The addition of
excess amounts (10 mol equiv) of potential N-donors such
as N-methylimidazole or pyridine showed no observable
change in the CV and UV-vis spectra of MeCN solutions
of 2 and 3, suggesting no affinity for these N-donors. The
immediate proximity of His1 to the Ni center in Ni-SOD,
however, may lead to immediate imidazole binding during
turnover, whereas our systems do not benefit from any
enhanced proximity via a built-in N-donor.
In addition to the binding of His1, another element in
regard to the mechanism of Ni-SOD is the source of protons
during the oxidative half reaction. One potential source may
arise from the thiolate-S of Cys2 or Cys6. Indeed, protonation
of Cys-S in Ni-SOD has been detected by S K-edge X-ray
absorption spectroscopy and also supported by DFT calcula-
tions.20 Thiolate protonation has also been suggested to
prevent sulfur oxidation via removal of S(π) interactions from
Acknowledgment. T.C.H. acknowledges support from
the Department of Chemistry at the University of Georgia
for startup funds and the University of Georgia Research
Foundation for a Faculty Research Grant.
(18) Grapperhaus, C. A.; Mullins, C. S.; Kozlowski, P. M.; Mashuta,
M. S. Inorg. Chem. 2004, 43, 2859.
(19) Due to the lack of aqueous solubility of 2 and 3, we were unable to
perform their electrochemistry measurements in aqueous solution. Accord-
ingly, superoxide disproportionation falls between -0.04 and +1.09 V,
versus Ag/AgCl in H2O see: (a) Sawyer, D. T.; Valentine, J. S. Acc. Chem.
Res. 1981, 14, 393. (b) Fridovich, I. Encyl. Biol. Chem. 2004, 4, 135.
(20) (a) Szilagyi, R. K.; Bryngelson, P. A.; Maroney, M. J.; Hedman, B.;
Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 3018.
(b) Pelmenschikov, V.; Siegbahn, P. E. M. J. Am. Chem. Soc. 2006, 128,
7466. (c) Prabhakar, R.; Morokuma, K.; Musaev, D. G. J. Comput. Chem.
2006, 27, 1438.
Supporting Information Available: Experimental details and
characterization of the ligand and complexes including tables
and CIF files. This material is available free of charge via the
Internet at http://pubs.acs.org.
(21) 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,
3354.