A nickel superoxide dismutase maquette that reproduces the spectroscopic and functional properties of the metalloenzyme

Article abstract of DOI:10.1021/ic0514344

Described herein is a nickel superoxide dismutase (NiSOD) maquette ([Ni(SOD^M1)]) based on the first 12 residues from the N-terminal sequence of Streptomyces coelicolor NiSOD. The apopeptide (SOD^M1) was prepared by standard solid-phase Fmoc peptide synthesis. SOD^M1 will readily coordinate Ni^II in a 1:1 ratio in slightly basic aqueous sodium phosphate buffer (0.1 M; pH = 7.2) forming a lightly colored beige/pink solution. Unlike NiSOD, which is isolated as a 1:1 mixture of oxidized (Ni ^III) and reduced (Ni^II) forms, [Ni(SOD^M1)] can only be isolated in the Ni^II oxidation state. The UV/vis, X-ray absorption, and CD spectra of [Ni^II(SOD^M1)] correspond well with those reported for the reduced form of NiSOD. Despite the fact that [Ni^III(SOD^M1)] is not isolable, [Ni(SOD^M1)] has an appropriate redox potential to act as an SOD (E_1/2 = 0.70(2) V vs Ag/AgCl) and in fact will catalytically disproportionate >40 000 equiv of KO₂.

Full text of DOI:10.1021/ic0514344

Inorg. Chem. 2006, 45, 2358−2360
A Nickel Superoxide Dismutase Maquette That Reproduces the
Spectroscopic and Functional Properties of the Metalloenzyme
Jason Shearer* and Linh M. Long
Department of Chemistry, UniVersity of NeVada, Reno, Reno, NeVada 89557
Received August 22, 2005
Described herein is a nickel superoxide dismutase (NiSOD)
maquette ([Ni(SODM1)]) based on the first 12 residues from the
N-terminal sequence of Streptomyces coelicolor NiSOD. The
apopeptide (SODM1) was prepared by standard solid-phase Fmoc
peptide synthesis. SODM1 will readily coordinate Ni in a 1:1 ratio
Scheme 1
II
in slightly basic aqueous sodium phosphate buffer (0.1 M; pH
)
7
.2) forming a lightly colored beige/pink solution. Unlike NiSOD,
III
which is isolated as a 1:1 mixture of oxidized (Ni ) and reduced
•-
loenzymes that catalyze O
2
disproportionation by cycling
Ni ) forms, [Ni(SODM1)] can only be isolated in the Ni oxidation
II
II
(
II
III
7-9
between the Ni and Ni oxidation states.
Crystal-
state. The UV/vis, X-ray absorption, and CD spectra of [Ni (SODM1)]
correspond well with those reported for the reduced form of NiSOD.
Despite the fact that [Ni (SODM1)] is not isolable, [Ni(SODM1)] has
II
7,8
9,10
lographic and spectroscopic studies have elucidated the
primary coordination sphere of NiSOD in both its oxidized
and reduced states. In the reduced state, the Ni center is
III
II
contained in a square-planar geometry ligated by two cis-
cysteinate sulfurs, one amide nitrogen derived from the
peptide backbone, and the N-terminus amine nitrogen from
an appropriate redox potential to act as an SOD (E
V vs Ag/AgCl) and in fact will catalytically disproportionate >40 000
equiv of KO
1
/
2
) 0.70(2)
2
.
III
His(1). Oxidation of NiSOD to the Ni oxidation state
produces a structural change about the metal center; the
III
N-terminal histidine imidazole coordinates to Ni in the axial
position (Scheme 1).
Aerobic organisms must utilize detoxification pathways
to protect themselves against oxidative damage resulting from
exposure to reactive oxygen species (ROS), which are
Despite the fact that the structures of reduced and oxidized
NiSOD have been elucidated, a number of questions
1,2
produced from the adventitious reduction of dioxygen. One
•
-
•
-
concerning both the detailed mechanism of O
2
dispropor-
ROS, superoxide (O
2
), is usually destroyed by metalloen-
1,3,4
tionation and how secondary coordination-sphere residues
influence reactivity remain unanswered. To better understand
zymes called superoxide dismutases (SODs).
catalyze the disproportionation of O
SODs
into dioxygen and
7
•
-
2
the fundamental chemistries of NiSODs, we have prepared
maquettes that closely mimic the spectroscopic and structural
properties of the parent metalloenzyme. Metalloprotein
maquettes are small metallopeptides that replicate some
aspect of their biological inspiration and can offer many
of the advantages of traditional metalloenzyme synthetic
hydrogen peroxide and, until recently, were thought to fall
4
into one of three classes: Fe, Mn, and Cu/Zn SODs. In the
late 1990s, a fourth class of SOD containing Ni (NiSOD)
was isolated from several Streptomyces species and cyano-
1
1
5
,6
bacteria. NiSODs are mononuclear Ni-containing metal-
1
2,13
models
while still utilizing a biological scaffold. Com-
*
To whom correspondence should be addressed. E-mail: shearer@
chem.unr.edu.
(
1) 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.
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3) Miller, A.-F. Curr. Opin. Chem. Biol. 2004, 8, 162-168.
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Biochem. J. 1996, 318, 889-896. (b) 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.
(7) Barondeau, D. P.; Kassmann, C. J.; Bruns, C. K.; Tainer, J. A.; Getzoff,
E. D. Biochemistry 2004, 43, 8038-8047.
(
(
(
(
(8) 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.
(9) 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.
(10) Fiedler, A. T.; Bryngelson, P. A.; Maroney, M. J.; Brunold, T. C. J.
Am. Chem. Soc. 2005, 127, 5449-5462.
(
6) 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 2003, 424, 1037-1042.
(11) Rabanal, F.; DeGrado, W. F.; Dutton, P. L. J. Am. Chem. Soc. 1996,
118, 473-474.
(12) Holm, R. H.; Kennepohl, P.; Solomon, E. I. Chem. ReV. 1996, 96,
2239-2314.
2358 Inorganic Chemistry, Vol. 45, No. 6, 2006
10.1021/ic0514344 CCC: $33.50
© 2006 American Chemical Society
Published on Web 02/11/2006

COMMUNICATION
municated herein are results from our first-generation NiSOD
M1
maquette, [Ni(SOD )].
M1
[
Ni(SOD )] is based on the first 12 residues from the
N-terminus of the active form of S. coelicolor NiSOD (H N-
2
7
M1
HCDLPCGVYDPA-COOH). The apopeptide (SOD ) was
synthesized via standard Fmoc solid-phase synthetic methods
M1
on Wang resin using HOBt/DIC coupling chemistry. SOD
was cleaved from the resin, washed with freshly distilled
diethyl ether, incubated at 45 °C with â-mercaptoethanol,
II
and purified by preparative reverse-phase HPLC. Ni was
then incorporated into the apopeptide using NiCl
anaerobic conditions forming [Ni(SOD )]. Incorporation
2
under
M1 14
Figure 1. UV/vis (bottom) and CD spectra (top) of [Ni(SODM1)] (20 °C,
0.1 M sodium phosphate buffer, pH ) 7.2).
II
M1
of Ni into SOD is highly pH dependent. When the pH is
II
less than ∼6, Ni will not coordinate to the peptide even
when a large excess of NiCl is utilized. However, at pH )
2
II
7
.2 (0.1 M sodium phosphate buffer) Ni will coordinate to
M1
SOD in an ∼1:1 ratio (based on spectrophotometric
titrations), forming a weakly colored beige-pink solution. [Ni-
M1
(
SOD )] exists in solution as a monomer as assessed by
gel permeation chromatography, while CD studies demon-
M1
strate that the peptide structure of [Ni(SOD )] is best
1
5
described as a random coil with some â-turn character.
II
Unlike NiSOD, which is isolated as a 1:1 mixture of Ni /
NiIII oxidation states, [Ni(SOD )] is obtained exclusively
7
M1
II
in the Ni oxidation state even when isolated in air. This is
1
evident from the 1D H NMR spectrum of the metallopep-
tide, which does not display any paramagnetically shifted
III
peaks or line-broadening, suggesting that paramagnetic Ni
is not present. Attempts were made to produce and isolate
III
M1
the Ni form of [Ni(SOD )] with no success. Not surpris-
ingly, the addition of dilute H or Oxone, which can
Figure 2. Cyclic voltammogram of [Ni(SODM1)] recorded as a “protein
thin film” on a carbon fiber ultramicroelectrode.14 The inset depicts the
data with the background subtracted.
2 2
O
16
oxidize coordinated thiolate-sulfurs, leads to the rapid and
complete decomposition of [Ni(SOD )]. We also found that
the addition of 1 equiv of ceric ammonium nitrate or [Fe -
M1
III
resolve many of these features (Figure 1), most of which
II
M1
(bipy)
3
]Cl
3
to anaerobic solutions of [Ni (SOD )] leads to
10
correspond well with reduced NiSOD. Most notable is the
the decomposition of the metallopeptide. In addition, we
-1
negative signed feature observed at 21 800 cm (∆ꢀmax
)
II
M1
observed that solutions of [Ni (SOD )] will decompose if
exposed to air over the course of several hours. In all cases,
the decomposition products are insoluble high-molecular-
weight polymers.
-1
-1
-
2.2 M cm ), which is also observed in the reduced
II
M1
metalloenzyme. One feature that we observe in [Ni (SOD )]
that was not observed in reduced NiSOD is a weak band at
-1
-1
-1
1
5 100 cm (ꢀ ) 70 M cm ), presumably a ligand-field
II
M1
Reduced [Ni (SOD )] displays relatively weak features
in the UV/vis spectrum (Figure 1). These features do not
transition, which is well resolved in the CD spectrum (∆ꢀmax
-1
-1
)
-2.0 M cm ).
The Ni K-edge X-ray absorption spectrum of [Ni -
II
change when dithionite is added to solutions of [Ni -
II
M1
II
(
SOD )], further supporting the Ni oxidation state. The
M1
(
SOD )] is also consistent with that reported for reduced
NiSOD. The pre-edge region displays a prominent peak at
337.4(2) eV corresponding to a 1s f 4p transition and a
most distinct feature in the UV/vis spectrum is a broad
9
-1
transition with a well-defined maximum at 21 800 cm (458
8
z
-
1
-1
-1
nm; ꢀ ) 510 M cm ) and a shoulder at 18 100 cm (552
weak peak at 8332.9(4) eV corresponding to a 1s f 3d
-1
-1
nm; ꢀ ) 240 M cm ). At higher energy, there is a shoulder
II
transition. These are both indicative of square planar Ni .
-
1
-1
-1
observed at 29 700 cm (337 nm; ꢀ ) 1060 M cm )
Best fits to the EXAFS region revealed two Ni-N scatterers
at 1.93(2) Å and two Ni-S scatterers at 2.180(2) Å, which
just before the onset of end absorbance. All three of these
1
0
features correspond to those observed in reduced NiSOD.
compares well with those reported for reduced NiSOD (Ni-
II
M1
The corresponding CD spectrum of [Ni (SOD )] helps to
7-9
N
ave, 1.89 Å; Ni-Save, 2.18 Å).
M1
The electrochemical behavior of [Ni(SOD )] was mea-
(
(
(
13) Karlin, K. D. Science 1993, 261, 701-708.
17
sured using “protein thin-film voltammetry” techniques on
14) See Supporting Information.
15) Woody, R. W. In Peptides, Polypeptides and Proteins; Blout, E. R.,
Bovey, F. A., Goodman, M., Lotan, N., Eds.; John Wiley: New York,
(17) (a) Vincent, K., A.; Armstrong, F., A. Inorg. Chem. 2005, 44, 798-
809. (b) L e´ ger, C.; Elliott, S. J.; Hoke, K. R.; Jeuken, L. J. C.; Jones
A. K.; Armstrong, F. A. Biochemistry 2003, 42, 8653-8662. (c)
Rusling, J. F. Acc. Chem. Res. 1998, 31, 363-369.
1
974, pp 338-348.
16) Grapperhaus, C. A.; Darensbourg, M. Y. Acc. Chem. Res. 1998, 31
51-459.
(
4
Inorganic Chemistry, Vol. 45, No. 6, 2006 2359

COMMUNICATION
M1
a carbon fiber ultramicroelectrode. [Ni(SOD )] has an Epc
)
Scheme 2
+
0.61(1) V and an Epa ) 0.80(2) V vs Ag/Ag . We note
that the relatively large peak separation (0.19 vs ∼0.02 V
typically obtained for fully reversible reactions recorded
M1
under similar conditions) observed for [Ni(SOD )] is
-
indicative of kinetic limitations to e transfer (i.e., a rate-
limiting change in coordination environment occurrs upon
-
e transfer). This is unexpected considering the rapid rate
The addition of 2800 equiv18 of KO
M1
2
to a [Ni(SOD )]/
•
-
9
of O
2
disproportionation observed for NiSOD and may
NBT solution produces a faint blue color (and a weak band
•
-
have implications concerning the mechanism of O
proportionation in the metalloenzyme and the maquette.
Although the midpoint potential observed for [Ni(SOD )]
2
dis-
M1
at 580 nm), indicating that [Ni(SOD )] is losing SOD
activity when exposed to a large excess of KO
2
. However,
are added to the
M1
18
it is not until ∼16 500 equiv of KO
2
4
is ∼0.4 V more positive than is usually observed for SODs,
M1
solution that [Ni(SOD )] is overwhelmed, as noted by the
19,20
it is not unprecedented for thiolate-ligated Ni complexes.
full formation of formazan from NBT. This suggests that
M1
Furthermore, the [Ni(SOD )] redox potential of 0.70(2) V
vs Ag/Ag suggests that it may be capable of behaving as
an SOD (Eox
In light of our electrochemical data, we investigated the
ability of [Ni(SOD )] to disproportionate superoxide in
aqueous solutions. We first examined the superoxide de-
composition products following the addition KO to solutions
2
of [Ni (SOD )]. Addition of solid KO to [Ni (SOD )]
2
results in the formation of bubbles, indicating the production
of dioxygen. Once bubbling had ceased, we assayed the
M1
[
Ni(SOD )] is capable of disproportionating up to 40 000
+
•-
equiv of O
2
before it can no longer offer NBT protection
O2•-
O2•-
+
) 0.04 V; Ered
) 1.09 V vs Ag/Ag ).
from oxidative damage. We note that neither the apopeptide
M1
SOD nor NiCl
2
will offer NBT protection from KO
2
.
M1
We also investigated the ability of two NiN
2 2
S
complexes
II
II
19,20
(
[Ni (bmedach)] (1) and [Ni (emi)](Et
4 2
N) (2), Scheme 2)
to protect NBT from KO . Neither 1 nor 2 displays SOD
2
II
M1
II
M1
activity under the conditions employed in the above experi-
ments; NBT was rapidly and fully converted to formazan
18
after the addition of 1400 equiv of KO
2
in each case. The
solution for H
2
O
2
II
content. For every mole of KO
2
added to
•-
M1
differences in the O
the two NiN
2
reactivity between [Ni(SOD )] and
M1
a solution of [Ni (SOD )] ∼0.5 mol of H
2 2
O are produced,
S
2 2
complexes investigated can be rationalized
M1
indicating that [Ni(SOD )] might be behaving as an SOD
M1
in terms of redox potential differences. [Ni(SOD )] has a
redox potential that is within the range necessary for SOD
activity. Both 1 and 2 do not have an appropriate redox
(
(
as opposed to a superoxide reductase). To address if [Ni-
SOD )] is truly catalyzing the disproportionation of O
we utilized the para-nitro blue tetrazolium chloride (NBT)/
M1
•-
2
II
potential to act as an SOD. Bis-amine-ligated 1 has a Ni /
5
a,14
formazan assay.
NBT reacts with O
max ≈ 580 nm (∼35 000 M cm )). The addition of 1400
equiv of KO to a 33 µM NBT solution produced an intense
III
+ 20
Ni redox potential (E1/2 > 1.2 V vs Ag/Ag ) far too
positive to reduce superoxide, while bis-amide-ligated 2 will
•-
2
forming the blue pigment formazan
-1
-1
(
λ
•-
III
be incapable of oxidizing O
2
after accessing the Ni
18
2
oxidation state (E1/2 ) -0.40 V vs Ag/Ag+).19 Therefore,
blue color with a band centered at 580 nm in the UV/vis. In
contrast, when the same reaction is performed with [Ni-
SOD )] (10 µM) present, formazan is not produced. This
suggests that [Ni(SOD ) is behaving as an SOD, protecting
NBT from oxidative damage. The fact that [Ni(SOD )]
displays SOD activity is surprising in light of our failed
attempts at preparing the Ni form of the maquette and
indicates that [Ni (SOD )] is at least quasi-stable. Prelimi-
both small-molecule NiN complexes are incapable of
2 2
S
acting as SODs on the basis of redox potential arguments.
M1
(
Altogether, these reactivity results suggest that the Ni-
M1
amine amide
(
S
2
N
N
) structural motif is electrochemically opti-
M1
mized to allow for Ni to display SOD activity.
III
Acknowledgment. We thank Prof. B. J. Frost and Ms.
III
M1
J. L. Irwin (University of Nevada, Reno) for supplying a
II
M1
II
nary UV/vis studies indicate that, although [Ni (SOD )]
will rapidly decompose following the addition of KO , it
sample of [Ni (bmedach)]. Financial support was provided
by the University of Nevada, Reno (Junior Faculty Research
Award). Use of the National Synchrotron Light Source,
Brookhaven National Laboratory, was supported by the U.S.
Department of Energy, Office of Science, Office of Basic
Energy Sciences, under Contract No. DE-AC02-98CH10886.
2
does not completely decompose until ∼90 s has passed. This
M1
indicates that [Ni(SOD )] will survive in solution long
•
-
enough to catalytically disproportionate O
2
.
(
18) Per mole of NBT.
19) Kr u¨ ger, H.-J.; Peng, G.; Holm, R. H. Inorg. Chem. 1991, 30, 734-
42.
(
Supporting Information Available: Experimental details;
7
M1
M1
analytical data for SOD and [Ni(SOD )]; and SOD activity
assays. This material is available free of charge via the Internet at
http://pubs.acs.org.
(
20) (a) Smee, J. J.; Miller, M. L.; Grapperhaus, C. A.; Reibenspies, J. H.;
Darensbourg, M. Y. Inorg. Chem. 2001, 40, 3601-3605. (b) We do
II
not observe reversible electrochemical behavior for [Ni (bmedach)]
in DMF/water solutions (0.1 M NaCl) over the scan range of -0.1 to
1.2 V vs Ag/AgCl.
IC0514344
2360 Inorganic Chemistry, Vol. 45, No. 6, 2006