Metal-binding and redox properties of substituted linear and cyclic ATCUN motifs

Article abstract of DOI:10.1016/j.jinorgbio.2014.06.004

The amino-terminal copper and nickel binding (ATCUN) motif is a short peptide sequence found in human serum albumin and other proteins. Synthetic ATCUN-metal complexes have been used to oxidatively cleave proteins and DNA, cross-link proteins, and damage cancer cells. The ATCUN motif consists of a tripeptide that coordinates Cu(II) and Ni(II) ions in a square planar geometry, anchored by chelation sites at the N-terminal amine, histidine imidazole and two backbone amides. Many studies have shown that the histidine is required for tight binding and square planar geometry. Previously, we showed that macrocyclization of the ATCUN motif can lead to high-affinity binding with altered metal ion selectivity and enhanced Cu(II)/Cu(III) redox cycling (Inorg. Chem. 2013, 52, 2729-2735). In this work, we synthesize and characterize several linear and cyclic ATCUN variants to explore how substitutions at the histidine alter the metal-binding and catalytic properties. UV-visible spectroscopy, EPR spectroscopy and mass spectrometry indicate that cyclization can promote the formation of ATCUN-like complexes even in the absence of imidazole. We also report several novel ATCUN-like complexes and quantify their redox properties. These findings further demonstrate the effects of conformational constraints on short, metal-binding peptides, and also provide novel redox-active metallopeptides suitable for testing as catalysts for stereoselective or regioselective oxidation reactions.

Full text of DOI:10.1016/j.jinorgbio.2014.06.004

Journal of Inorganic Biochemistry 139 (2014) 65–76
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Metal-binding and redox properties of substituted linear and cyclic
ATCUN motifs
⁎
Kosh P. Neupane, Amanda R. Aldous, Joshua A. Kritzer
Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, MA 02155, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 April 2014
Received in revised form 29 May 2014
Accepted 2 June 2014
Available online 12 June 2014
The amino-terminal copper and nickel binding (ATCUN) motif is a short peptide sequence found in human serum
albumin and other proteins. Synthetic ATCUN–metal complexes have been used to oxidatively cleave proteins
and DNA, cross-link proteins, and damage cancer cells. The ATCUN motif consists of a tripeptide that coordinates
Cu(II) and Ni(II) ions in a square planar geometry, anchored by chelation sites at the N-terminal amine, histidine
imidazole and two backbone amides. Many studies have shown that the histidine is required for tight binding
and square planar geometry. Previously, we showed that macrocyclization of the ATCUN motif can lead to
high-affinity binding with altered metal ion selectivity and enhanced Cu(II)/Cu(III) redox cycling (Inorg. Chem.
2013, 52, 2729–2735). In this work, we synthesize and characterize several linear and cyclic ATCUN variants to
explore how substitutions at the histidine alter the metal-binding and catalytic properties. UV–visible spectros-
copy, EPR spectroscopy and mass spectrometry indicate that cyclization can promote the formation of ATCUN-
like complexes even in the absence of imidazole. We also report several novel ATCUN-like complexes and
quantify their redox properties. These findings further demonstrate the effects of conformational constraints
on short, metal-binding peptides, and also provide novel redox-active metallopeptides suitable for testing as
catalysts for stereoselective or regioselective oxidation reactions.
Keywords:
ATCUN
Catalysis
Copper
Cyclic peptides
Metallopeptides
Nickel
© 2014 Elsevier Inc. All rights reserved.
1. Introduction
Despite the importance of the imidazole, the ATCUN histidine has
been substituted with other amino acids in order to design altered
The amino-terminal Cu- and Ni-binding (ATCUN) motif is a sequence
derived from the N-termini of mammalian serum albumins and other
proteins. ATCUN motifs are tripeptides with a free N-terminus and histi-
dine in the third position, and these motifs bind several divalent transi-
tion metal ions with high affinity [1]. Cu(II) and Ni(II) complexes of
ATCUN peptides have been used as oxidation catalysts for site-specific
DNA cleavage and protein cleavage [1–13], protein–protein cross linking
[14–16], enzyme inhibitors [17,18], and metallodrugs [19–21]. ATCUN
motifs bind Cu(II) or Ni(II) in a square planar geometry using nitrogens
from the N-terminal amine, imidazole, and two backbone amides. The
imidazole and the amine are the major contributors to the high thermo-
dynamic stability of ATCUN–metal complexes [22]. Amino acid substitu-
tions at the first and second positions can affect DNA cleavage and other
intermolecular processes, but have more modest effects on affinity,
stoichiometry, and redox characteristics of the peptide–metal complex
[11,23]. Adding additional histidines at the first [24] or second position
[25] reduces the binding affinity and alters the coordination geometry
of metal complexes, demonstrating that imidazole positioning within
short peptides has an overriding effect on metal complexation.
metal–peptide complexes. Several studies have described complexes
between tripeptides and metal ions that are mediated through the
N-terminal amine, amide nitrogens, backbone carbonyl oxygens, the
carboxy terminal group, and/or various non-imidazole side chains. For
instance, Arg-Lys-Asp binds Cu(II) and Ni(II), though only at more alka-
line conditions than Gly-Gly-His [26,27]. Similar results were observed
for Gly-Gly-Cys and Ala-Ala-Cys [28,29]. Spectroscopic and potentio-
metric studies using Gly-Gly-Met suggested that the thioether of Met
is not the primary binding site for 3d transition metal ions due to the
rapid oxygenation of sulfur [22,30]. Ultimately, most of these tripeptide
ligands do not form 1:1, square planar complexes with divalent metal
ions (an “ATCUN-like complex”). Instead, the intrinsic flexibility of the
peptide backbone and the lack of the strongly chelating imidazole
group allow other binding modes to compete with the square planar,
1:1 complex characteristic of high-affinity ATCUN motifs.
While complexes between linear peptides and metals have been
broadly explored, there are fewer studies on metal binding by designed
cyclic peptides [22,31–37]. Macrocyclization has powerful effects on
metal-binding behavior, and the design of cyclic ligands has been re-
ported for selective metal ion recognition, ion transport, metalloenzyme
modeling, catalysis, MRI contrast agents, luminescence probes, and car-
riers for drug delivery [38–44]. We recently reported macrocyclization
of the ATCUN motif in a manner that maintains a high-affinity complex
⁎
Corresponding author. Tel.: +1 617 627 0451; fax: +1 617 627 3443.
E-mail address: joshua.kritzer@tufts.edu (J.A. Kritzer).
http://dx.doi.org/10.1016/j.jinorgbio.2014.06.004
0162-0134/© 2014 Elsevier Inc. All rights reserved.

66
K.P. Neupane et al. / Journal of Inorganic Biochemistry 139 (2014) 65–76
Scheme 1. Structures of linear and cyclic ATCUN peptides. Linear peptides used in this study include GGHL, GGDL, GGXL, GGCL, GGhCL, and GGML, where X represents 2-pyridylalanine and
hC represents homocysteine. Cyclic peptides used in this study include peptide 1 containing D-His, 1D with D-Asp, 1X with D-Pal, 1C with D-Cys, 1hC with D-hCys, and 1M with D-Met.
Cyclic peptides used D-amino acids whereas linear analogs used L-amino acids at the same position. In prior work, we showed that the glycines' lack of chirality makes L- and D-amino
acids interchangeable within these short, linear tetrapeptides [45]. Structures are shown as the predominant species at pH 7.0. Donor atoms are shown in blue boldface. (For interpretation
of the references to color in this scheme legend, the reader is referred to the web version of this article.)
with Cu(II) or Ni(II) [45]. By characterizing several diastereomers and
linear analogs, we demonstrated that the binding of the macrocyclic
ATCUN peptide (peptide 1, shown in Scheme 1) to Cu(II) and Ni(II)
was altered due to its cyclic structure. Considering the limitations of
non-imidazole-containing, linear tripeptides as metal ligands, we hy-
pothesized that the cyclic scaffold could enforce the square planar, 1:1
complex even in the absence of the imidazole group. This would allow
direct substitution of other metal-binding side chains in order to pro-
duce metallopeptides with unique metal-binding selectivities and
redox properties.
Peptide 1 consists of the ATCUN motif Lys-Asp-D-His (where D-His
refers to the D-enantiomer of natural L-histidine) with two modifica-
tions. First, the side-chain of Lys is linked via amide bond to the C-
terminus of the D-His. Second, the side-chain of the Asp is linked via
amide bond to an amide-capped Leu residue to provide for a linkage
to solid-phase synthesis resin and to allow for reverse-phase HPLC puri-
fication. Peptide 1 binds Cu(II) and Ni(II) in an ATCUN-like, 1:1, square
planar complex with unique metal-binding and redox properties [45].
In this article, we expand this class of macrocyclic metal ligands by
synthesizing and characterizing analogs that substitute the D-His of 1
with D-aspartate (D-Asp), D-pyridylalanine (D-Pal), D-cysteine (D-Cys),
D-homocysteine (D-hCys), and D-methionine (D-Met) (Scheme 1). We
synthesized linear and cyclic versions of each peptide in order to com-
pare the roles of His within linear and cyclic metallopeptides, to explore
how other ligand sets bind Cu(II) and Ni(II), and to produce a diverse set
of metal–peptide complexes for use in ongoing catalysis investigations.
OH, Fmoc-DHcy(Trt)-OH, and Fmoc-DMet-OH. Diisopropylethylamine
(DIPEA), trifluoroacetic acid (TFA), acetic anhydride, Pd(PPh3)4, 2′,7′-
dichlorofluorescein diacetate, hydrogen peroxide, CuCl2 and NiCl2 were
purchased from Sigma-Aldrich. N,N-dimethylformamide (DMF) and ace-
tonitrile were purchased from VWR. DTT was purchased from Ultra Pure.
All chemicals were used as received without any further purification.
2.2. Peptide synthesis and purification
Peptides were synthesized manually using 25 mL reaction vessels
and wrist shakers using standard Fmoc-based protection strategies on
Rink amide methylbenzhydrylamine (MBHA) resin (0.20 mmol scale)
with PyBop/HOBt/DIPEA coupling conditions [46]. Linear and cyclic
peptides were synthesized as previously reported [45]. For linear pep-
tides, Fmoc was deprotected with 20% piperidine, but for cyclic peptides
attached to the resin through Leu-Asp linkages, 20% piperidine was ob-
served to promote aspartimide formation. Aspartimide formation was
minimized through screening of deprotection conditions. Briefly, a
small aliquot of resin was cleaved (TFA/TIPS/H2O), ether precipitated,
and analyzed by ESI-MS (electrospray ionization-mass spectrometry)
and HPLC to quantitate aspartimide formation. For cyclic peptides,
Fmoc removal with 5% piperazine and 0.1 M HOBt (5 min, twice at
room temperature) limited aspartimide formation to 10–15%. After syn-
thesis of the tetrapeptide, the allyl-protected C-terminus of Asp was
deprotected using [Pd(PPh3)4/PhSiH3] (0.25:10 eq) in dry DCM for
2 h. In order to remove Pd(PPh3)4, the resin was washed with sodium
N,N-diethyldithiocarbamate (0.5% w/v in DMF, 5 × 5 min) [47]. The pep-
tides were then cyclized using PyBOP/HOBt/DIEA (5:5:10 eq) for 2 h.
Peptides were cleaved off the resin using TFA/TIPS/H2O (95:3:2) or
TFA/TIPS/EDT/H2O (92:3:3:2) for thiol-containing peptides. The volume
was reduced by evaporation and peptides were ether-precipitated.
Crude peptides were dissolved in 3–5 mL of 50:50 water:acetonitrile.
Thiol-containing crude peptides were reduced by dithiothreitol (DTT)
at this stage, prior to HPLC purification. Peptides were purified using
reverse-phase HPLC on a preparative-scale C8 or C18 column (solvent
A: water/0.1% TFA, solvent B: acetonitrile/0.1% TFA, linear gradient of
5–40% solvent B over 20 min was used). The fraction containing desired
product was collected and lyophilized. The purities and identities of the
peptides were assessed by analytical HPLC (linear gradient: 5–40% B
2. Experimental
2.1. Materials
N-α-(9-fluorenyl methyloxycarbonyl) (Fmoc) protected amino acids,
MBHA Rink Amide resin, N-hydroxybenzotriazole (HOBt), and
Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(PyBop) were purchased from AnaSpec. Protected amino acids used in
this work included Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Pal-OH, Fmoc-Cys(Trt)-OH, Fmoc-Hcy(Trt)-
OH, Fmoc-Met-OH, Fmoc-Asp-OAll, Boc-Lys(Fmoc)-OH, Fmoc-
DHis(Trt)-OH, Fmoc-DAsp(OtBu)-OH, Fmoc-DPal-OH, Fmoc-DCys(Trt)-

K.P. Neupane et al. / Journal of Inorganic Biochemistry 139 (2014) 65–76
67
over 30 min) and ESI-MS. HPLC retention times and ESI-MS data for apo-
2.6. Fluorescence assay for detection of hydroxyl radicals
peptides, Cu(II)–peptide complexes, and Ni(II)–peptide complexes are
given in Table S1 (supporting information).
Concentrated stocks of 0.9 mM peptide–metal complexes were
prepared using 1 mM peptide and 0.9 eq of CuCl2 or NiCl2, in order to
ensure complete complexation of metal ion. Thus, following dilution, as-
says included final concentrations of 0.9 μM metal(II)–peptide complex
with 0.1 μM apo-peptide (as shown below, all apo-peptides were shown
to have no effect). Metal salts without peptide were tested at 0.9 µM.
Assays also included 0.9 mM H2O2 and 100 mM NaCl in 20 mM 2-[4-
(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), pH 8.5
or 9.5. Solutions were incubated at room temperature for 60 min, after
which 10 μM 2′,7′-dichlorofluorescein diacetate was added. After an ad-
ditional 15 minute incubation, total fluorescence intensity was read in
96-well plates using a TECAN infiniTE 200 plate reader with excitation
at 485 nm and emission at 535.
2.3. pH dependence of metal binding and metal binding stoichiometry
Each lyophilized peptide was freshly dissolved in deionized Milli-Q
water (≥18 MΩ cm−1) purged with argon prior to metal complexation.
Concentrations of thiol-containing peptides were measured by
dithionitrobenzoic acid (DTNB) assay as described [48,49]. Concentra-
tions of other peptides were initially estimated by mass and then calcu-
lated more exactly using metal-binding titrations [50]. To measure the
pH dependence of metal binding, 1.0 mM peptide solutions were pre-
pared and 1.0 eq of metal ion (CuCl2 or NiCl2) was added to the solution.
The pH of the resulting solution was lowered to roughly 2.5–3.0 using
dilute HCl. UV–vis spectroscopy (Cary 100, Agilent) was used to verify
that no metal complexation occurred at this low starting pH. As small al-
iquots of dilute KOH were slowly added to the solution, pH was mea-
sured using a microelectrode (3 mm, Mettler Toledo) and absorption
spectra were recorded. d–d transition bands near 525 and 425 nm
were observed for ATCUN-like Cu(II)–peptide and Ni(II)–peptide
complexes, respectively. KOH was added until a saturation point was
observed. For plotting pH dependence curves, the absorption was nor-
malized to unity at the upper bound, and percent formation of each
metallopeptide complex was plotted against pH.
For titrations at constant pH to determine metal-binding stoichiome-
try, 1.0 mM peptide solution was prepared in 50 mM N-ethylmorpholine
(NEM) buffer at appropriate pH. Background absorption due to the pep-
tide was normalized to zero, and 0.2 eq of CuCl2 or NiCl2 was added from
a 200 mM aqueous stock solution. The samples were mixed well and ab-
sorption spectra were recorded. The titration was repeated until there
was no further change in absorbance other than scattering due to forma-
tion of metal-hydroxide precipitate.
3. Results and discussion
3.1. UV–visible (UV-vis) spectroscopy of complexes between Cu(II) and
linear peptides
For these peptide ligands, formation of the metal complex involves
competition between protonation and metal coordination at several
donor atoms. The more stable the metal–peptide complex, the lower
the pH at which it can form [51]. In the past, the pH at which 50% of
metal ions are complexed (or precipitated, in cases for which free
metal ions precipitate) has been used to judge the relative stabilities
of related complexes [51]. In this work, we use pH50 (the pH at which
50% of the metal ions are complexed) to assess complex stability in a
semiquantitative manner.
Peptide–metal complexes were initially characterized by UV–vis
spectroscopy as pH was gradually raised from 2.5. Fig. 1A shows select-
ed UV–vis spectra and pH dependencies of Cu(II) binding to linear pep-
tides Gly-Gly-His-Leu (GGHL), Gly-Gly-Asp-Leu (GGDL), Gly-Gly-Pal-
Leu (GGXL), Gly-Gly-Cys-Leu (GGCL), Gly-Gly-hCys-Leu (GGhCL), and
Gly-Gly-Met-Leu (GGML). As pH was raised, a discrete, two-state tran-
sition between the aqua complex (λmax near 800 nm, observed at low
pH) and a complex with λmax at 530 to 545 nm was observed for
GGHL, GGXL, and GGDL (Fig. 1B and Fig. S1). The d–d transition band
at 530–545 nm is consistent with the formation of a square-planar
complex with an N4 or N3O donor atom set, and the wavelengths, inten-
sities, and cooperative transitions are all identical to classical ATCUN
motifs [1,27,52–55]. This led us to conclude that GGDL and GGXL form
ATCUN-like complexes with Cu(II).
2.4. EPR spectroscopy
Fresh Cu(II)–peptide complexes (0.9 mM CuCl2 and 1.0 mM peptide
in water with 10% glycerol) were prepared at the specified pH by adding
small aliquots of dilute KOH/HCl. These were transferred into capillary
tubes and inserted into a quartz EPR tube, then slowly frozen in liquid
nitrogen. X-band EPR data were recorded using a Bruker EMX instru-
ment at a microwave frequency of 9.32 GHz. All spectra were recorded
at −150 °C (123 K) using microwave power of 0.64 mW and modula-
tion frequency of 100 kHz. Other instrumental parameters include a
sweep width of 1500 G (2250 to 3750 G) for a total of 1024 data points,
time constant 655.36 ms, conversion time 163.84 ms, sweep time
167.77 s, and receiver gain 1 × 104 to 2 × 104. All spectra were average
of 5 scans.
For the sulfur-containing linear peptides GGCL, GGhCL, and GGML,
more complicated behavior was observed above pH 5.0 consistent
with the formation of different metal–peptide complexes. Since d–d
transition bands cannot reliably distinguish among N, O or S coordina-
tion to Cu(II), we also examined the charge transfer bands at lower
wavelengths for sulfur-containing peptides. N → Cu(II) and O → Cu(II)
charge transfers produce a characteristic band at 260–270 nm,
while S → Cu(II) charge transfer produces a characteristic band at
290–330 nm [28,29]. For GGCL, Cu(II) binding to the cysteine thiolate
starts near pH 4.6, as indicated by a shoulder near 350 nm due to the
S → Cu(II) transfer band (Fig. 1C). Between pH 4.6 and 5.2 a d–d transi-
tion band at 570 nm (amine, thiolate, and two H2O ligand set) appears,
then gradually shifts to 540 nm (amine, thiolate, amide and H2O
ligand set) when pH is increased up to 5.5. At pH 6.5, a band centered
at 517 nm is observed due to the amine, two amide and thiolate ligand
set, which is the ATCUN-like configuration for this peptide. These inter-
pretations are consistent with known absorption bands for these ligand
sets [56,57], and are supported by EPR data (see below). The d–d transi-
tion band centered at 517 nm appears at all pHs above 6.5. At higher pH,
the S → Cu(II) charge transfer band is more pronounced and shifts from
350 nm to 320 nm (2400 M−1 cm−1) (Fig. 1C).
2.5. Cyclic voltammetry
A standard three-electrode cell (glassy carbon electrode as a working
electrode, platinum wire as an auxiliary electrode, and saturated calomel
electrode as a reference electrode) was used to perform the electrochem-
ical measurements on a CHI830 Electrochemical Workstation (CH
Instruments Inc., USA). All metallopeptide samples were prepared
freshly in degassed water and 200 mM KCl was added as supporting
electrolyte. The pH was adjusted as required with KOH and HCl. The
sample was purged with nitrogen gas for 5 min before data collection.
Scan velocity was 100 mV/s for each scan. Cyclic voltammograms
presented are the average of three scans that were then background-
subtracted. The half-wave potential (E1/2) was determined from the
expression E1/2 = (Eap + Ecp) / 2.

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K.P. Neupane et al. / Journal of Inorganic Biochemistry 139 (2014) 65–76
Fig. 1. UV–vis spectroscopy of Cu(II) binding to linear peptides. (A) Percentage formation of Cu(II)–peptide complexes versus pH. (B) UV–vis spectra of Cu(II) complexes of linear peptides
GGHL, GGDL and GGXL at pH 6.5. Inset shows a magnification of the d–d transition bands near 550 nm. (C) UV–vis spectra at selected pHs for GGCL. (D) UV–vis spectra at selected pHs for
GGhCL. (E) UV–vis spectra at selected pHs for GGML. For (C–E), the insets show a magnification of the d–d transition bands between 550 and 800 nm. (F) pH dependencies of the wave-
length of maximal intensity (λmax) for sulfur-containing peptides, demonstrating the presence of multiple metal–peptide complexes at intermediate pHs.
Cu(II) binding to GGhCL also starts at pH N 4.5, but the S → Cu(II)
charge transfer band near 325 nm only starts appearing at pH near 7.0
(Fig. 1D). This shows that homocysteine is less able to coordinate
Cu(II) in the context of the ATCUN-like architecture compared to cyste-
ine. A band centered at 625 nm was observed at pH near 6.0, and this
band persisted as the more characteristic 515 nm band appeared near
pH 7.4. The 625 nm band gradually diminished through pH 10.0. At
this high pH, both the 515 nm band and the 325 nm band intensified, in-
dicating the dominance of a discrete N3S-coordinated Cu(II) complex
(likely the ATCUN-like, square-planar complex). For Cu(II) complexa-
tion with GGML (Fig. 1E), the d–d transition bands and S → Cu(II)
charge transfer bands are somewhat similar to those observed for
GGhCL. One important difference is that the band at 625 nm is promi-
nent only up to a pH of 6.0, and by 7.6 the spectrum is dominated by a
band at 545 nm. In agreement with the lower pH transition observed
for GGML, the S → Cu(II) charge transfer band at 295 is more prominent
at pH 6.0 for GGML compared to GGhCL.
Overall, in contrast to the discrete, two-state transitions observed for
GGHL, GGDL and GGXL, all three sulfur-containing linear peptides have
a variety of intermediate complexes below neutral pH and oxidation
products at basic pH (Figs. S2–S4). All three sulfur-containing linear
peptides showed an absorption band at roughly 625–650 nm at mid-
range pH values, either as a very minor contributor (GGCL) or a signifi-
cant contributor to the UV–vis spectrum (GGhCL and GGML) (Fig. 1F).
Based on prior results and on additional EPR data (see below),
the 625–650 nm band may arise from Cu(II) coordinated to the
N-terminal amine, the sulfur, one amide nitrogen, and one oxygen
from a carbonyl group or water [58]. Hydroxide or chloride coordina-
tion at this fourth site also cannot be ruled out. The 545 nm band ob-
served for GGML is most likely the ATCUN-like Cu(II) complex with
the N-terminal amine, two amide nitrogens and the thioether as the li-
gand set, an interpretation supported by the observed charge transition
band, our own EPR data (see below), and by other observations of short,
methionine-containing peptides [59].

K.P. Neupane et al. / Journal of Inorganic Biochemistry 139 (2014) 65–76
69
3.2. UV–vis spectroscopy of complexes between Cu(II) and cyclic peptides
characteristics as GGHL, the cyclic analogs containing Asp and Pal (1D
and 1X) differ greatly from the His-containing peptide 1. 1D and 1X
have an initial pH transition from the aqua complex (d–d transition
band at 800 nm) to another complex with a λmax at 550 nm for 1D
and 555 nm for 1X. This band matches the typical ATCUN–Cu(II) square
planar complex, and EPR data (see below) support the conclusion that
this represents a square-planar complex with the Cu(II) coordinated
by the N-terminal amine, two amide nitrogens, and the carboxylate
(for 1D) or pyridyl group (for 1X). For both cyclic peptides, this
ATCUN-like complex is the predominant species at pH 8.6 to 8.8, but
at higher pH values a blue-shift is observed to a band near 500 nm.
This is not observed for peptide 1 (Figs. S5–S7).
Cyclic peptides with sulfur-containing coordination groups have
similarly complicated Cu(II)-binding behavior, but for 1C, 1hC, and 1M
the additional information from S → Cu(II) charge transfer bands al-
lows further insight (Fig. S11). Specifically, the thiol and thioether
groups are not involved in Cu(II) coordination until pH approaches
We previously reported that cyclic peptide 1 binds Cu(II) using the
amine, imidazole, and two amide nitrogens in a square planar geometry
at neutral pH [45]. Cyclic analogs of 1 that replace the imidazole with a
carboxylic acid (1D), pyridine (1X), thiol (1C and 1hC) or methyl
thioether group (1M) were each prepared, and Cu(II) complexes of
these cyclic peptides were characterized by UV–vis spectroscopy. All
these cyclic peptides show d–d transition bands (and, for sulfur-
containing peptides, S → Cu(II) charge transfer bands) that indicate
Cu(II) complexation between pH 4.0 and pH 11.5. However, for all cyclic
peptides except 1, several shifts among d–d transition bands between
800 nm (Cu(II)-aqua complex) and 500 nm are observed, indicating
multiple competing complexes at mid-range pH values (Fig. 2 and
Figs. S5–S10).
First, we examined the effects of cyclization within ATCUN peptides
without sulfur. While GGDL and GGXL showed similar Cu(II) binding
Fig. 2. UV–vis spectroscopy of Cu(II) binding to cyclic peptides. Plots show full spectra at selected pHs for 1 (A), 1D (B), 1X (C), 1C (D), 1hC (E), and 1M (F). Insets show the pH dependencies
of the wavelength of maximal intensity (λmax) for Cu(II) complexes with each peptide, demonstrating the presence of multiple metal–peptide complexes at intermediate pHs. For addi-
tional spectra at a wide range of pH values, see Figs. S5–S10 in the supporting information.

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K.P. Neupane et al. / Journal of Inorganic Biochemistry 139 (2014) 65–76
6.0, implying that the Cu(II) complexes with d–d transition bands at
650–700 nm observed near pH 6.0 involve coordination to N-terminal
amines and the side-chain sulfurs. The transition from that complex to
one with a d–d transition band near 550 nm is consistent with depro-
tonation of two amide nitrogens and formation of an ATCUN-like,
square planar complex; EPR data support this conclusion (see below).
At pH above 8.0, a blue-shift to a band near 500 nm is observed for
Cu(II) complexed to 1C, 1hC, and 1M (Figs. S8–S10). Cu(II)–1D and
Cu(II)–1X show similar high-pH shifts, while these shifts are not ob-
served for Cu(II)–1 or for any Cu(II) complexes of linear peptides that
contain His at the third position [45]. This implies that these shifts are
not due to sulfur oxidation, which might be expected at elevated pH,
but are due to some other structural or electronic reason. At higher
pH, it may be possible for coordination to exchange between the non-
imidazole side chain and an amide from the macrocycle linker or the
Leu outside of the macrocyclic ring. A similar switch between Cu(II) co-
ordination by side chains and backbone amides was observed at alkaline
pH in a cyclic peptide with multiple imidazoles, cyclo-(Gly-His-Gly-His-
Gly-His-Gly) [52]. In a different example of the same phenomenon, a
blue shift was observed for the non-ATCUN Cu(II)–GGGH complex at
high pH due to the coordination of a third amide nitrogen to Cu(II)
[60]. Taken together, all these data suggest that alternative ligand sets
become available at higher pH, but that strong coordination by the
amino terminus and the side chain at the third position (the hallmarks
of the ATCUN complex) can thermodynamically favor ATCUN-like com-
plexes even at high pH [1,31,61,62]. Since the blue shift is missing for 1,
minimal for 1X, moderate for 1D, and pronounced for the sulfur-
containing peptides, this suggests that D-Pal and D-Asp behave as
stronger anchoring groups within cyclic ATCUN peptides, but not as
strong as D-His.
Fig. 4. EPR spectra of Cu(II) complexes with linear and cyclic peptides containing Cys, hCys
or Met. The EPR spectra for Cu(II)–GGHL and Cu(II)–1 are shown for comparison. All spec-
tra were recorded using 0.9 mM Cu(II)–peptide complex at 123 K in water with 10%
glycerol.
titrations using UV–vis spectroscopy confirmed saturation of the
ATCUN-like band at 525–550 nm at 1:1 stoichiometry (see Fig. S12). How-
ever, for sulfur-containing linear and cyclic peptides, the Cu(II)-binding
stoichiometry was observed at less than 1:1 due to metal-catalyzed thiol
oxidation; still, all UV–vis spectroscopy, mass spectrometry, and EPR spec-
troscopy data (see below) are consistent with the formation of 1:1 stoichi-
ometries for these Cu(II)–peptide complexes prior to sulfur oxidation.
3.3. Stoichiometry of Cu(II) binding
The stoichiometries of the observed Cu(II)–peptide complexes were
investigated using UV–vis spectroscopy and ESI-MS spectrometry. Stoi-
chiometric titrations were performed at the lowest pH values for which
maximum ATCUN-like binding was observed for each peptide–Cu(II)
complex. For most peptides, ESI-MS revealed masses consistent with
metal-binding complexes with 1:1 stoichiometry (see Table S1) and
3.4. EPR spectroscopy of Cu(II)–peptide complexes
EPR spectroscopy is a useful technique for studying Cu(II)–peptide
complexes with various ligand donor sets in solution. EPR spectra of
Cu(II)–complexes of linear and cyclic peptides at different pHs are
shown in Figs. 3 and 4, with measured EPR parameters summarized in
Table 1. The EPR parameters obtained for all complexes follow the
trend g N g⊥ N ge suggesting that all Cu(II)–peptide complexes have a
dx2
ground state. This is a characteristic feature of square planar
−y2
(D4h), square pyramidal (C4v), or axially elongated tetragonal octahe-
dral geometries with D4h symmetry [63–65]. Thus, the d–d transitions
observed for these complexes are due to the Cu(II) dx2
→ dz2 transi-
−y2
tion. The magnitude of g in four-coordinate Cu(II) complexes is depen-
dent on the nature of the ligand atoms and the coordination geometry
[66]. A detailed study by Peisach and Blumberg using a variety of ligand
sets suggests that g decreases in the series O4 N N2O2 N N3O N N4
N2S2 N S4 [67]. This reflects that the higher the covalency in the
Cu(II)–ligand bond, the more the electron from dx2 delocalizes to-
N
−y2
wards the ligand and thus the less it will interact with the Cu(II) nucle-
us. Also, for a given set of ligands, the g will increase with the degree of
distortion from square planar towards tetragonal or tetrahedral geome-
try. According to these trends, we would expect the g value for the Asp-
containing ligands to be highest, followed by His- and Pal-containing
ligands, followed by Cys-, hCys- and Met-containing ligands. The g
values obtained for the Cu(II) complexes of GGDL (g = 2.20), GGXL
(g = 2.19) and GGHL (g = 2.18) at pH 7.0 followed the Peisach and
Blumberg trend (Fig. 3 and Table 1). In agreement with the electronic
absorption spectra, the low-temperature (123 K) X-band EPR spectra
of the Cu(II)–peptide complexes of GGDL, GGXL, 1D, and 1X are all high-
ly similar to their imidazole-containing analogs GGHL and 1 (Fig. 3 and
Fig. 3. EPR spectra of Cu(II) complexes with linear and cyclic peptides containing
D-His, D-Asp or D-Pal. All spectra were recorded with 0.9 mM Cu(II)–peptide complex
at 123 K in water with 10% glycerol.

K.P. Neupane et al. / Journal of Inorganic Biochemistry 139 (2014) 65–76
71
Table 1
nature of the blue-shifted species, we obtained EPR spectra for these
Cu(II) complexes at pH 11.0. EPR spectra of Cu(II)–1X showed broad-
ened peaks that were difficult to interpret, but EPR spectra of Cu(II)–
1D showed two distinct signals in the g region (Figs. S6 & S7). One
signal had a g = 2.21, and is the same as the ATCUN-like complex ob-
served at lower pH. The second signal has a lower g of 2.18, and could
indicate a square planar, N4 donor set. The fourth nitrogen could arise
from deprotonation of the Leu amide, linker amide, OH− or possibly
from an intermolecular interaction. We did not observe any hydroly-
sis or oxidation of these non-thiol-containing peptides at any pH
(Fig. S13).
Next, we examined EPR spectra of thiol-containing Cu(II)–peptide
complexes, which showed multiple species during pH titrations. The
EPR spectra of Cu(II)–peptide complexes of GGCL, 1C, GGhCL, 1hC,
GGML, and 1M are overlayed in Fig. 4. The EPR spectra of Cu(II) bound
to each of these sulfur-containing linear and cyclic peptides are all sim-
ilar except for Cu(II)–GGML, suggesting similar coordination environ-
ments. From the electronic absorption spectra, it was evident that
thiols in GGCL and GGhCL start to coordinate Cu(II) at pH 4.6, and are
fully engaged in Cu(II) binding at pH 9.0. For these complexes, the g
region of the EPR spectrum clearly shows a single species at pH 7.0
with g = 2.22 and A between 179 and 182 × 10−4 cm−1. The ratio of
g /A , denoted f, is a parameter that is empirically correlated with the co-
ordination environment of the Cu(II) complex, with the range of 105–
120 cm for square planar, 130–150 cm for slight-to-moderate distortion
from square planar, and 180–250 cm for a large distortion [66]. The f
values obtained for Cu(II)–GGCL and Cu(II)–GGhCL are 122–124 cm,
which indicate that the geometry for these complexes is nearly square
planar. The g = 2.21 and A = 186 × 10−4 cm−1 values observed for
Cu(II)–1C and Cu(II)–1hC at neutral pH are slightly different from the
analogous linear peptide complexes. The corresponding value of f =
119 cm suggests that cyclization enforces a planar geometry for
cysteine- and homocysteine-containing, ATCUN-like peptides.
EPR data of Cu(II)–linear and Cu(II)–cyclic peptide complexes. Multiple entries indicate
the presence of multiple sets of peaks.
Cu(II)–peptide
pH
7.0
7.5
7.0
8.5
11.0
g
A ×10−4 (cm−1
)
f (g /A ) (cm)
GGHL
1
GGDL
1D
2.18
2.18
2.20
2.21
2.21
2.18
2.19
2.19
2.18
2.22
2.22
2.16
2.22
2.16
2.21
2.24
2.21
2.18
2.22
2.22
2.15
2.15
2.21
2.24
2.21
2.17
2.32
2.22
2.32
2.17
2.20
2.21
2.20
2.18
202
204
204
193
194
198
204
197
204
179
179
204
204
204
186
195
186
194
182
182
191
191
186
196
186
201
146
197
146
201
188
204
188
196
108
107
108
115
114
110
107
112
107
124
124
106
124
106
119
115
119
112
122
122
113
112
119
115
119
108
159
113
159
108
117
108
117
112
GGXL
1X
7.0
8.5
11.0
7.0
GGCL
8.5
12.0
1C
7.0
12.0
GGhCL
1hC
7.0
9.5
12.0
7.0
12.0
GGML
1M
6.0
8.0
7.5
11.0
When pH was raised to 8.5 or higher, an additional Cu(II) complex
was observed by UV–vis spectroscopy (Table 1 and Figs. S8–S10).
Cu(II) and Ni(II) can catalyze sulfur oxidation in short peptides contain-
ing thiol or thioether groups, so we suspected these represented com-
plexes between Cu(II) and oxidized peptides [68,69]. The two broad
EPR signals observed at higher pHs suggest that these oxidation prod-
ucts still form complexes with Cu(II), which would be expected for
sulfoxo species or for disulfide-bridged dimers [70]. Analytical HPLC
and ESI-MS analysis of Cu(II)–GGCL and Cu(II)–GGhCL at pH 9.0 clearly
showed species with oxygenated sulfurs (~40% sulfoxide and sulfone),
with ~40% covalent dimers (Fig. S13). All together, the spectroscopic
and mass spectrometric data show that the thiol-containing linear and
cyclic peptides form ATCUN-like complexes, but that Cu(II) can irrevers-
ibly oxidize the thiol, especially at pHs above 8.5. These findings validate
the ability to switch out the imidazole for a thiol group within the cyclic
Table 1). As expected, g for Cu(II)–1D (g = 2.21) at pH 8.5 is slightly
higher than Cu(II)–1 (g = 2.18) and Cu(II)–1X (g = 2.19). The EPR
data suggest that cyclic ATCUN motifs with His and Pal (1 and 1X)
form square-planar Cu(II) complexes nearly identical to those formed
by their linear analogs.
For sulfur-containing linear and cyclic peptides, we would expect
lower g values compared to peptides containing His, Asp or Pal. Howev-
er, higher g values were observed at pH 7.0 to 7.5, indicating that the
complexes may be distorted from square-planar geometry, or that the
thiol is oxidized, and/or that a coordination site is occupied by a
water, hydroxide, or chloride.
As noted above, the electronic absorption spectra indicated a shift to
a different species at elevated pH for Cu(II) complexes with 1D and 1X,
but not with 1 or with linear analog GGDL or GGXL. To investigate the
Fig. 5. pH dependence of Ni(II) binding to linear peptides (A) and UV–vis spectra of Ni(II)-linear peptide complexes (B). Percentage formation was calculated using absorption at 530 nm
for GGHL, 545 nm for GGDL, 535 nm for GGXL, 517 nm for GGCL, 515 nm for GGhCL, and 545 nm for GGML. Additional full spectra at different pHs are shown in Fig. S14.

72
K.P. Neupane et al. / Journal of Inorganic Biochemistry 139 (2014) 65–76
Table 2
UV–vis data for Cu(II) and Ni(II) binding to linear and cyclic peptides.
Name
Sequence (linear peptides)
Cu(II) binding
pH50
Ni(II) binding
pH50
λ
max (ε, M−1 cm−1
)
λ )
max (ε, M−1 cm−1
GGHL
GGDL
GGXL
GGCL
GGhCL
GGML
Gly-Gly-His-Leu
Gly-Gly-Asp-Leu
Gly-Gly-Pal-Leu
Gly-Gly-Cys-Leu
Gly-Gly-hCys-Leu
Gly-Gly-Met-Leu
4.5
5.5
4.5
6.5
7.5
6.2
530 (95)
6.0
7.2
6.4
6.4
6.4
8.3
425 (122)
442 (220)
427 (150)
427 (290)
440 (115)
420 (175)
545 (130)
535 (100)
517 (200)
515 (150)
545 (120)
Name
Sequence (cyclic peptides)
Cu(II) binding
pH50
Ni(II) binding
pH50
λ
max (ε, M−1 cm−1
)
λ )
max (ε, M−1 cm−1
1
Lys-Asp(Leu)-D-His
Lys-Asp(Leu)-D-Asp
Lys-Asp(Leu)-D-Pal
Lys-Asp(Leu)-D-Cys
Lys-Asp(Leu)-D-hCys
Lys-Asp(Leu)-D-Met
6.0
525 (98)
570 (84)
555 (87)
590 (47)
590 (47)
570 (62)
8.8
–
430 (98)
–
1D
1X
1C
1hC
1M
6.6a
6.4a
6.3a
6.3a
6.5a
−
−
7.4
7.4
−
420 (875)
435 (490)
−
a
Indicates there are at least two species present at this pH.
scaffold, but will complicate future applications with thiol-containing
cyclic peptides.
a discrete, two-state transition from a Ni(II)-aqua complex to a species
with a d–d transition band between 420 and 442 nm. As shown in
Fig. 5A, the pH profiles showed that Ni(II) binding is most favorable
for the imidazole-containing ATCUN sequence GGHL, which has a
pH50 of 6.0 (50% complexation at pH 6.0). Binding of Ni(II) to GGCL,
GGhCL, and GGXL is also observed at relatively low pHs, with pH50
values near 6.4 and complete binding by pH 7.2. pH50 values were 7.2
for Ni(II)–GGDL and pH 8.3 for Ni(II)–GGML (Fig. 5A, Table 2), and the
electronic absorption bands observed for these complexes are very sim-
ilar to values reported in the literature for analogous peptides [1,28,29,
59]. For all sulfur-containing Ni(II)–peptide complexes, electronic
transition bands at 255 nm (ε = ~4300 M−1 cm−1, N−, Sσ → Ni(II))
and two d–d transition bands at 420 to 440 nm (dxy →dx2−y2 ) and 500
to 550 nm (dzx,dyz →dx2−y2 ) are observed (Table 2) [29]. The ligand-
to-metal charge transfer band characteristic for Sπ → Ni(II) is not very
distinct for the linear peptides, probably due to the overlap with the
O/N− → Ni(II) transition. Finally, not only do all linear peptides form
ATCUN-like complexes with Ni(II), but also Ni(II) complexes with
thiol-containing linear peptides are observed to be quite stable for
hours at neutral pH, with no sulfur oxidation products as observed for
Cu(II) complexes.
Next, we examined the EPR spectra for Cu(II)–GGML to better un-
derstand the several species observed in the electronic absorption spec-
tra. At pH 6.0, there are at least two species of the Cu(II)–GGML complex
(Figs. 4 and S4, Table 1). One species has EPR parameters that correlate
well to values for Cu(II)–N2O2 complexes or highly distorted Cu(II)–
NSO2 complexes (g = 2.32, A =146 × 10−4 cm−1 and f = 159), and
the other species has parameters more consistent with an ATCUN-like
Cu(II)–N3S complex (g = 2.22, A = 197 × 10−4 cm−1, f = 112).
These interconverting species have been previously observed for com-
plexes of Cu(II) and methionine-containing peptides [71]. By pH 8.0
the ATCUN-like species dominates. Interestingly, the EPR spectra show
that the Cu(II)-binding environment for 1M at pH 7.5 is similar to the
Cu(II)-binding environment of Cys- and hCys-containing linear and cy-
clic peptides, but not that of GGML (Fig. 4). This indicates that the mac-
rocyclic constraint pre-organizes the N3S ligand set within 1M into a
square-planar geometry similar to those formed by thiol-containing
peptides, a geometry which is not necessarily preferred by the linear
analog.
3.5. UV–vis spectroscopy of complexes between Ni(II) and linear peptides
3.6. UV–vis spectroscopy of complexes between Ni(II) and cyclic peptides
In a sharp contrast to Cu(II) binding, Ni(II) binding by the linear pep-
tides GGCL, GGhCL and GGML showed no evidence of multiple species at
any pH. Binding behavior for all the linear peptides was consistent with
The original, imidazole-containing cyclic peptide 1 formed an ATCUN-
like Ni(II) complex with a pH50 of 8.8 [45]. This represents a somewhat
Fig. 6. pH dependence of Ni(II) binding to cyclic peptides 1, 1C and 1hC (A). UV–vis spectra of Ni(II) complexes with cyclic peptides 1 (black trace, pH 9.5), 1C (light gray trace, pH 9.0) and
1hC (gray trace, pH 8.4) (B). Percentage formation was calculated using absorption at 430 nm for Ni(II)–1, 420 nm for Ni(II)–1C and 435 nm for Ni(II)–1hC. A distinct charge transfer band at
335 nm was seen for Ni(II) complexes of 1C and 1hC which was not observed for Ni(II)–GGCL or Ni(II)–GGhCL. Additional full spectra at different pHs are shown in Fig. S15.

K.P. Neupane et al. / Journal of Inorganic Biochemistry 139 (2014) 65–76
73
destabilized form of the linear ATCUN complex formed by Ni(II)–GGHL,
which has a pH50 of 5.8. Interestingly, while linear peptides GGDL and
GGXL show very similar Ni(II) binding properties to GGHL, cyclic peptides
1D and 1X do not bind Ni(II) up to pH 8.0, at which point Ni(OH)2 was ob-
served to precipitate from solution. This demonstrates that the shape
enforced by the macrocyclic linker can support square planar complexa-
tion of Ni(II) in the context of the imidazole ligand, but not for pyridyl
or carboxyl ligands at the same position.
Not surprisingly, we observed stable complexation of Ni(II) with
thiol-containing cyclic peptides, with pH50 values near 7.4 for Ni(II)
binding to 1C and 1hC. This is a lower pH50 value than the Ni(II)–1 com-
plex (pH50 8.8), implying greater complex stability (Fig. 6A). In fact,
complexation of Ni(II) to thiol-containing cyclic peptides is nearly as fa-
vorable as complexation to linear thiol-containing peptides, which have
pH50 values of 6.4. Thus, we conclude that the cyclic architecture is
compatible with high-affinity Ni(II) binding, particularly when the
imidazole is switched out for a thiol group.
There are several additional interesting differences among the elec-
tronic absorption spectra of Ni(II) complexes with linear and cyclic
thiol-containing peptides. Specifically, the spectra of Ni(II)–1C and
Ni(II)–1hC have an extra band at 335 nm and higher overall extinction
coefficients for all bands (Fig. 6B, Table 2). Ni(II) complexes with
thiolate ligands usually produce an intense absorption band in the
250–350 nm range. The band at 335 nm is thus attributed to a charge
transfer transition from S−π → Ni(II) [29,72]. The higher extinction coef-
ficient of the band at 335 nm for Ni(II)–1C and Ni(II)–1hC may originate
from one of two effects. First, more favorable ligand orientation and en-
ergy match between the sulfur π-orbitals and metal ion d-orbitals may
lead to better orbital overlap and thus a higher extinction coefficient
[73]. Second, the higher extinction coefficients of the 420–440 nm d–d
bands for Ni(II)–1C and Ni(II)–1hC suggest that the Ni(II)–N3S plane is
distorted from square planar, making the d–d transition more favorable.
The extinction coefficient for Ni(II)–GGhCL is lowest, followed by Ni(II)–
GGCL, then Ni(II)–1hC, and then Ni(II)–1C. This is probably due to the
ring strain created by 5,5,5-membered chelate rings in Ni(II)–GGCL
and Ni(II)–1C, which would distort the planar geometry of Ni(II) center
and result in higher extinction coefficients [74].
3.7. Stoichiometry of Ni binding
The stoichiometries of the observed Ni(II)–peptide complexes were
investigated using UV–vis spectroscopy and ESI-MS spectrometry. Stoi-
chiometric titrations were performed at the lowest pH values for which
maximum ATCUN-like binding was observed for each peptide–Ni(II)
complex. For all peptides, ESI-MS revealed masses consistent with
metal-binding complexes with 1:1 stoichiometry (see Table S1) and
titrations using UV–vis spectroscopy confirmed saturation of the
ATCUN-like band at 425–450 nm at 1:1 stoichiometry (see Fig. S12).
3.8. Cyclic voltammetry
We next examined how substitution of the imidazole group affects
the redox properties of linear and cyclic ATCUN peptides. Unsurprising-
ly, cyclic voltammetry (CV) of thiol- and thioether-containing peptides
was obscured due to the irreversible oxidation of sulfur atoms. Cyclic
voltammograms for Cu(II)/Cu(III) and Ni(II)/Ni(III) couples for com-
plexes of GGHL, GGDL, and GGXL are shown in Fig. 7A–B and the elec-
trochemical data are summarized in Table 3. All three linear
metallopeptides undergo one-electron redox processes that are quasi-
reversible, and the Cu(II)/Cu(III) couples for Cu(II)–GGHL and Cu(II)–
GGDL are nearly reversible (ΔE = 0.070 V and Iopx/Ipred = 1.07 for
Cu(II)–GGHL, ΔE = 0.064 V and Iopx/Ipred = 0.954 for Cu(II)–GGDL).
Substitution of His to Asp reduces the redox potential of Cu(II)–GGDL
(E1/2 = 0.758 V). This is consistent with the more electron-rich carbox-
ylate coordinated to the metal center. This implies that these complexes
are more easily oxidized to corresponding M(III) species, and thus may
Fig. 7. Cyclic voltammetry of Cu(II) and Ni(II) complexes. (A) Cu(II)–linear peptide com-
plexes, (B) Ni(II)–linear peptide complexes, and (C) Cu(II)–cyclic peptide complexes.
The voltammograms were recorded using 1.0 mM peptide–metal complex in 200 mM
KCl with a scan rate of 100 mV/s at pH 7.5 for Cu(II)–linear peptide complexes, pH 8.0
for Ni(II)–linear peptide complexes, and pH 8.5 for Cu(II)–cyclic peptide complexes.
Each trace is average of 3 scans and is background-subtracted.
make more potent catalysts for M(III)-mediated transformations. By
contrast, substitution of the imidazole with pyridine increases
the redox potential of Cu(II)–GGXL (E1/2 = 0.853 V) and Ni(II)–GGXL
(E1/2 = 0.748) relative to corresponding GGHL complexes. A similar in-
crease in redox potential was observed when a His to Pal substitution
was made in a heme protein [75,76].
CV of Cu(II)–1D and Cu(II)–1X complexes showed similar trends
as Cu(II) complexes with linear peptides. The substitution of D-His to

74
K.P. Neupane et al. / Journal of Inorganic Biochemistry 139 (2014) 65–76
Table 3
Electrochemical data for Cu(II)– and Ni(II)–peptide complexes.
Peptides
Cu(II)–peptides
Eopx (V)
Ni(II)–peptides
Eopx (V)
Erped (V)
ΔEp (V)
Iopx/Irped
E1/2 (V)
Erped (V)
ΔEp (V)
Iopx/Irped
E1/2 (V)
GGHL
1
GGDL
1D
GGXL
1X
0.830
0.778
0.790
0.712
0.889
0.875
0.760
0.704
0.726
0.641
0.818
0.765
0.070
0.078
0.064
0.071
0.071
0.110
1.07
1.56
0.954
1.55
1.24
18.2
0.795
0.739
0.758
0.677
0.853
0.820
0.761
0.888
0.689
–
0.678
Irrev
0.605
–
0.083
–
1.19
–
0.719
–
0.084
–
1.21
–
0.647
–
0.792
–
0.705
–
0.087
–
1.24
–
0.748
–
Note: 1D and 1X do not bind Ni(II) up to pH 8.0, above which Ni(OH)2 precipitate was observed.
D-Asp lowered the redox potential from 0.739 V for Cu(II)–1 to 0.677 V
for Cu(II)–1D, and the substitution of D-His to D-Pal raised the redox po-
tential from 0.739 V for Cu(II)–1 to 0.812 V for Cu(II)–1X (Fig. 7C and
Table 3). These data indicate that Asp-containing linear and cyclic pep-
tides may have the best potential as catalysts for processes that involve
a Cu(II)/Cu(III) or Ni(II)/Ni(III) redox cycle. Since the UV–vis spectra of
Cu(II)–cyclic peptide complexes indicated the formation of alternate
species at high pH, we measured the redox potential of Cu(II)–1D at
pH 8.5 to ensure complete metal complexation for all peptides (see
Figs. 1 and 2). Nickel complexes were similarly tested at pH 9.2, at
which all peptides are fully complexed, and also at pH 8.5, at which all
peptides except for 1 were fully complexed (Figs. 5 and 6). Peptides
with sulfur-containing side chains are rapidly oxidized in the presence
of H2O2, and were thus omitted from these assays.
In accordance with previous studies, Cu(II)–1 produces diffusible
hydroxyl radicals [45], though to a lesser extent than unliganded copper
ions at pH 8.5. We were interested in the effects of substitution of the
imidazole with a carboxylic acid or pyridyl group on catalytic activity
for linear and cyclic peptides, and whether cyclization would
consistently boost catalytic activity. At pH 8.5, Cu(II)–1D and Cu(II)–1X
produced hydroxyl radicals to a greater extent than Cu(II)–1, with aver-
age intensities similar to that of unliganded copper (CuCl2) (Fig. 8). Linear
analogs Cu(II)–GGDL and Cu(II)–GGXL showed no measurable formation
of hydroxyl radicals, reinforcing that macrocyclization promotes Cu(II)/
Cu(III) cycling. We conclude that copper complexes of cyclic ATCUN mo-
tifs containing carboxylate and pyridyl groups have potential as oxidation
agents, and that this growing collection of copper–peptide complexes
represents a potentially useful set of redox catalysts.
pH 11.4. The lower redox potential of Cu(II)–1D at pH 11.4 (E1/2
=
0.426 V) indicates stronger donor ligands involved in Cu(II) coordina-
tion at this pH (Fig. S16) [77]. A similar phenomenon is also observed
for Cu(II)–1X.
Also, Cu(II)–1X was observed to have a particularly high ΔEp value,
with anodic current (oxidation) higher than cathodic current (reduc-
tion). This can be attributed to one of two factors. Either the presence
of a non-ATCUN, more poorly defined complex at this pH is causing
slower electron transfer and a broadening of oxidation and reduction
peaks, or the Cu(II)–1X complex must undergo a pronounced structural
rearrangement between the two redox states.
3.9. Fluorescence assay for detection of hydroxyl radicals
We then examined nickel–peptide complexes to evaluate their util-
ity as redox catalysts. Nickel–peptide complexes were tested at pH 8.5
(Fig. 8A) and pH 9.2 (Fig. 8B). At both pHs, it is clear that Ni(II) alone
has no activity above baseline and that Ni(II)–1 has no activity as well.
However, the other four complexes tested (Ni(II)–GGH, Ni(II)–GGHL,
Ni(II)–GGDL, and Ni(II)–GGXL) all produce diffusible hydroxyl radicals.
These data show that complexes of Ni(II) and linear ATCUN-like pep-
tides also have potential as oxidation catalysts.
Interestingly, cyclization of the canonical, imidazole-containing
ATCUN motif appears to enhance hydroxyl radical production under
Fenton conditions for copper complexes, but diminishes hydroxyl radi-
cal production for nickel complexes. Also, based on E1/2 values calculat-
ed from CV data, one might expect Cu(II)–1D to be the most redox-
active catalyst, followed by Cu(II)–1 and then Cu(II)–1X. Under Fenton
conditions at pH 8.5, we observe Cu(II)–1D and Cu(II)–1X to be equally
In order to directly measure catalytic activity, we incubated
metallopeptide complexes with 2′,7′-dichlorofluorescein diacetate in
the presence of hydrogen peroxide. This profluorescent dye reacts with
hydroxyl radicals (but not peroxides or other radical oxygen species) to
form 2′,7′-dichlorofluorescein in solution [78]. This and similar dyes
allow quantitative measurement of each complex's ability to produce hy-
droxyl radicals via a Fenton-like reaction [5,45]. The assay was unable to
detect hydroxyl radical formation by all peptides tested in the absence
of metal ion (Fig. 8, white bars), demonstrating that the redox-active
metal ion is required for producing hydroxyl radicals under these condi-
tions. In adapting this assay for a range of linear and cyclic peptides, we
found that background signal increased with increasing pH of solution.
To control for these variations, all copper complexes were tested at
Fig. 8. (A) Production of diffusible hydroxyl radicals by unliganded metal ions, apo-peptides, Cu(II)–peptide complexes, and Ni(II)–peptide complexes at pH 8.5. Fluorescence arises from
the reaction of diffusible hydroxyl radicals at 25 °C with 10 μM 2′,7′-dichlorofluorescein diacetate in 20 mM HEPES, 100 mM NaCl, and 0.9 mM H2O2. (B) Experiments identical to (A) were
performed at pH 9.2 with NiCl2, apo-peptides and Ni(II)–peptide complexes. Metal-free assays are shown as white bars, copper-containing assays are shown as light gray bars, and nickel-
containing assays are shown as dark gray bars. Error bars show standard deviations from 3 independent trials. Metal salts and metal–peptide complexes were each tested at 0.9 μM, with
apo-peptides tested at 1.0 μM. **At pH 8.5, the Ni(II)–1 complex accounts for only ~10% of the Ni(II) ions present (Fig. 6A).

K.P. Neupane et al. / Journal of Inorganic Biochemistry 139 (2014) 65–76
75
active at producing hydroxyl radicals, and Cu(II)–1 to have less activity.
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GGDL
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GGCL
GGhCL
GGML
1
NH2-Gly-Gly-His-Leu-CONH2
NH2-Gly-Gly-Asp-Leu-CONH2
NH2-Gly-Gly-Pal-Leu-CONH2
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