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Intermediate Cu(II)-Thiolate Species in the Reduction of Cu(II)GHK by Glutathione: A Handy Chelate for Biological Cu(II) Reduction

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Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV–vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter.
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Intermediate Cu(II)-Thiolate Species in the Reduction of Cu(II)GHK
by Glutathione: A Handy Chelate for Biological Cu(II) Reduction
Iwona Ufnalska, Simon C. Drew, Igor Zhukov, Kosma Szutkowski, Urszula E. Wawrzyniak,
Wojciech Wróblewski, Tomasz Frączyk,*and Wojciech Bal*
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ABSTRACT: Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions.
Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the
mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest
reductant naturally occurring in human plasma. Spectroscopic techniques (UVvis, CD, EPR, and NMR) and cyclic voltammetry
helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary
ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate.
The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may
provide a missing link in copper tracking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular
copper transporter.
INTRODUCTION
Gly-His-Lys (GHK) is a high-anity copper chelator naturally
occurring in human blood (log cK7.4 = 12.62).
1
The origin of
the tripeptide is still not clear. It is most likely released through
proteolytic degradation of extracellular proteins, such as
SPARC (secreted protein acidic and rich in cysteine) or type
I collagen, in response to the process of tissue damage.
2,3
GHK
was discovered in 1973 by L. Pickart and M. Thaler, who
noted that liver tissue from an elderly donor treated with blood
plasma from a young volunteer regained the ability to produce
proteins specic to the young tissue.
4
This rejuvenating eect
was attributed to a higher level of GHK since a decline in the
tripeptide concentration with age has been noted (from 200 ng
mL1at the age of 20 to 80 ng mL1by the age of 60).
5
GHK
was isolated from blood plasma as a Cu(II) complex. This
suggested the involvement of Cu(II) in its biological activity,
which includes stimulation of growth and proliferation of
various cultured cell types, leading to tissue regeneration,
wound healing, angiogenesis, radiation damage recovery, and
even anxiety reduction in laboratory animals.
614
The skin
regenerating activity, stimulation of collagen synthesis, and
anti-inammatory, antioxidant, and anti-aging properties
5,15
prompted the widespread use of Cu(II)/GHK in skin care
(due to the often limited knowledge on specic complexes, we
used a slash in the formula where the components are known,
but they may represent various stoichiometries or the exact
stoichiometry is not known). As a regulator of expression of a
number of genes,
16
it has been proposed as a therapeutic agent
for certain cancers, chronic obstructive pulmonary disease
(COPD), and bleomycin-induced pulmonary brosis.
1720
The mechanistic aspects of this multitude of activities remain,
however, obscure, and it is tempting to speculate that they
include copper handling on the tissue level.
Received: August 28, 2021
Published: November 15, 2021
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The binary Cu(II)GHK complex was extensively studied
using spectroscopic methods, potentiometry, calorimetry, and
crystallography.
1,2127
The Cu(II) chelation occurs via the
glycine amino nitrogen, the deprotonated amide nitrogen of
the GlyHis peptide bond, and the His side-chain imidazole
nitrogen, yielding two fused chelate rings. In this respect, GHK
is a representative of the class of H2NXxxHis (XH)
peptides, known as particularly ecient Cu(II) chelators.
28,29
Importantly, the fourth equatorial binding site in the
Cu(II)GHK structure is available for ternary complex
formation (here noted as LCu(II)GHK, where L represents
a monodentate ligand).
1,21,27
The ability to bind a transient
partner and a fast Cu(II) exchange rate between the holo and
apo forms reinforced a postulated Cu(II) carrier role of GHK,
consistent with oxidative conditions in blood.
30
Indeed, the
copper-binding sites in known copper carriers (e.g., human
serum albumin, HSA, and cellular copper transporter Ctr1),
belonging to the amino-terminal Cu and Ni binding
(ATCUN) family,
31
strongly prefer Cu(II). However, the
transfer of copper to cells requires its prior reduction to Cu(I)
due to the specicity of the Ctr1 transmembrane channel.
32
While the site(s) and mechanism(s) of this reduction in
humans are a matter of debate,
33
the GHK participation would
require a facile mechanism of reduction of GHK-bound Cu(II)
to Cu(I). Previous studies showed that Cu(II)GHK is inert
versus the physiological levels of ascorbate,
27
the major
reducing agent in blood.
34
Nevertheless, body uids are rich
in other antioxidants, such as retinol, α-tocopherol, β-carotene,
and reduced glutathione (GSH), protecting against free
radicals.
35
In our studies, we focused on GSH, which is a
stronger reductant than ascorbate.
36,37
GSH (γ-glutamyl-cysteinyl-glycine) is the most abundant
thiol in nature.
38
It is present in millimolar concentrations in
most human cells and plays a crucial role in cellular redox
homeostasis. Its extracellular level in healthy humans is
micromolar.
39,40
Apart from being a reductant, GSH is also a
complexing agent for heavy metals, participating in their
cellular detoxication via its thiolate and, additionally, the
nitrogen and oxygen donors.
41
The reaction between GSH and
Cu(II) cations includes a swift Cu(II) reduction, yielding
Cu(I)/GSH and Cu(II)GSSG complexes, depending on the
metal-to-ligand ratio and oxygen availability.
4245
The reaction
is slowed down for strongly chelated Cu(II), as in complexes
with products of enzymatic hydrolysis of Aβpeptides, but with
the same copper reduction products.
42
In this work, we targeted the redox behavior of Cu(II)GHK
in the presence of glutathione using UVvis and circular
dichroism (CD) spectroscopies, aided by EPR, NMR, and
electrochemical studies. The model ATCUN peptide, C-
terminally amidated GGH, was used as a reference,
representing biorelevant Cu(II) complexes bearing the
saturated 4N coordination plane (such as in HSA and Ctr1).
The results allowed us to discuss the mechanism of
glutathione-mediated reduction of GHK-bound Cu(II) and
its putative physiological role.
RESULTS AND DISCUSSION
The reaction of Cu(II)GHK with GSH was initially monitored
by electronic spectroscopy. The addition of 1 mM GSH to the
sample containing 0.50 mM GHK with 0.45 mM Cu(NO3)2in
50 mM HEPES, pH 7.4, 37 °C, under aerobic conditions
resulted in an immediate dd band decrease, accompanied by
an increase of absorbance in the 250350 nm region. These
changes were reverting over a longer period, as revealed by
time-dependent measurements (Figure S1).
Next, the eect of experimental conditions on the reaction
was studied by comparing its course in 50 mM phosphate
buer, 50 mM HEPES, and unbuered (water) solution, each
at pH 7.4. The comparison of the kinetic traces for selected
wavelengths, provided in Figure S2, revealed that each buer
aected the course of the reaction, although the eects of
HEPES were subtler. To keep the studied system unambig-
uous, all further experiments were conducted in pure water.
This could be done because, once the sample was prepared, its
pH remained stable within 0.2 pH unit during the experiment.
The reaction at 37 °C in water, monitored by UVvis and
CD spectra, is presented in Figures 1A and S3, respectively.
The CD bands in the vis and near-UV range exhibited only a
rapid decrease after GSH addition, followed by a gradual
return of the spectral intensity, whereas the UVvis spectral
changes also involved new bands appearing in the near-UV
range. A slight blue shift of the dd band from the initial 606
nm to ca. 595 nm was also noted. Wavelengths for kinetic
tracing were selected according to the major intensity
dierences revealed by dierential spectra (Figure S4). In
UVvis, the decrease of the main dd band was accompanied
by the immediate rise of absorbance at 300 and 255 nm,
assigned to S-to-Cu(I) charge-transfer (CT) transitions in a
Cu(I)/GSH thiolate complex (Figure 1).
46
The low-intensity
band at 410 nm, emerging later in the reaction, originated from
the interaction of Cu(II)/Cu(I) with GSH in the presence of
oxygen, as conrmed by the control experiments (Figure S5)
as well as reported previously.
42
Figure 1. (A) UVvis spectra collected with 5 min intervals for 0.50 mM GHK with 0.45 mM Cu(II) in the presence of 1.0 mM GSH at pH = 7.4,
T=37°C and (B) absorbance changes at 255 nm (black squares), 300 nm (red circles), and 606 nm (blue triangles), plotted as a function of time;
absorbance values for the binary complex are presented in the shaded eld; dashed line represents the spectrum of Cu(II)GHK.
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Then, the kinetic prole of the reaction at 37 °C was studied
by single-wavelength measurements at 606 nm to improve its
time resolution from 5 min to 5 s (Figure S6). This allowed us
to detect the dd band intensity minimum between the
second and fourth minute of observation, which separated the
Cu(II) reduction and Cu(I) re-oxidation phases of the
reaction.
For the 2.2-fold excess of GSH used in these experiments,
the Cu(II) reduction process was incomplete, typically
reaching 60%, despite the fact that just 1 mol. equiv. of GSH
is required to reduce Cu(II) to Cu(I). Full Cu(II) reduction
was achieved at the 4.4- and 6.6-fold GSH excess (2 and 3
mM, Figure S7). At these GSH concentrations, the recovery of
the Cu(II)GHK complex also slowed down but was not
prevented. Repetitions of the reduction/re-oxidation cycle by
periodic additions of adequate GSH amounts indicated a high
reproducibility of the examined process (Figure S8). The
availability of ambient oxygen was the limiting factor of the re-
oxidation step, as demonstrated in Figure S9, analogously to
the previously studied ATCUN systems. The Cu(I)/GSH
complex is stable under anaerobic conditions.
42,47
The HPLC separation of reaction products collected after
the re-oxidation step, followed by ESI-MS analysis, allowed us
to identify GSSG as the only covalent product of the studied
reaction (signals at 613.2 and 307.1 m/z represent [M + H]+
and [M + 2H]2+ forms, respectively). No MS-detectable
covalent GHK modications appeared in the reaction (as
revealed by the presence of m/z = 341.2 for [M + H]+GHK
species and the absence of identiable products of its covalent
modication), in agreement with the reproducibility of the
reaction cycle (Figures S10 and S11).
Spectroscopic measurements did not indicate a contribution
of the Cu(II)GSSG binary complex (characterized by an
absorption band at 625 nm
36,42,48
) to the overall Cu(II)
equilibrium at the completion of copper re-oxidation. This was
expected based on the respective anity constants at pH 7.4
for Cu(II)GHK versus Cu(II)GSSG, log K= 12.62 versus
10.37.
1,48
The control titration of 0.45 mM Cu(II)GHK with
up to 5 mM GSSG did not alter the dd band position of the
former, additionally conrming the absence of any ternary
GSSG-Cu(II)GHK species (Figure S12).
Having thus demonstrated that the re-oxidation reaction
consisted of oxidation of the Cu(I)/GSH complex into GSSG
and Cu(II) ions, which were promptly scavenged by GHK to
re-form Cu(II)GHK, we followed the reduction step in greater
detail. First, we repeated the reaction at lower temperatures, 20
and 5 °C, whose results are summarized in Figure S13. The
reduction phase at 5 °C is presented in detail in Figure 2. The
reduction endpoint, measured by the minimum of the dd
absorption peak and the maximum of CT bands, increased
from 60% at 37 °C to 70% at 5 °C, while the reaction rate,
measured as the time required to reach the minimum,
decreased from 3 min to 15 min to 45 min, for 37, 20, and
5°C, respectively. The re-oxidation phase was also
correspondingly slower.
The variation of dd band position became clearly visible at
5°C(Figure 2). During the reduction step, a prominent blue
shift of the signal from 606 nm down to 595 nm occurred. This
lasted for about 10 min and was followed by a partial backshift
to ca. 600 nm. This observation strongly suggested that two
dierent phenomena occurred in this system in the given time
window. The presence of an additional spectral component at
the very beginning of the reaction was further supported by the
analysis of dierential spectra (Figure S14A). The absorbance
decrease around 615 nm parallel to the increase around 505
nm indicated two overlapping Cu(II) species, with one being
formed at the expense of another (Figure S14A, inset). These
changes were accompanied by a transient band at ca. 345 nm.
Such phenomena were not seen in the re-oxidation phase
(Figure S14B). Therefore, a short-lived species was character-
istic only for the reduction phase.
To further conrm the observed spectral eects that could
be misinterpreted due to a low signal-to-noise ratio, another
experiment, with 4-fold higher concentrations of reagents, was
carried out (Figure S15). Not only the signicant blue shift of
the dd band was conrmed but also a profound eect of air
oxygen supply on the kinetic prole of the reaction was
demonstrated (Figure S15AC).Theincreaseinthe
concentration of the binary complex and GSH with limited
oxygen access inuenced the redox balance in the examined
redox system, resulting in the inhibition of the reoxidation
phase, as revealed by a comparison to previous results (Figure
S15D).
The observed dd band blue shift indicated that the Cu(II)-
coordinated water molecule in Cu(II)GHK was replaced by a
stronger eld ligand. Large blue shifts (typically 4060 nm)
were observed in 3 + 1N ternary Cu(II) complexes formed by
GHK and other XH-type peptides with imidazole rings, with
much smaller shifts observed for 3N + 1O complexes.
1,4951
An intermediate shift detected here (1012 nm) could be due
to a partial formation of a 3 + 1N ternary species with another
GHK molecule, GHK-Cu(II)GHK,
1
as a consequence of an
increased excess of GHK over Cu(II) ions, depleted by partial
reduction to Cu(I).
Alternatively, these spectral changes could result from the
engagement of GSH thiolate in the ternary complex, as in
previous studies, no evidence was found for the participation of
amino acid or peptide amines in 3 + 1N ternary complexes
with XH peptides.
1,4952
The key evidence for the GSH
thiolate binding was provided by EPR measurements taken at
given time points for the sample stored on ice. The stepwise
isolation of individual signals (Figures S16 and S17) revealed
the presence of a short-lived GSH-Cu(II)GHK species within
the rst minutes of reaction in addition to the Cu(II)GHK and
GHKCu(II)GHK complexes (Figure 3). The Cu(II)GHK
and GHKCu(II)GHK spectra were obtained from controls
in the absence of GSH, while that of GSH-Cu(II)GHK was
derived from the rst spectrum of the reaction. The shift of the
Figure 2. Selected UVvis spectra collected with 5 min intervals for
0.50 mM GHK with 0.45 mM Cu(II) in the presence of 1.0 mM GSH
at 5 °C, pH = 7.4, demonstrating the dd band blue shift
concomitant with the absorbance decrease; the dashed line represents
the spectrum of Cu(II)GHK; restoration of the signal for the same
reaction is presented in Figure S14B.
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g parallel value upon GSH addition (from 2.233 to 2.175,
Table S2), as well as the change within the superhyperne
structure, is very similar to that observed for analogous
thiosemicarbazone complexes, where GSH is bound equato-
rially to a tridentate ligand
53,54
It was further armed by the
consistency of the simulated and experimental EPR data
(Figure S18 and Table S2). Using these data, the time course
of the reaction on ice was reconstructed in Figure S19, with the
full spectral analysis provided in Figure S20. It should be
emphasized, however, that freezing the samples for EPR
measurement alters the relative binding anities of binary and
ternary species, and according to our previous experience, the
apparent stability of GHKCu(II)GHK is much higher at 77
K than at room temperature.
1
This freezing artifact will be
investigated in detail and presented elsewhere. On the other
hand, the swiftness of the examined reaction required the
measurement in a frozen solution mode. Therefore, the EPR-
based species distribution ought to be regarded as qualitative
but rm evidence for the transient GSH-Cu(II)GHK species.
Despite the uncertain speciation of Cu(II) complexes, EPR
provided a reliable quantitation of total Cu(II) (Figure S19),
which, along with the published stability constants for
Cu(II)GHK and GHKCu(II)GHK,
1
allowed us to calculate
the concentrations of these complexes corresponding to the
UVvis spectra at given reaction times. This, in turn, was the
basis for the decomposition of dd signals collected at the
beginning of the reaction performed at 5 °C. This analysis,
presented in Figure S21, revealed the third Cu(II) component
with the dd maximum at 570 nm, which declined over time.
This nding correlates well with a transient signal at 345 nm
revealed by dierential analysis and the short-lived GSH-
Cu(II)GHK species detected by EPR.
To learn more about the mechanism of formation of thiolate
ternary complexes of Cu(II)GHK, we reacted it with two other
thiols, 2-mercaptoethanol and sodium methanethiolate
(NaSCH3). The main spectral changes triggered by the former
reected those observed for GSH; however, the dd band
disappearance was accompanied by the precipitation of an
apparently insoluble Cu(I) species (Figure S22A). In the
presence of NaSCH3, a 9 nm shift concomitant with a slight
dd band enhancement was observed initially, followed by the
standard pattern of partial Cu(II) reduction to a Cu(I) species,
followed by re-oxidation (Figure S22B,C). Noteworthy, in
both cases, a characteristic band at 345 nm could be
distinguished at the beginning of the reaction (Figure 4).
These results indicate that all the three examined thiols formed
transient ternary complexes with Cu(II)GHK via a sulfur atom,
giving rise to a new absorption band at 345 nm, ascribed to S
Cu(II) LMCT,
55,56
and the dd band blue shift (Figure
S23). A dynamic relationship between ternary Cu(II) complex
formation and Cu(II) reduction depends on the reduction
potential of the thiol in the order NaSCH3<2-
mercaptoethanol < GSH. Notably, the formation of the
thiolate ternary Cu(II) complex was ecient for monodentate
thiols, not requiring the chelate ring formation described for
cysteine complexes by Hanaki et al.
55,57,58
Figure 3. X-band (9.42 GHz) frozen solution (77 K) EPR spectra showing the isolation of the spectrum corresponding to GSH-Cu(II)GHK by
weighted subtraction of the spectrum of Cu(II)GHK (Figure S16) and GHKCu(II)GHK (Figure S17) from the spectrum obtained for 0.50 mM
GHK with 0.45 mM Cu(II) in the presence of 1.0 mM GSH at 4 °C. Dashed vertical lines indicate the approximate position of the prominent
Cu(II) hyperne features of each coordination mode as a guide to the eye.
Figure 4. Dierential absorption spectra calculated for 0.50 mM GHK
with 0.45 mM Cu(II) in the presence of 2.0 mM 2-mercaptoethanol,
T=10°C (black line), 1.0 mM NaSCH3,T=15°C (red line), and
1.0 mM GSH, T=5°C (blue line) expressed as a dierence between
the rst spectrum recorded after thiol introduction and Cu(II)GHK
spectrum.
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Having conrmed the existence of the transient S
Cu(II)GHK species (S represents the monodentate thiolate
coordination), we proceeded to establish its role in the Cu(II)
reduction process. First, we employed NMR to check if GHK
remained coordinated with Cu(I) after reduction. The reaction
mixture, consisting of 2 mM GSH with 0.5 mM GHK and 0.45
mM Cu(NO3)2, was prepared in 50 mM sodium phosphate
buer, pH 7.4, under anaerobic conditions to ensure complete
reduction of Cu(II) ions. The reaction was followed over 4
days in the NMR tube at 20 °C(Figure S24). The selected
GHK signals were integrated and compared to the signal of the
methyl group of acetate, which was a GHK counterion. They
indicated that as long as oxygen-free conditions were present in
the NMR tubes, the GHK apopeptide remained at the same
level throughout the experiment. A control reaction without
GHK was also performed under the same conditions (Figure
S25). The separately recorded spectra of GSH, GSSG, and
GHK in the absence of copper are presented in Figure S26 to
aid the interpretation of the results. As shown in Figure S27,
GSSG (determined directly) and Cu(II)GHK (inferred by the
loss of GHK signals due to paramagnetic broadening) were the
only chemical species at the end of the re-oxidation step. To
better characterize the products, DOSY experiments were
collected for the Cu(II)GHK reaction with GSH immediately
after the GSH addition and after 22.5 and 93 h. These spectra
are presented and analyzed in Figure S28. Two sets of diusion
parameters could be discerned. The one with the slower
diusion, at 1.3 ×1010 m2·s1, corresponded to a Cu(I)/GSH
species and was absent at 93 h, while the other, at 3 ×1010
m2·s1, contained the GHK and GSSG apopeptide signals. The
singlet of the acetate methyl group diused faster, at 7 ×1010
m2·s1, as expected for a small non-interacting molecule. No
signals from the GHK molecule bound to Cu(I) were seen. In
particular, the His imidazole signals [the primary binding site
expected for both Cu(I) and Cu(II)] exhibited single diusion
peaks aligned with other signals of the monomeric GHK
apopeptide. Figure S29 presents the calculation of molecular
volumes based on the DOSY signals of Glu βprotons from the
Cu(I)/GSH and GSSG spectra. The apparent volume of
Cu(I)/GSH was 3 times larger than that of GSSG,
corresponding well with the Cu(I)4GSH6stoichiometry
indicated by Fahrni et al.
47
The above analysis clearly
conrmed this complex as the only stable Cu(I) species
throughout the reaction.
The role of GSH-Cu(II)GHK in the redox process was
further studied in the presence of a 100-fold excess of
imidazole (Im) at 37 °C(Figure S30A). Im is able to displace
water from the fourth equatorial site of Cu(II)GHK with a log
cKof 2.86 at pH 7.4.
1
The calculated initial composition of
Cu(II) species in this experiment, prior to GSH addition, was
96.6% Im-Cu(II)GHK and 3.4% Cu(II)GHK, conrmed by a
blue shift of the dd band maximum down to 565 nm,
expected for the eective saturation of the fourth Cu(II)
coordination site with Im. The addition of GSH to this ternary
complex resulted in the partial Cu(II) reduction within the
sample mixing time, exactly as in the absence of Im, followed
by a similarly slow recovery of the Cu(II) species (Figure 5).
No spectroscopic eects of the addition of GSH were noted in
the dd band region.
Since the ternary Im complex did not block the reduction of
Cu(II) bound to GHK by GSH, control experiments for GGH
amidated at the C-terminus were carried out. This peptide,
together with its non-amidated counterpart, is the simplest
ATCUN model, with amidation providing a better mimic of
the peptide chain extension in ATCUN peptides/proteins.
31,59
The GSH addition to Cu(II)GGH caused a partial Cu(II)
reduction manifested by a decrease of the dd band at 525 nm
(Figure S30B), analogous to Cu(II)GHK and Im-Cu(II)GHK,
but about 50-fold slower (Figure 5). In turn, the timespan of
re-oxidation was similar for all the three systems, about 1 hour
in our experimental conditions. There was no eect of GSH on
the dd band, as in Im-Cu(II)GHK. The features and course
of Cu(II)GGH reaction with GSH were similar to other
ATCUN systems studied in similar conditions.
42
The impact of Im on the rate of Cu(II)GHK reduction was
further studied at 20 °C. First, the rates recorded in the
presence of varied Im concentrations were compared (Figure
S31). A moderate ca. 2-fold drop of reduction rate was
observed for 30 and 50 mM Im versus the sample without Im
(see Table S3). Considering the near saturation of the fourth
equatorial site in Cu(II)GHK at such Im excess, this suggested
that GSH-Cu(II)GHK could not be a key species in the
reduction. In another experiment, the reduction stage of the
reaction without Im was followed at single wavelengths of 300,
345, and 606 nm, thereby shortening the dead time of signal
detection to 10 s. The signals at 345 nm, characteristic for
GSH-Cu(II)GHK, and at 606 nm reached their maximum
within the shortened dead time (Figure S32). This was
followed by an absorbance drop, reaching its minimum after 5
min and 10 min for CT and dd band, respectively. The
reduction progress monitored by dd signal decay (represent-
ing all Cu(II) species) was in a perfect match with the
absorbance changes at 300 nm (Figure S33).
A more precise analysis of these experiments required the
knowledge of stability constant for GSH-Cu(II)GHK. It was
determined on the basis of its CT band at 345 nm clearly
recorded at low temperatures, where the reaction was
suciently slow to enable the sequential collection of full
UVvis spectra. The data were obtained from a series of
kinetic experiments where 0.45 mM Cu(II) and 0.5 mM GHK
were reacted with 0.410 mM GSH (Figure S33). The
sequentially recorded full UVvis spectra revealed an
isosbestic point at 320 nm in the initial reaction phase,
indicating that Cu(I)4GSH6(CT at 300 nm) was formed from
GSH-Cu(II)GHK (CT at 345 nm) in a quantitative fashion in
the given time window. This nding allowed us to calculate the
absorption coecients for GSH-Cu(II)GHK (ε300II,ε345II,
Table S4) needed to determine its concentration along with
Figure 5. Absorbance changes of the dd band triggered by GSH
introduction into a solution of 0.50 mM GHK with 0.45 mM Cu(II)
(606 nm, black squares), 50 mM imidazole (565 nm, green triangles),
and 0.50 mM GGH-am with 0.45 mM Cu(II) (525 nm, blue circles).
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the reaction progress. Details are described in the Supporting
Information section Calculation of the GSH-Cu(II)GHK
stability constant. The concentrations of all relevant complex
species and the conditional stability constant CK7.4 for GSH-
Cu(II)GHK (eq 1) were obtained from extrapolation of A300
and A345 to the zero time point for each reaction using an
empiric rst-order reaction equation (Table S5). This was
necessary to make valid assumptions about the concentration
of GSH, which was consumed during the reaction in a non-
linear fashion.
=[− ]
[]·[ ]
ΙΙ
ΙΙ
KGSH Cu GHK
GSH Cu GHK
c
7.4 (1)
The nal value of log cK7.4, averaged over the four lowest
GSH concentrations, was 2.91 ±0.06. The limitation of this
data range for calculations was dictated by the lowest extent of
side reactions such as GSSG production, which inuenced the
absorbance intensities in the CT region.
The CK7.4 for GSH-Cu(II)GHK enabled us to calculate the
initial compositions of reaction solutions, immediately after the
GSH additions. Such calculations are provided in Table S6 for
the kinetic experiments in the absence and presence of Im. The
CK7.4 values for GSH-Cu(II)GHK and Im-Cu(II)GHK are
identical within the experimental error,
1
conrming that a 30-
and 50-fold excess of Im over GSH diminished the ternary
complex share to single percentage points versus its estimated
initial presence at 40% of Cu(II) in the absence of Im. These
ndings indicated that the ternary GSH-Cu(II)GHK complex
was not necessary for the Cu(II) reduction but participated in
this process in the absence of alternative ternary ligands.
The quantitative redox aspects of the examined system were
investigated by cyclic voltammetry (CV). The representative
cyclic curves of Cu(II)GHK are presented in Figure S34A. The
main electrochemical feature of the binary complex is an
irreversible metal ion reduction at around 0.60 V (vs AgCl/
Ag), followed by a redox center oxidation near 0.1 V. A
signicant shift in the position of the anodic response relative
to the one expected upon the assumption of one-electron
transfer (i.e., ΔEis higher than 60 mV) suggests that a
considerable structural change occurs in the copper coordina-
tion sphere upon reduction.
27,60,61
The reorganized structure
of the complex does not seem to meet the stereochemical
requirements of the Cu(I) cation, as evidenced by the shift of
anodic response along with successive scans, accompanied by a
peak shape alteration alongside the scan rate decrease (Figure
S34A, inset). A likely explanation is an exclusion of the amide
nitrogen from the coordination sphere upon reduction, as
Cu(I) is unable to deprotonate and coordinate it.
62
This leads
to the release of free Cu(I) ions, which tends to adsorb and
accumulate on the electrode surface during the redox cycling.
This feature corresponds to the absence of Cu(I)-GHK species
demonstrated by NMR. Irreversible oxidation of Cu(II)GHK
to a Cu(II)Ispecies was seen at 1.3 V.
27,63
A high potential
value makes this process irrelevant for the studied reaction.
Tracing the course of the Cu(II)GHK reaction with GSH by
electrochemical methods is a dicult task since the thiol group
alone undergoes a series of oxidation reactions in the range of
positive potentials.
64
The situation gets even more complicated
when an interaction with copper is examined due to multiple
interconnected chemical and electrochemical redox events,
yielding a continuous change of electrode signals. Therefore,
we focused on the range of negative potentials, where only the
processes specic to the Cu(II)GHK system occurred (Figure
6). No reduction signal was detected in the above potential
window in the GHK absence under anaerobic conditions
enabled by an argon blanket; hence, the observed electro-
chemical eects originated in the GHK complex (Figure
S34B). The GSH addition inuenced both the position and
the current of the cathodic peak. First, a dynamic cathodic
current drop along with the signal shift toward less negative
potentials was observed (Figure 6A), indicating that
coordination changes caused by GSH favored the electron
transfer. The Cu(I) species formed electrochemically was
rapidly captured by GSH, causing an immediate disappearance
of the follow-up anodic peak (ca. 0.1 V). After reaching the
minimum value of the current, the reduction peak shifted back
to negative values and remained unaected unless the oxygen
access was provided (Figure 6B). This prole of electro-
chemical changes is consistent with the spectroscopic results
discussed above. Since the cathodic signal shift toward less
negative potentials was observed previously as a consequence
of ternary complex formation by 3N species,
65
such temporary
uctuations in the reduction peak position along with a
signicant current decrease t well with the transient GSH-
Cu(II)GHK species. A longer-term signal stabilization at a
potential slightly higher than for Cu(II)GHK correlates with
the accumulation of GHK-Cu(II)GHK, accompanying the loss
of Cu(II) due to reduction, as explained above.
Figure 6. Selected CV curves recorded for 0.50 mM GHK with 0.45 mM Cu(II) in the presence of 1.0 mM GSH in 100 mM KNO3;υ= 100 mV/
s; panel (A) depicts current responses for the Cu(II)GHK binary complex (blue line) and three rst measurements after GSH addition (red, green,
and black line, respectively); panel (B) in addition to the voltammogram of Cu(II)GHK shows the last curve from panel (A) and the one
representing steady state; pH = 7.4, room temperature.
Inorganic Chemistry pubs.acs.org/IC Article
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Inorg. Chem. 2021, 60, 1804818057
18053
The above results allowed us to propose the scheme of
Cu(II)GHK interaction with GSH. Reaction 2 provides the
overall stoichiometry of Cu(II) reduction, where [CuGHK]*
is a postulated reduction intermediate detected by electro-
chemistry but not visible to spectroscopic methods due to its
very short lifetime, with the follow-up rapid formation of
Cu(I)4GSH6driven by a huge dierence of Cu(I) anities to
nitrogen ligands (nano- to picomolar)
66
and GSH (attomo-
lar).
47
Ι
→[ ]*
→+Ι +
10GSH 4Cu( )GHK
CuGHK
2GSSG Cu( )4GSH6 4GHK (2)
However, the corresponding oxidation of GSH to GSSG was
suprastoichiometric, as indicated by the correlation of the
extent of Cu(II) reduction with the initial GSH concentration
(see Figure S33). A complete reduction was achieved for 2
mM GSH under aerobic conditions, but measurements under
near-anaerobic conditions (0.51% O2) clearly indicate a
prominent impact of oxygen already in the reduction phase.
For 1 mM GSH, the reduction endpoint calculated from the
minimum of dd absorption band was ca. 60% for aerobic and
75% for low oxygen conditions (Figure S9). Comparing the
pace of this process with a low oxidation susceptibility of
control GSH solutions, one can conclude that the additional
GSH oxidation was catalyzed by Cu(II)/GHK complexes
rather than the free Cu2+ ion, limited to less than picomolar
concentrations by very strong chelation by GHK. This
observation is corroborated by electrochemistry, which
demonstrated the redox capability of Cu(II)/GHK complexes.
It is also in agreement with the ndings presented by Compton
et al.
67
The parallel GSH oxidation mechanism established in the
literature includes the electron transfer from Cu(I) to GSH by
a transient superoxide anion-radical, which yields the thiyl
radical, nally recombining into GSSG, according to the
following reactions
45,68,69
[
−]+ − +
·
Cu(I) X O Cu(II) X O
22
(3)
+→
·− ·
OGSHGS
2(4)
+→
··
GS GS GSSG
(5)
where [Cu(I)X] represents all intermediate Cu(I) species
prone to an oxygen-induced oxidation process.
What remains to be established is the nature of the transient
[CuGHK]*species in reaction 2 and the pathway of its
formation. A plausible mechanism can be inferred from the
recent nding that the redox activity of 4N species of GGH
and other ATCUN complexes
70,71
involved a 2N kinetic
intermediate, providing a quasi-reversible Cu(II)/Cu(I) redox
pair. An analogous 2N species with a Nim +NH
2donor set was
also observed in stopped-ow studies of Cu(II) binding to the
GHTD-am peptide, which shares the Cu(II) coordination
mode with GHK.
72
This structure is inferred for [Cu(II)-
GHK]*, as presented in Scheme 1. The peptide nitrogen
provides eective stabilization of higher copper oxidation states
[Cu(II) over Cu(I) and Cu(III) over Cu(II)] by electrostatic
interactions.
63,73,74
Therefore, its exclusion from the coordi-
nation sphere in [Cu(II)GHK]*enables its susceptibility to
reduction by GSH according to reaction 6.
[
]* + → [ ]* + ·
Cu(II)GHK GSH Cu(I)GHK GS (6)
Furthermore, a steric hindrance from the GHK peptide
chain may actually exclude a water molecule from the vacated
equatorial position, making this complex three-coordinate in a
roughly T-shaped ligand arrangement. This hypothetical steric
hindrance is marked with a star in Scheme 1. It should be
noted that [Cu(II)GHK]*is actually a generic term
comprising the ligand L, which may be a water molecule, an
equatorially coordinated GSH, or another ternary partner, such
as Im.
Such species can be universally approached by an axial
GSH or other thiol, as illustrated in Scheme 1. This proposal
readily explains the merely ca. 2-fold slowdown of the Cu(II)
reduction reaction in the Im ternary complex. For L being the
non-thiol, there is only one (axial) pathway of the inner
sphere electron delivery to Cu(II) by the reducing thiolate,
while for L being GSH or other thiol, there are two possible
ways, the axialand the equatorialones. The respective
reduction rates are additionally modulated by the nucleophilic
properties of ternary ligands L, which is indicated by
electrochemical measurements, and by thermodynamic
stability of [Cu(I)GHK]*with various L. The inner sphere
electron transfer from the thiol, demonstrated for similar
systems by Holwerda et al.,
75
is followed by detachment of
Scheme 1. Proposed Structural Scheme of the Cu(II) Reduction Reaction at pH 7.4 in an Aqueous Solution
a
a
The initial L-Cu(II)GHK complex (L = H2O, a thiolate including GSH or Im) (A) spontaneously rearranges to a minor reactive intermediate
[Cu(II)GHK]*by the peptide nitrogen detachment and reprotonation (B), where the steric hindrance from the peptide chain (*) makes it a T-
shaped three-coordinate transient susceptible to the axialapproach by the thiolate reductant enabling the inner sphere electron transfer (C).
Inorganic Chemistry pubs.acs.org/IC Article
https://doi.org/10.1021/acs.inorgchem.1c02669
Inorg. Chem. 2021, 60, 1804818057
18054
GHK assisted by an additional thiol moleculetoward the
nal Cu(I)/thiolate structure, for example, Cu4GSH6.
Under near-anaerobic conditions, the Cu(II) reduction
progressed until GSH was exhausted by oxidation to GSSG
and tight complexation of generated Cu(I). Then, a steady-
state phase followed, co-habitated by Cu(I) and Cu(II)
complexes. This indicates that GSH in the Cu(I)4GSH6
complex is not able to reduce the external Cu(II) ions. This
phase was terminated by allowing the access of dioxygen. The
Cu(I) re-oxidation to Cu(II) driven by it also has a radical
reaction mechanism. The similarity of its rate in the presence
of various Cu(II)-chelating His peptides and the absence of
any covalent oxidation products except GSSG indicated that
Cu(I) was oxidized within the Cu(I)4GSH6complex, followed
by rapid Cu(II) capture by His peptides. In their absence,
Cu(II)GSSG and GSSG were the sole nal reaction
products.
42
The re-oxidation mechanism is described by
reactions 35presented above.
45,68,69
The relevance of GSH-Cu(II)GHK strongly depends on the
biological compartment, limited by its relatively low stability
constant but oset by its relatively long lifetime. Bearing in
mind the extracellular presence of GHK and the absence of
covalent reaction side products, we imply that this complex
may be a missing link in the process of clean delivery of Cu(I)
ions for transmembrane transporters of the CTR family
because extraneous radical species are eciently scavenged by
GSH as in reactions 4 and 5. Other Cu(II) complexes in blood,
such ATCUN motifs present, for example, in HSA, or amino
acid complexes, for example, Cu(II)His2, are virtually
impossible to reduce in blood serum conditions. Nevertheless,
we tend to advocate a small-molecule Cu(I) delivery system as
more compatible with the large extracellular domain of human
CTR1, as opposed to putative cell surface Cu(II) reductases,
which are known in yeast whose CTR1 has only a very small
extracellular part.
76
This idea is further supported by the ability
of ternary ligands, both imidazoles and thiols, to tune its rate.
Analogous species could also form and act intracellularly, given
the abundance of XH motifs in cellular proteins,
77
and the
known strict homology of various XH motifs in terms of
Cu(II) coordination properties.
4952
CONCLUSIONS
In the present work, we reported data on the glutathione-
mediated reduction of Cu(II) bound to GHK and GGH
peptides, followed by spectroscopic methods (UVvis, CD,
EPR, and NMR) and cyclic voltammetry. Our comprehensive
study sheds new light on the biological role of the widely
studied Cu(II)GHK complex, revealing it as a good candidate
for extracellular and intracellular Cu(I) supply via ternary
complexes with thiol compounds, such as GSH.
ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c02669.
Experimental details, additional spectroscopic and
voltammetric experiments, mass spectrometry data, and
detailed description of performed calculations (PDF)
AUTHOR INFORMATION
Corresponding Authors
Tomasz Frączyk Polish Academy of Sciences Institute of
Biochemistry and Biophysics, Warsaw 02-106, Poland;
orcid.org/0000-0003-2084-3446; Email: tfraczyk@
ibb.waw.pl
Wojciech Bal Polish Academy of Sciences Institute of
Biochemistry and Biophysics, Warsaw 02-106, Poland;
orcid.org/0000-0003-3780-083X; Email: wbal@
ibb.waw.pl
Authors
Iwona Ufnalska Chair of Medical Biotechnology, Faculty of
Chemistry, Warsaw University of Technology, Warsaw 00-
664, Poland
Simon C. Drew Department of Medicine (Royal Melbourne
Hospital), The University of Melbourne, Victoria 3010,
Australia; orcid.org/0000-0002-1459-9865
Igor Zhukov Polish Academy of Sciences Institute of
Biochemistry and Biophysics, Warsaw 02-106, Poland;
orcid.org/0000-0002-9912-1018
Kosma Szutkowski NanoBioMedical Centre, Adam
Mickiewicz University, Poznań61-614, Poland;
orcid.org/0000-0002-6091-9049
Urszula E. Wawrzyniak Chair of Medical Biotechnology,
Faculty of Chemistry, Warsaw University of Technology,
Warsaw 00-664, Poland
Wojciech Wróblewski Chair of Medical Biotechnology,
Faculty of Chemistry, Warsaw University of Technology,
Warsaw 00-664, Poland
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.1c02669
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
This research was nanced by the National Science Centre of
Poland (NCN) grants 2016/23/B/ST5/02253 and 2018/29/
B/ST4/01634 to W.B. The equipment used at IBB PAS was
sponsored, in part, by the Centre for Preclinical Research and
Technology (CePT), a project co-sponsored by the European
Regional Development Fund and Innovative Economy, The
National Cohesion Strategy of Poland. Electrochemical
research was nancially supported by Warsaw University of
Technology.
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Inorganic Chemistry pubs.acs.org/IC Article
https://doi.org/10.1021/acs.inorgchem.1c02669
Inorg. Chem. 2021, 60, 1804818057
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... Copper as a cofactor is involved in enzymes active sides by facilitation enzymatic redox processes. The most prominent function of Cu as static cofactor is related to cytochrome C oxidase [13,14], and tripeptide glutathione involved in antioxidant response [15]. Copper role includes enzymes' synthesis and activation e.g. ...
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Introduction Coronary artery disease possess inflammatory background related to enzymatic processes with trace elements involvements as co-factors. The aim of the study was to compare serum, urine and salivary copper, magnesium, calcium and zinc levels with inflammatory indices obtained from the whole blood count in patients with complex coronary artery disease. Material and method Fifty-two (42(81%) males, 10 (19%) females) consecutive patients (mean (SD) age 68 (9) years with symptomatic complex coronary artery disease were enrolled into prospective single center study in 2021. Serum, saliva and urine samples were collected at the day of admission for trace elements concentration (copper, zinc, magnesium, calcium) and compared with inflammatory indexes obtained from preoperative and perioperative period. Results Multivariable regression analysis revealed relation between the copper serum concentration and neutrophil to lymphocyte ratio (NLR) and systemic inflammatory index (SII). Conclusion Serum copper concentration interplay with preoperative inflammatory activation in complex coronary disease measured by NLR and SII. The copper serum concentration possesses the strongest relation to preoperative inflammatory activation in patients reffered for off-pump coronary artery disease.
... Copper accepts and donates single electrons as it transitions between its two redox states, Cu + and Cu 2+ . Extracellular oxidized Cu 2+ bound to a tripeptide (gly-his-lys) and complexed with glutathione (GSH) is purported to provide reduced copper (Cu + ) to the human copper transporter (hCTR1) for entry into the cell [84]. Once in the cell, Cu + is bound to an array of chaperones, storage and transport proteins to maintain optimal copper for oxidative phosphorylation, antioxidant protection by superoxide dismutase, and other essential activities [85]. ...
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Purpose of Review An epidemic of age-associated cognitive decline, most commonly ascribed to neurodegenerative conditions such as Alzheimer’s and Parkinson’s disease, is causing healthcare costs to soar and devastating caregivers. An estimated 6.5 million Americans are living today with Alzheimer’s disease, with 13.8 million cases projected by mid-century. Although genetic mutations are known to cause neurodegeneration, autosomal dominant disease is very rare and most sporadic cases can be attributed, at least in part, to modifiable risk factors. Recent Findings Diet is a potential modifiable risk factor in cognitive decline. Food communicates with the brain through a complex signaling web involving multiple cells, mediators and receptors. Gut-brain communication is modulated by microorganisms including bacteria, archaea, viruses, and unicellular eukaryotes, which together constitute the microbiota. Microbes not only play major roles in the digestion and fermentation of the food, providing nutrients and bioactive metabolites, but also reflect the type and amount of food consumed and food-borne toxic exposures. Food components modify the diversity and abundance of the microbial populations, maintain the integrity of the gut barrier, and regulate the passage of microbes and their metabolites into the blood stream where they modulate the immune system and communicate with body systems including the brain. Summary This paper will focus on selected mechanisms through which interactions between diet and the gut microbiota can modify brain integrity and cognitive function with emphasis on the pathogenesis of the most common dementia, Alzheimer’s disease.
... Interestingly, the importance of such partial decoordination on the reduction of Cu II by GSH was recently shown with the tridentate peptide ligand GHK. 25 It is also worth mentioning that the change in charge from the negative thiolate to a neutral thione is expected to decrease the electron density on Cu II and hence favor reduction to Cu I . Thus, considering that the Cu II −Dp44mT complex can exist in equilibrium with its protonated form Cu II −HDp44mT in solution (see Scheme 1), we have simulated the reprotonation of the N 2 atom (see Scheme 1) using the hydronium ion as a protonating agent. ...
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Glutathione (GSH) is the most abundant thiol in mammalian cells and plays a crucial role in maintaining redox cellular homeostasis. The thiols of two GSH molecules can be oxidized to the disulfide GSSG. The cytosolic GSH/GSSG ratio is very high (>100), and its reduction can lead to apoptosis or necrosis, which are of interest in cancer research. CuII ions are very efficient oxidants of thiols, but with an excess of GSH, CuIn(GS)m clusters are formed, in which CuI is very slowly reoxidized by O2 at pH 7.4 and even more slowly at lower pH. Here, the aerobic oxidation of GSH by CuII was investigated at different pH values in the presence of the anticancer thiosemicarbazone Dp44mT, which accumulates in lysosomes and induces lysosomal membrane permeabilization in a Cu-dependent manner. The results showed that CuII-Dp44mT catalyzes GSH oxidation faster than CuII alone at pH 7.4 and hence accelerates the production of very reactive hydroxyl radicals. Moreover, GSH oxidation and hydroxyl radical production by CuII-Dp44mT were accelerated at the acidic pH found in lysosomes. To decipher this unusually faster thiol oxidation at lower pH, density functional theory (DFT) calculations, electrochemical and spectroscopic studies were performed. The results suggest that the acceleration is due to the protonation of CuII-Dp44mT on the hydrazinic nitrogen, which favors the rate-limiting reduction step without subsequent dissociation of the CuI intermediate. Furthermore, preliminary biological studies in cell culture using the proton pump inhibitor bafilomycin A1 indicated that the lysosomal pH plays a role in the activity of CuII-Dp44mT.
... e g//factor equals 2.20, while the hyperfine splitting equals 185 ± 5 10 −4 cm −1 (dotted lines). e parameters are in line with a equatorial donor set of [2N,S,O] [53] and strongly reminiscent with those of a Cu(II) complex bound to three nitrogen atoms and one thiolate ligand [54]. In the perpendicular region, superhyperfine lines are observed that mirror the interaction of the Cu(II) center with nitrogen atoms form the ligand. ...
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A series of zinc(II) ([Zn(H2O)(L)Cl] (1)), copper (II) ([Cu(L)Cl] (2), [Cu(L)Br] (3), [Cu2(L)2(CH3COO)2]·4H2O (4)), nickel(II) ([Ni(HL)2]Cl2·H2O (5)), and cobalt(III) ([Co(L)2]Cl (6)) complexes were obtained with 2-formylpyridine N⁴-allylthiosemicarbazone (HL). In addition another two thiosemicarbazones (3-formylpyridine N⁴-allylthiosemicarbazone (HLa) and 4-formylpyridine N⁴-allylthiosemicarbazone (HLb)) have been obtained. The synthesized thiosemicarbazones have been studied using ¹H and ¹³C NMR spectroscopy, IR spectroscopy, and X-ray diffraction analysis. The composition and structure of complexes were studied using elemental analysis, IR and UV-Vis spectroscopies, molar conductivity, and magnetic susceptibility measurements. Single crystal X-ray diffraction analysis elucidated the structure of thiosemicarbazones HL, HLa, and HLb, as well as complexes 4 and 5. The antiproliferative properties of these compounds toward a series of cancer cell lines (HL-60, HeLa, BxPC-3, RD) and a normal cell line (MDCK) have been investigated. The nickel complex shows high selectivity (SI > 1000) toward HL-60 cell line and is the least toxic. The zinc complex shows the highest selectivity toward RD cell line (SI = 640). The copper complexes (2–4) are the most active molecular inhibitors of proliferation of cancer cells, but exhibit not such a high selectivity and are significantly more toxic. Zinc and copper complexes manifest high antibacterial activity. It was found that calculated at B3LYP level of theory different reactivity descriptors of studied compounds strongly correlate with their biological activity.
... 22 Likewise, frozen-solution EPR spectra yield comparable static (g ⊥ , g ∥ , A ⊥ , A ∥ ) EPR parameters ( Figures S4 and S5); moreover, the stability of low-molecular-weight Cu(GHK)N Im complexes is enhanced by orders of magnitude upon freezing. 22,25 Therefore, attempts to decompose the CD and UV−vis spectra of Cu/GHK/HSA mixtures ( Figure 2) using the above methods yield only the total concentration of Cu(GHK)N Im and not the absolute concentrations of Cu(GHK)N Im GHK and Cu(GHK)N Im HSA . In contrast to the above methods, room-temperature EPR is sensitive to the rotational motion of paramagnetic centers, which enables further discrimination of Cu 2+ complexes based on differences in their molecular mass. ...
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Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu2+ as part of their normal functions. GHK is found at its highest concentration in the albumin-rich fraction of plasma, leading to speculation that HSA and GHK form a ternary Cu2+ complex. Although preliminary evidence was presented 40 years ago, the structure and stability of such a complex have remained elusive. Here, we show that two ternary Cu(GHK)NImHSA complexes are formed between GHK and the imino nitrogen (NIm) of His side chains of HSA. We identified His3 as one site of ternary complex formation (conditional binding constant cKCu(GHK)NImHis3Cu(GHK) = 2900 M–1 at pH 7.4), with the second site (cKCu(GHK)NImHisXCu(GHK) = 1700 M–1) likely being supplied by either His128 or His510. Together with the established role of HSA as a molecular shuttle in the blood, these complexes may aid the transport of the exchangeable Cu2+ pool and the functional form of GHK.
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Copper(II) complexes of peptides with a histidine residue at the second position (His2 peptides) provide a unique profile of electrochemical behavior, offering signals of both Cu(II) reduction and Cu(II) oxidation. Furthermore, their structures with vacant positions in the equatorial coordination plane could facilitate interactions with other biomolecules. In this work, we designed a library of His2 peptides based on the sequence of Aβ5-9 (RHDSG), an amyloid beta peptide derivative. The changes in the Aβ5-9 sequence highly affect the Cu(II) oxidation signals, altered further by anionic species. As a result, Cu(II) complexes of Arg1 peptides without Asp residues were chosen as the most promising peptide-based molecular receptors for phosphates. The voltammetric data on Cu(II) oxidation for binary Cu(II)-His2 peptide complexes and ternary Cu(II)-His2 peptide/phosphate systems were also tested for His2 peptide recognition. We achieved a highly promising identification of subtle modifications in the peptide sequence. Thus, we introduce voltammetric measurement as a potential novel tool for peptide sequence recognition.
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Link to Free Full Text: ............................................................................................................................................ https://onlinelibrary.wiley.com/share/author/J5YPUTRMDWJNMENJYC8K?target=10.1002/cbdv.202100043 ............................................................................................................................................ Proteins anchor copper(II) ions mainly by imidazole from histidine residues located in different positions in the primary protein structures. However, the motifs with histidine in the first three N-terminal positions (His1, His2, and His3) show unique Cu(II)-binding properties, such as availability from the surface of the protein, high flexibility, and high Cu(II) exchangeability with other ligands. It makes such sequences beneficial for the fast exchange of Cu(II) between ligands. Furthermore, sequences with His1 and His2, thus, non-saturating the Cu(II) coordination sphere, are redox-active and may play a role in Cu(II) reduction to Cu(I). All human protein sequences deposited in UniProt Knowledgebase were browsed for those containing His1, His2, or His3. Proteolytically modified sequences (with the removal of a propeptide or Met residue) were taken for the analysis. Finally, the sequences were sorted out according to the subcellular localization of the proteins to match the respective sequences with the probability of interaction with divalent copper.
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Copper is crucial for carrying out normal physiological functions in all higher life forms. Copper Transporter 1 (CTR1) is the high-affinity copper importer found in all eukaryotic organisms. The copper transporter family primarily comprises ~ six members (CTR1-6) and the related members share high sequence homology with CTR. However, with the exception of CTR1, not all six CTRs are present in every organism. Despite having a simple trimeric channel structure, CTR1 and other members exhibit some unique regulatory properties. In the present review, we attempt to understand the diversity and similarity of regulation and functioning of the members of this copper transporter family. Graphic Abstract
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The Aβ5–x peptides (x = 38, 40, 42) are minor Aβ species in normal brains but elevated upon the application of inhibitors of Aβ processing enzymes. They are interesting from the point of view of coordination chemistry for the presence of an Arg-His metal binding sequence at their N-terminus capable of forming a 3-nitrogen (3N) three-coordinate chelate system. Similar sequences in other bioactive peptides were shown to bind Cu(II) ions in biological systems. Therefore, we investigated Cu(II) complex formation and reactivity of a series of truncated Aβ5–x peptide models comprising the metal binding site: Aβ5–9, Aβ5–12, Aβ5–12Y10F, and Aβ5–16. Using CD and UV–vis spectroscopies and potentiometry, we found that all peptides coordinated the Cu(II) ion with substantial affinities higher than 3 × 1012 M–1 at pH 7.4 for Aβ5–9 and Aβ5–12. This affinity was elevated 3-fold in Aβ5–16 by the formation of the internal macrochelate with the fourth coordination site occupied by the imidazole nitrogen of the His13 or His14 residue. A much higher boost of affinity could be achieved in Aβ5–9 and Aβ5–12 by adding appropriate amounts of the external imidazole ligand. The 3N Cu-Aβ5–x complexes could be irreversibly reduced to Cu(I) at about −0.6 V vs Ag/AgCl and oxidized to Cu(III) at about 1.2 V vs Ag/AgCl. The internal or external imidazole coordination to the 3N core resulted in a slight destabilization of the Cu(I) state and stabilization of the Cu(III) state. Taken together these results indicate that Aβ5–x peptides, which bind Cu(II) ions much more strongly than Aβ1–x peptides and only slightly weaker than Aβ4–x peptides could interfere with Cu(II) handling by these peptides, adding to copper dyshomeostasis in Alzheimer brains.
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The tripeptide NH2–Gly–His–Lys–COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature.
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The amino‐terminal copper and nickel/N‐terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards Cu II ions and have been implicated in human copper physiology. Little is known, however, about the rate and exact mechanism of formation of such complexes. We used the stopped‐flow and microsecond freeze‐hyperquenching (MHQ) techniques supported by steady‐state spectroscopic and electrochemical data to demonstrate the formation of partially coordinated intermediate Cu II complexes formed by glycyl‐glycyl‐histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two‐nitrogen coordination, t ½ ∼100 ms at pH = 6.0 and the ability to maintain the Cu II /Cu I redox pair is the best candidate for the long‐sought reactive species in extracellular copper transport.
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Aβ4-42 is the major subspecies of Aβ peptides characterized by avid Cu(II) binding via the ATCUN/NTS motif. It is thought to be produced in vivo proteolytically by neprilysin, but in vitro experiments in the presence of Cu(II) ions indicated preferable formation of C-terminally truncated ATCUN/NTS species including CuIIAβ4-16, CuIIAβ4-9, and also CuIIAβ12-16, all with nearly femtomolar affinities at neutral pH. Such small complexes may serve as shuttles for copper clearance from extracellular brain spaces, on condition they could survive intracellular conditions upon crossing biological barriers. In order to ascertain such possibility, we studied the reactions of CuIIAβ4-16, CuIIAβ4-9, CuIIAβ12-16, and CuIIAβ1-16 with reduced glutathione (GSH) under aerobic and anaerobic conditions using absorption spectroscopy and mass spectrometry. We found CuIIAβ4-16 and CuIIAβ4-9 to be strongly resistant to reduction and concomitant formation of Cu(I)-GSH complexes, with reaction times ∼10 h, while CuIIAβ12-16 was reduced within minutes and CuIIAβ1-16 within seconds of incubation. Upon GSH exhaustion by molecular oxygen, the CuIIAβ complexes were reformed with no concomitant oxidative damage to peptides. These finding reinforce the concept of Aβ4-x peptides as physiological trafficking partners of brain copper.
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Significant changes observed in the electrochemical response of the Cu( ii )-Aβ 5–9 complex upon phosphates addition provided a new insight into the design of a promising class of peptide-based molecular receptors selective for phosphate species.
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Amyloid beta (Aβ) peptides are notorious for their involvement in Alzheimer’s disease (AD), by virtue of their propensity to aggregate to form oligomers, fibrils, and eventually plaques in the brain. Nevertheless, they appear to be essential for correct neurophysiology on the synaptic level and may have additional functions including antimicrobial activity, sealing the blood–brain barrier, promotion of recovery from brain injury, and even tumor suppression. Aβ peptides are also avid copper chelators, and coincidentally copper is significantly dysregulated in the AD brain. Copper (Cu) is released in significant amounts during calcium signaling at the synaptic membrane. Aβ peptides may have a role in maintaining synaptic Cu homeostasis, including as a scavenger for redox-active Cu and as a chaperone for clearing Cu from the synaptic cleft. Here, we employed the Aβ1–16 and Aβ4–16 peptides as well-established non-aggregating models of major Aβ species in healthy and AD brains, and the Ctr1–14 peptide as a model for the extracellular domain of the human cellular copper transporter protein (Ctr1). With these model peptides and a number of spectroscopic techniques, we investigated whether the Cu complexes of Aβ peptides could provide Ctr1 with either Cu(II) or Cu(I). We found that Aβ1–16 fully and rapidly delivered Cu(II) to Ctr1–14 along the affinity gradient. Such delivery was only partial for the Aβ4–16/Ctr1–14 pair, in agreement with the higher complex stability for the former peptide. Moreover, the reaction was very slow and took ca. 40 h to reach equilibrium under the given experimental conditions. In either case of Cu(II) exchange, no intermediate (ternary) species were present in detectable amounts. In contrast, both Aβ species released Cu(I) to Ctr1–14 rapidly and in a quantitative fashion, but ternary intermediate species were detected in the analysis of XAS data. The results presented here are the first direct evidence of a Cu(I) and Cu(II) transfer between the human Ctr1 and Aβ model peptides. These results are discussed in terms of the fundamental difference between the peptides’ Cu(II) complexes (pleiotropic ensemble of open structures of Aβ1–16 vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aβ4–16) and the similarity of their Cu(I) complexes (both anchored at the tandem His13/His14, bis-His motif). These results indicate that Cu(I) may be more feasible than Cu(II) as the cargo for copper clearance from the synaptic cleft by Aβ peptides and its delivery to Ctr1. The arguments in favor of Cu(I) include the fact that cellular Cu export and uptake proteins (ATPase7A/B and Ctr1, respectively) specifically transport Cu(I), the abundance of extracellular ascorbate reducing agent in the brain, and evidence of a potential associative (hand-off) mechanism of Cu(I) transfer that may mirror the mechanisms of intracellular Cu chaperone proteins.
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Multiple intermediates were found in Cu( ii ) binding to Aβ 4–16 before the formation of a tight complex.
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Copper(II) binding properties of Saccharomyces cerevisiae pheromone (α-factor) analogues, namely WHWSKNR-am, FHWSKNR-am and WHFSKNR-am amino acid sequences, were studied using voltammetry techniques, fluorescence and UV–Vis spectroscopy. Despite the same 3N binding mode attributed to XHZ-type oligopeptides, the obtained results demonstrated high dependence of the electrochemical properties of their copper(II) complexes on the metal ion coordination environment. Changes triggered by ternary complex formation with imidazole molecule, leading to 4N structure formation, were also examined. The investigated derivatives underwent reduction and oxidation processes as well, however the coordination sphere affected mainly the anodic signal. Different electrochemical responses of α-factor analogues revealed significant influence of tryptophan residue position in a peptide sequence and indicated the complexity of ongoing redox processes. That stems from the presence of two different redox active centers and the possibility of the occurrence of multiple electrostatic interactions engaging charged groups, metal cation and indole moiety.