Low catalytic activity of the Cu(II)-binding motif (Xxx-Zzz-His; ATCUN) in reactive oxygen species production and inhibition by the Cu(i)-chelator BCS

Article abstract of DOI:10.1039/c8cc06040a

The catalytic redox activity of Cu(ii) bound to the motif NH₂-Xxx-Zzz-His (ATCUN) with ascorbate and H₂O₂/O₂ is very low and can be stopped via Cu(i)-chelation. This impacts its application as an artificial Cu-enzyme to degrade biomolecules via production of reactive oxygen species in a Cu(i)-chelator rich environment like the cytosol.

Full text of DOI:10.1039/c8cc06040a

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Low catalytic activity of the Cu(II)-binding motif
(Xxx-Zzz-His; ATCUN) in reactive oxygen species
production and inhibition by the Cu(I)-chelator
BCS†
Cite this: Chem. Commun., 2018,
54, 11945
Received 25th July 2018,
Accepted 27th September 2018
Alice Santoro,‡a Gulshan Walke, ‡§ab Bertrand Vileno,ac Prasad P. Kulkarni,b
DOI: 10.1039/c8cc06040a
a
Laurent Raibauta and Peter Faller
*
rsc.li/chemcomm
The catalytic redox activity of Cu(II) bound to the motif NH2–Xxx-
Zzz-His (ATCUN) with ascorbate and H2O2/O2 is very low and can
be stopped via Cu(I)-chelation. This impacts its application as an
artificial Cu–enzyme to degrade biomolecules via production of
reactive oxygen species in a Cu(I)-chelator rich environment like the
cytosol.
Fig. 1 (A) Mechanism for Cu catalysed ROS production in the presence of
ascorbate (AscHꢀ) and dioxygen. The electron flow is from the AscHꢀ to
the oxygen species, catalyzed by Cu. Step 1: Cu undergoes redox cycling,
classically between reduction of Cu(II) to Cu(I) through the oxidation of
AscHꢀ to Ascꢁꢀ. Step 2: dioxygen is reduced in one electron events by Cu(I)
Copper (Cu) is an essential element for most living organisms
being involved in various biological functions, mainly by play-
ing a redox catalytic role (essentially as Cu(I) and Cu(II)) in
enzymes. Free or loosely bound Cu ions are very efficient
catalysts in the production of reactive oxygen species (ROS),
including the Fenton type reaction.1,2 Hence, Cu-metabolism is
tightly controlled and almost all Cu ions are tightly bound to
proteins or peptides. Also during transport or storage, Cu is
bound to proteins.3,4 Serum albumin is implicated in the
to superoxide (O2 ꢀ), hydrogen peroxide (H2O2) and finally hydroxyl
ꢁ
radicals (HOꢁ). In the presence of AscHꢀ and H2O2 the reaction is mainly
limited to the last part highlighted by a red frame. (B) Schematic structure
of a Cu(II)–XZH complex. In the present study their activity as catalysts for
ROS production is studied.
transport of Cu in the blood stream via Cu(II)-binding to the of 10ꢀ12 to 10ꢀ15 Mꢀ1 10 Thus, by adding just three amino acids
.
N-terminal sequence Asp-Ala-His (for humans). This Cu(II)-site (XZH) to any peptide/protein at the N-terminus, or by mutating
is an example of the so called amino-terminal Cu and Ni-binding the third amino acid to a His, a strong Cu(II)-binding site can be
(ATCUN) motif, with the general sequence H2N–Xxx-Zzz-His introduced. Indeed, this strategy has been used by several
(XZH).5–7
groups in different contexts for anticancer or antimicrobial appli-
Cu(II) is bound to XZH in a square planar complex coordi- cations. The Cu(II)–XZH motif was introduced to add a ROS
nated by four nitrogens, the N-terminal amine, the two depro- catalytic unit playing the role of an artificial enzyme to degrade
tonated N of the amide (between X-Z and Z-H), and the N(Pi) of biomolecules (such as DNA, RNA, proteins or sugars).11–14
the imidazole (Fig. 1).8,9 The conditional dissociation constants
The cleavage of biomolecules catalyzed by Cu is a redox
at pH 7.4 for Cu(II) are sequence dependent but are in the range dependent mechanism (Fig. 1) involving Cu(I) and Cu(II) (Cu(III)
was also suggested15), and needs a reducing agent (like
ascorbate (AscHꢀ)) and an oxidant (mostly O2 or H2O2).12
a
´
Institut de Chimie, UMR 7177, CNRS-Universite de Strasbourg,
However, the same motif XZH was also used in chelation
therapy based on the redox silencing of Cu.8,16 For instance
peptides/proteins with an XZH motif have been used to suppress
the ROS production by Cu-amyloid-b in the presence of AscHꢀ and
O2.17 This is based on the retrieval of Cu from amyloid-b and strong
stabilization of Cu(II) bound to XZH. Also, the motif was used for
64Cu imaging, with the idea of a redox-inert Cu(II)–XZH complex.18
Hence, there seems to be a discrepancy. On the one hand
Cu(II)–XZH is used to produce ROS, for which an efficient redox
4 rue Blaise Pascal, 67000, Strasbourg, France. E-mail: pfaller@unistra.fr
b Bioprospecting Group, Agharkar Research Institute,
Savitribai Phule Pune University, Pune-411004, India
c French EPR Federation of Research (REseau NAtional de Rpe interDisciplinaire
´ ´
(RENARD), Federation IR-RPE CNRS #3443), Strasbourg, France
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8cc06040a
‡ Contributed equally to the work.
§ Present address: Department of Chemistry, Faculty of Exact Science, Bar Ilan
University, Ramat Gan, 5290002, Israel.
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cycling of Cu is warranted, on the other hand, the XZH motif is for Cu(II), Cu(II)–DAHK, Cu(II)–KGHK and Cu(II)–FRHD with 250 mM
used to redox silence Cu, based on an arrest of its redox cycling AscHꢀ and H2O2. The measurements with AscHꢀ or H2O2 only are
once Cu is bound to XZH. In order to gain insight into this shown in Fig. S2 (ESI†). Several observations can be made.
discrepancy, we studied three common variants of XZH, i.e. DAHK (i) Cu in the buffer is much more efficient in HOꢁ generation
(the motif from serum albumin19), KGHK (one of the most used than Cu(II)–XZH complexes (by about 2 orders of magnitude),
and most efficient to perform cleavage of biomolecules11) and (ii) in line with the literature the HOꢁ production is more
FRHD (the motif found in a truncated variant of amyloid-b with a efficient with H2O2 and AscHꢀ, about twice as fast as with
redox-silencing activity20). The redox activity and ROS production AscHꢀ alone. H2O2 alone produced HOꢁ very slowly, at least an
of these Cu(II)–XZH complexes were evaluated under the most order of magnitude slower than AscHꢀ alone (Fig. S2, ESI†).
classical conditions, i.e. with O2, AscHꢀ and/or H2O2. Even under Note, the concentrations of hundred mM used here are physio-
the most favorable conditions, the ROS production is very low.
logically relevant for AscHꢀ,21 but H2O2 is normally much
First, the HOꢁ production catalysed by the three Cu(II)–XZH lower.22 (iii) Although Cu bound to the XZH peptides showed
complexes was measured (for complex formation see Fig. S1, ESI†). very little activity, in all repetitions of the experiment Cu(II)–KGHK
The HOꢁ production was monitored by following the kinetics of was slightly more active than Cu(II)–DAHK and Cu(II)–FRHD.
fluorescence of 7-HO-CCA (7-hydroxycoumarin-3-carboxylic acid). However, this difference was around the statistical error, and
Coumarin-3-carboxylic acid (CCA) reacts with HOꢁ to produce hence is just a tendency.
7-HO-CCA. Fig. 2A shows the kinetics of 7-HO-CCA fluorescence
We confirmed the low HOꢁ production of Cu(II)–KGHK com-
pared with Cu in the buffer at different ratios of Cu(II):peptide
(1 : 1, 1 : 1.2, 1 : 2, 1 : 3), by EPR through a spin-trapping investiga-
tion in the presence of AscHꢀ and H2O2 (Fig. S3, ESI†).
The measurement of HOꢁ production via CCA or POBN
(a-(4-pyridyl N-oxide)-N-tert-butylnitrone) used to evaluate the redox
activity of Cu(II)–XZH complexes is indirect since it needs the
trapping of HOꢁ. HOꢁ can also be trapped by other molecules in
the solution (e.g. peptides), thus the measurement is not quanti-
tative. Hence, we also measured the consumption of the substrate,
i.e. AscHꢀ, via absorbance spectroscopy at lmax = 265 nm.
Indeed, Fig. 2B and Fig. S4 (ESI†) show that Cu in the buffer
oxidizes AscHꢀ rapidly (with or without H2O2) with an initial
rate of 12.3 ꢂ 1.9 mM minꢀ1 under the given conditions (Table 1). In
contrast Cu bound to the XZH peptides almost completely blocked
the AscHꢀ oxidation, with rates of 0.08–0.11 mM minꢀ1 (Table 1).
These rates are similar to the background of AscHꢀ oxidation (in
which no Cu and peptide are present). This parallels the HOꢁ
trapping experiments shown above. Moreover, Cu(II)–KGHK was
slightly more active than DAHK/FRHD. The AscHꢀ oxidation rate of
Cu(II)–KGHK at a 1 : 1.2 ratio was the same in phosphate buffer and
HEPES buffer (Fig. S9 and S10, ESI†), although Cu alone showed
higher activity in HEPES than phosphate (18.9 mM minꢀ1).
For comparison, we also selected well known redox active
Cu(II)-complexes, i.e. Cu(II)DmBipy2 (5,50-DmBipy: 5,50-dimethyl-
2,20-dipyridyl), and Cu(II)Phen2 (Phen: 1,10-phenantroline) that
Table 1 Molar AscHꢀ oxidation rate (mM minꢀ1), calculated for the different
Cu(II)-complexes tested in this work (Fig. 2B)
Fig. 2 (A) Time course of HOꢁ production: evolution of fluorescence
of the HOꢁ adduct of CCA (HO–CCA; excitation: l = 390 nm; emission
l = 450 nm) as a function of time in the presence of AscHꢀ and H2O2.
Cu(II)–XZH (H–DAHK–OH, H–KGHK–OH, H–FRHD–NH2) complexes
were pre-formed at the desired ratio (Cu(II) : peptide, 1 : 1.2 to avoid the
presence of free Cu in phosphate buffer (50 mM, pH 7.4)). The final
concentrations of Cu(II), peptide/ligands, AscHꢀ, H2O2 and CCA used were
25 mM, 30 mM, 250 mM, 250 mM and 0.5 mM respectively in 50 mM
phosphate buffer, pH 7.4. (B) Time course of AscHꢀ oxidation: evolution of
the AscHꢀ absorption (lmax = 265 nm) as a function of time after exposure
to free Cu(II), Cu(II)–XZH, Cu–His2, Cu(II)–5,50-DmBipy2, and Cu(II)–Phen2
complexes with H2O2. AscHꢀ oxidation was started by the addiction of free
Cu(II) or the preformed Cu(II)–X complexes after 10 min. The final concentrations
of Cu(II), peptide/ligands, AscHꢀ and H2O2 were respectively 10 mM, 12 mM/
24 mM, 100 mM and 100 mM in 50 mM phosphate buffer, pH 7.4.
a
Cu(II)-complex
robs (mM minꢀ1
)
Background
Cu(II)
0.11 ꢂ 0.06
12.3 ꢂ 1.9
Cu(II)–KGHK (1 : 1.2)
Cu(II)–FRHD (1 : 1.2)
Cu(II)–DAHK (1 : 1.2)
Cu(II)–His (1 : 2.4)
Cu(II)–5,50-DmBipy (1 : 2.4)
Cu(II)–Phen (1 : 2.4)
0.11 ꢂ 0.01
0.06 ꢂ 0.02
0.08 ꢂ 0.03
0.89 ꢂ 0.02
6.16 ꢂ 1.07
8.29 ꢂ 0.61
a
Measurements were performed in triplicate with different solutions at
different days and the average values of robs (mM minꢀ1) with standard
deviations are reported.
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have been used to produce ROS via a Cu dependent mechanism
(Fig. 2B).23,24 Both Cu(II)–5,50-DmBipy2 and Cu(II)–Phen2 were
about as active as free Cu(II) in AscHꢀ oxidation. Also Cu(II)–His2
was more active than Cu(II)–XZH. All three complexes were one or
two orders of magnitude faster than Cu(II)–XZH (Table 1). If one
subtracts the background of AscHꢀ oxidation the difference is
even larger. Hence comparison with these Cu(II)-complexes con-
firms that Cu(II)–XZH complexes are very slow catalysts for ROS
production, even under the most favorable conditions. Even in the
most active case, i.e. Cu(II)–KGHK in the presence of AscHꢀ and
H2O2, less than 7 mM AscHꢀ was consumed over 1 h (Fig. 2B). This
corresponds to a maximal turnover rate of about 0.7 per hour with
100 mM AscHꢀ and H2O2. Subtracting the background AscHꢀ
oxidation yields even lower activity.
This very low reactivity was also confirmed by measuring the
effect of AscHꢀ or H2O2 on the Cu(II) d–d bands of Cu(II)–KGHK,
Cu(II)–DAHK and Cu(II)–FRHD. In the case of a strong reactivity
with the substrate one would expect the disappearance of
the d–d bands, either due to reduction to Cu(I) by AscHꢀ or
oxidation to Cu(III) by H2O2. But even at a 100 times excess, no
significant changes in the d–d bands typical for Cu(II) could be
observed (V Fig. S5 and S6, ESI†).
Fig. 3 Kinetics representing the tendency of formation of free or loosely
bound Cu(I) from Cu(II)–KGHK in the presence of (i) AscHꢀ (black squares
ꢀ
profile), (ii) AscHꢀ and H2O2 (red circles profile), (iii) H2O2 (blue triangle
profile), and (iv) blank, i.e. no AscHꢀ and H2O2 (inversed grey triangle) using
BCS as a Cu(I) chelator (lmax [Cu(I)–BCS2] = 483 nm). Inset: Corresponding
UV-vis spectra for (iv) (left) and (iii) (right) conditions: concentrations of
Cu(II), peptide, AscHꢀ, H2O2 and BCS were respectively 100 mM, 120 mM,
10 mM, 10 mM and 200 mM; 50 mM phosphate buffer, pH 7.4.
Next, we investigated the redox state(s) of Cu that is/are important to note that BCS can complex the formed Cu(I),
involved in the slow catalytic reaction of Cu(II)–XZH with AscHꢀ indicating that Cu(I) is not strongly bound and accessible for
and/or H2O2. In the presence of AscHꢀ, it is generally assumed a ligand.
that the ROS production by Cu(II)–XZH complexes takes place
To confirm the relation of Cu(I) formation to the HOꢁ
via a redox cycling between Cu(II) and Cu(I), as AscHꢀ is a production, we measured HOꢁ in the presence of BCS. If the
reducing agent. It is known from electrochemical studies that formed Cu(I) is the key species for the HOꢁ formation, in the
Cu(II)–XZH is very difficult to reduce and that Cu(I) does not presence of BCS no HOꢁ should be produced, because it is known
bind to the XZH motif, as soft Cu(I) is not acidic enough to that Cu(I)–BCS2 is very redox inert and hence does not react with
deprotonate amides and it prefers a tetrahedral and not a oxygen under aerobic conditions. As BCS could interfere with CCA
square planar coordination geometry.10 Hence, we hypothe- fluorescence because of its absorption at the same spot we used
sized that if the HOꢁ production of the Cu(II)–XZH complexes EPR Spectroscopy. Fig. 4 shows the HOꢁ production by Cu(II)–
passes via Cu(I), this Cu(I) is not strongly bound and could be KGHK measured using POBN as a spin trap. After 4 hours of
retrieved from the peptide. To address this, we used batho- incubation using AscHꢀ and H2O2, a signal originating from
cuproinedisulfonate (BCS), a well-known Cu(I)–ligand/chromo- trapped HOꢁ could be detected. However, in the presence of
phore (Fig. 3). Adding BCS to Cu(II)–KGHK, Cu(II)–DAHK or BCS, after 4 h no signal was detected. This supports the mecha-
Cu(II)–FRHD did lead to a very small increase of less than 0.003 nism that indeed when the formed Cu(I) from Cu(II)–XZH is
corresponding to the typical Cu(I)–BCS2 complex (lmax
=
chelated by BCS no HOꢁ is detected anymore.
483 nm) (left inset Fig. 3). This corresponds to 0.2 mM Cu(I)–
In conclusion, our data indicate that the ROS production of
BCS2 formed after 1 h. However, upon AscHꢀ addition the band Cu(II)–XZH is very slow, with KGHK being tentatively the most
at 483 nm increased steadily due to the Cu(I)-binding to BCS. active peptide, and that in the presence of both AscHꢀ and H2O2 it
This indicates that the formation of Cu(I) is directly linked to is less than 0.7 turnover per hour under the present conditions.
HOꢁ production. Analogous experiments were conducted with Other well known Cu(II)-complexes (Cu(II)–DmBipy2/Cu(II)–Phen2)
H2O2 alone. As H2O2 is an oxidant, we were surprised, that or free Cu had two orders of magnitude higher initial turn-over
Cu(I)–BCS2 was also formed and clearly more than the back- rates. Moreover, the test with the Cu(I) specific chelator BCS
ground (right inset Fig. 3), at least for Cu(II)–KGHK. Again, the suggests that Cu(I) is involved in the mechanism, indicating that
amount of Cu(I)–BCS2 formed paralleled the HOꢁ production the redox couple Cu(I)/Cu(II) is predominant and not Cu(II)/Cu(III).
efficiency. This indicates that also in the case of H2O2 only, a This is also supported by the much lower HOꢁ production activity
strong oxidant, the HOꢁ production takes place via Cu(I) and in the presence of H2O2 alone. If Cu(III) would easily be reached, a
not Cu(III). Cu(III) could have been expected, because the square fast reaction of Cu(II) + H2O2 - Cu(III) + HOꢁ could occur (at least
planar coordination in XZH (Fig. 1) is well adapted for Cu(III), one turnover). This is not observed. This is also in line with the
but not for Cu(I).1,2,10 However, this seems not to be the case as redox potential of around 1 V (NHE) for Cu(II)–XZH (H2O2/HOꢁ
the BCS test shows the formation of Cu(I)–BCS2. An explana- with 0.32 V).
tion, in line with the very slow kinetics, is that H2O2 is reducing
Overall this indicates that the cleavage of biomolecules by
Cu(II)–XZH, as it is known for Cu in the buffer.25 Moreover, it is Cu(II)–XZH with AscHꢀ and H2O2/O2 is catalytically not very
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(ASTF 505-2016), respectively. The Frontier Research in Chemistry
Foundation of Strasbourg (P. F.), the University of Strasbourg
Institute for Advanced Study (USIAS) (P. F.), IDEX attractivity
program of the University of Strasbourg (L. R.) and the Reseau
´ ´
NAtional de Rpe interDisciplinaire (RENARD, Federation IR-RPE
CNRS #3443) are acknowledged. P. F. would like to thank
Dr C. Hureau (LCC, Toulouse) for helpful discussions. P. K. would
like to acknowledge funds from the Department of Science and
Technology, India (EMR/2014/001235). We would like to thank
Dr Romain Ruppert (UMR7177 – Institut de Chimie, Strasbourg)
for the gift of the Phen and DmBipy compound.
Conflicts of interest
Fig. 4 Indirect evidence of HOꢁ production by Cu(II)–KGHK measured by
EPR spin trapping with POBN in the presence (right panel) and absence
(left panel) of BCS. A POBN–CH3 spin adduct (g = 2.0056, AH = 2.7 G,
AN = 16 G) was observed after 4 h of mixing Cu(II)–KGHK (at 1 : 1.2 ratio)
with AscHꢀ and H2O2. It results from the reaction between the spin trap
and a carbon centred radical originating from the decomposition of EtOH
with HOꢁ. EtOH was added here as an efficient hydroxyl scavenger (5% v/v)
to enhance the detection threshold and thus the EPR S/N. The two lines
observed at t = 0 are ascribed to the ascorbyl radical. Experimental
conditions: KGHK 120 mM, Cu(II) 100 mM (1.2 : 1), AscHꢀ 1 mM, H2O2
1 mM, PB 100 mM, pH 7.4, POBN 50 mM, ETOH 5%, ꢂBCS 300 mM.
There are no conflicts to declare.
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A. S. and G. W were supported by the IDEX PhD program
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11948 | Chem. Commun., 2018, 54, 11945--11948
This journal is ©The Royal Society of Chemistry 2018