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Metal complexes with valuable biomolecules

Cite this: Dalton Trans., 2023, 52,
produced by Pseudomonas aeruginosa: a review of
4276 the coordination properties of pyocyanin,
pyochelin and pyoverdines
Tina P. Andrejević, a Darko P. Ašanin, b Bojana V. Pantović, a
Nevena Lj. Stevanović, a Violeta R. Marković, a Miloš I. Djuran c
and
Biljana Đ. Glišić *a

Pseudomonas aeruginosa is an opportunistic, Gram-negative bacterium, involved in severe infections

Received 30th January 2023,
Accepted 14th March 2023
DOI: 10.1039/d3dt00287j
rsc.li/dalton
associated with cystic fibrosis, pneumonia, burn wounds, ocular diseases, and immunosuppressive ill-
nesses, and is a major cause of intrahospital infections. This bacterium is also one of the most commer-
cially and biotechnologically significant microorganisms, since it can produce valuable biomolecules
which represent a rich source of potential drug candidates. On the other hand, metal complexes have
been used in medicine for both therapeutic and diagnostic purposes since ancient times. This class of
compounds can adopt different geometries and generally have a three-dimensional shape, contributing
to their higher clinical success compared to flat purely organic compounds. In the present review article,
attention has been devoted to the three natural products derived from P. aeruginosa, namely pyocyanin,
pyochelin, and pyoverdine(s) and their ability to form complexes with different metal ions, including
iron(II/III), manganese(II/III), gallium(III), chromium(III), nickel(II), copper(II), zinc(II) and cadmium(II).
Investigation of the coordination properties of pyocyanin, pyochelin, and pyoverdine(s) towards these
metal ions is important because the resulting bacterially derived natural product-metal complex can serve
as a model for the study of metal ion metabolism (transport and storage) in living systems and might also
be considered as a novel therapeutic agent for potential use in medicine.

Introduction
Pseudomonas aeruginosa is one of the members of the Gamma
a
University of Kragujevac, Faculty of Science, Department of Chemistry, R. Proteobacterium class of bacteria, a Gram-negative and
Domanovića 12, 34000 Kragujevac, Serbia. E-mail: biljana.glisic@pmf.kg.ac.rs
b
aerobic rod-shaped bacterium, which can be found in soil,
University of Kragujevac, Institute for Information Technologies Kragujevac,
Department of Science, Jovana Cvijića bb, 34000 Kragujevac, Serbia
water, and many other environments.1 This bacterium has
c
Serbian Academy of Sciences and Arts, Knez Mihailova 35, 11000 Belgrade, Serbia gained attention due to its pathogenicity, and also because of

Tina P. Andrejević was born in 1993 in Kragujevac (Serbia). She
finished her MSc studies in 2017 under the supervision of
Professor Miloš I. Djuran, at the Faculty of Science, University of
Kragujevac. She started her PhD studies in Chemistry in 2017.
Since 2021, Tina Andrejević has been a Research Assistant at the
Department of Chemistry (University of Kragujevac, Serbia). Her
research interests are focused on the synthesis and characteriz-
ation of metal complexes, as well as their biological evaluation.
She has also been interested in the investigation of interactions of
the synthesized complexes with biologically important molecules
( proteins and nucleic acids).
Dr Darko P. Ašanin was born in 1976 in Kragujevac, Serbia. In
2003, he received his BSc in Chemistry from the University of
Kragujevac, Faculty of Science. He finished his PhD studies in
2015 at the Faculty of Science, University of Kragujevac. Dr Ašanin
is a Research Associate at the Department of Science, Institute for
Information Technologies, University of Kragujevac, Serbia. The
main research interests of Dr Ašanin are related to synthesis and
structural characterization of different transition metal complexes
as potential therapeutic agents, and investigation of their inter-
actions with biologically important molecules.

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Dalton Transactions
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Fig. 1 Structural formulas of three natural products produced by
P. aeruginosa.
its color and ability to produce different pigments. It is worth
mentioning that no other species of Gram-negative non-fer-
menting bacteria can produce a pigment called pyocyanin that
can be used in the identification of P. aeruginosa.1 Pyocyanin
(Fig. 1) is a blue-green pigment that acts as both a virulence
factor and a quorum sensing signaling molecule for
P. aeruginosa.1 It is an aromatic nitrogen-containing com-
pound, which belongs to the tricyclic phenazine class of com-
pounds. Pyocyanin is a zwitterion that contains a phenol
group, which contributes to its weak acidic characteristics ( pKa
of 4.9).2 It can exist in three states, ionized at physiological pH
(blue color), protonated in an acidic solution (red color) and
neutral solution (blue color).2 Pyocyanin is involved in various
important biological processes, such as gene expression, main-
taining the fitness of bacterial cells, and biofilm formation,
and it is also recognized as an electron shuttle for bacterial
respiration. The phenazine family, to which pyocyanin
belongs, has shown a variety of significant biological effects,
including antimicrobial and antitumor activities.3,4 Due to the
broad-spectrum antimicrobial activity against various bacterial
and fungal pathogens, several phenazines, such as phenazine-
1-carboxylic acid and phenazine 5-oxide, have been approved
Bojana V. Pantović was born in 1997 in Kragujevac, Serbia. In
2021, she received her MSc in Chemistry from the University of
Kragujevac, Faculty of Science. She was awarded by the Serbian
Chemical Society for great success during her studies. In 2021, she
enrolled in PhD studies under the mentorship of Dr Biljana Đ.
Glišić. Since 2022, Bojana has been a Junior Research Assistant at
the Faculty of Science, University of Kragujevac. She is interested
in the field of Medicinal Inorganic Chemistry. Her research is
focused on the synthesis and structural characterization of various
transition metal complexes with biologically important ligands.
This journal is © The Royal Society of Chemistry 2023
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as pesticides in agricultural crops.5,6 Phenazines have low
molecular weight and zwitterionic properties, allowing them
to easily penetrate cell membranes and cross through the
blood–brain barrier, resulting in cell damage.7 Moreover, the
redox-active tricyclic zwitterion forms reactive oxygen species
(ROS), a process which can influence the cardiovascular, res-
piratory, urological, and central nervous systems.8,9 In the case
of pyocyanin, no toxicity or oxidative stress was observed in mice
upon its application at low concentrations,10 while a higher con-
centration of pyocyanin (50–100 μM) may lead to ROS formation,
oxidative stress, and toxicity, as well as ATP depletion and cell
death.11 Pyocyanin is produced in a large amount in media with
a low concentration of iron and plays an important role in the
metabolism of this metal, which is required for the growth of
P. aeruginosa.12 Pyocyanin also participates in the mechanism
related to the reduction and release of iron from its transport
protein, transferrin. Considering this, the study of its reactions
with different metal ions, including Fe(II), could be of great
importance for gaining knowledge on the subject of metal ion
transport and storage in living systems.
As an aerobic bacterium, P. aeruginosa has also developed
systems by which it fulfills its needs for iron,13 including the
biosynthesis of molecules known as siderophores, which have
an affinity to bind Fe(III) ions.14 P. aeruginosa produces two
major siderophores to satisfy its needs for iron, namely pyo-
chelin and pyoverdine (Fig. 1). These siderophores are iron
chelating agents, which solubilize and transport iron through
the bacterial membranes via specific receptor proteins FptA
and FpvA, respectively, and have a TonB-like system for iron
translocation through the cytoplasmic membrane.13 Both are
also responsible for P. aeruginosa infection and are virulence
factors.15 Pyoverdine appears to be more essential for the
growth of P. aeruginosa, under iron-depleted conditions, while
pyochelin is only moderately synthesized.16,17 Moreover, it was
shown that P. aeruginosa possesses a regulatory switching
mechanism which enables it to produce its two major sidero-
phores as a function of iron concentration.18 Thus, it syn-
thesizes pyochelin first and switches to pyoverdine when the
concentration of iron is severely limited since the production
of pyoverdine is more energy consuming. Pyoverdine has a
much higher binding affinity for Fe(III) than pyochelin ( pFe =
27.0 and 16.0, respectively),19,20 but it is also produced at least
Nevena Lj. Stevanović was born in 1993 in Jagodina, Serbia. In
2018, she received her MSc in Chemistry from the University of
Kragujevac, Faculty of Science under the supervision of Dr Biljana
Đ. Glišić. She started her PhD studies (Inorganic Chemistry) in
2018 in the same research group. During her PhD studies, she
spent five months in the group of Prof. Iztok Turel at the
University of Ljubljana. Her research has been focused on the syn-
thesis and characterization of silver(I), gold(III), copper(II) and
zinc(II) complexes with antifungal azoles and investigation of their
reactions with nucleic acids and proteins.
Dalton Trans., 2023, 52, 4276–4289 | 4277

Perspective
10-fold more than pyochelin.21 As chelating ligands, both pyo-
chelin and pyoverdine can form stable complexes with
different metal ions, which could be further considered as
potential therapeutics.
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Pyochelin is a hydrophobic siderophore, containing a
hydroxyphenyl–thiazolinyl–thiazolidin scaffold (Fig. 1).22
Structurally, it is a monocarboxylic acid that belongs to the
group of phenolic compounds and it is a member of thiazoli-
dines and is biosynthetically derived from one salicylate mole-
cule, and two cysteine molecules, which initially gave salicyl-
cysteinylcysteine that is later dehydrated, methylated, and
reduced to pyochelin.22 Pyochelin represents an equilibrium
mixture of two stereoisomers differing in the configuration at
C-2″ (Fig. 1): pyochelin I which has the absolute configuration
4′R, 2″R, 4″S and pyochelin II with the following configuration:
4′R, 2″S, 4″S.23–25 Besides this, two additional stereoisomers,
neopyochelin I and II, were obtained during synthesis and
have a C-4′S configuration.25
Pyoverdine is a yellow-green, fluorescent pigment produced
by the fluorescent Pseudomonas species. It acts as a marker for
bacterial differentiation and, as a siderophore, it is an efficient
scavenger and transporter of the Fe(III) ion.26 Although the dis-
covery of pyoverdine (due to its fluorescence properties) dates
to the end of the 19th century, its structure remained unknown
until 1981.27 Until now, more than 100 pyoverdines, which are
produced by different strains and species of the Pseudomonas
bacterium, have been identified.28 Pyoverdines contain a
hydroxyquinoline moiety attached to a short peptide with 6–14
amino acid residues.29 The structural formula of the pyover-
dine produced by P. aeruginosa is presented in Fig. 1.30 Three
structural segments can be differentiated: (1) a quinoline
moiety, which is the chromophore, responsible for the color
and fluorescence of the biomolecule, (2) an acyl chain attached
to the amino group of the chromophore, and (3) a peptide
chain linked by its N-terminus to the carboxyl group of the
chromophore. The chromophore is (1S)-5-amino-2,3-dihydro-
8,9-dihydroxy-1H-pyrimido[1,2-a]quinoline-1-carboxylic acid
and is identical for all pyoverdines.29 The second segment, an
acyl chain, is the residue of a dicarboxylic acid, which can be a
succinate or malate and their amide forms, or α-ketoglutarate
or glutamate, depending on the strain that produced pyover-
dines, as well as growth conditions.26 A few forms of pyover-
dines (isoforms), differing only in the type of acyl chain, are
Dr Violeta Marković was born in 1985 in Meulan, France. She
defended her PhD thesis titled: “Synthesis, spectral characteriz-
ation and mechanistic studies of new pyrazole and pyrazolone
derivatives”, in 2012 at the Faculty of Science, the University of
Kragujevac. She has been a postdoctoral researcher at Sapienza,
University of Rome and Wrocław University of Science and
Technology. Since 2018, Dr Marković has been an Assistant
Professor at the Faculty of Science, University of Kragujevac. Her
research interest is focused on the synthesis and structural charac-
terization of potentially bioactive organic molecules.
4278 | Dalton Trans., 2023, 52, 4276–4289
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Dalton Transactions
usually present in the growth medium and can be separated
by ion-exchange chromatography or by isoelectrophoresis.
Nevertheless, pyoverdines differing only in the type of acyl
chain show an identical efficiency in iron uptake.26 The most
important part of pyoverdines is the peptide chain, which
can involve unusual amino acids, such as δN-acyl(acetyl or
formyl or β-hydroxybutyryl)-δN-hydroxyornithine, cyclized δN-
hydroxyornithine, β-hydroxyaspartic acid, β-hydroxyhistidine,
diaminobutyric acid, and common amino acids, which can
also have a D-configuration.26 Pyoverdines are perfectly
designed for the iron chelation, since the chromophore can
coordinate to the Fe(III) ion via a catecholate function, while
a linear or cyclic peptide moiety can bind to this metal ion
through two hydroxamate and/or carboxylate functional
groups.29
Considering the great importance of pyocyanin, pyochelin
and pyoverdines, all produced from P. aeruginosa, in the
present review article, the overview of their coordination behav-
iour toward different metal ions (including Fe(II/III), Mn(II/III),
Ga(III), Cr(III), Ni(II), Cu(II), Zn(II) and Cd(II)) has been given.
The study of the reactions between these three P. aeruginosa-
derived natural products and the metal ions is important for
the following reasons. First, these metal ions play an essential
role in cellular function owing to their redox and coordination
chemistry. As cofactors for different enzymes, they participate
in key processes such as respiration, neural transmission, elec-
tron transfer processes, photosynthesis, oxygen transport,
tissue maturation, defence against oxidative stress and iron
metabolism.31 Furthermore, the compounds resulting from
the complexation reactions of the natural products and metal
ions could be considered as model-molecules for the expla-
nation of the role of these ions in biological systems, and
potential drug candidates with some crucial advantages com-
pared to purely organic compounds, as they preferably have
different modes of action (ligand exchange or release, ROS for-
mation, redox activation and catalytic generation of toxic
species) and a three-dimensional shape.32 Based on the above-
mentioned facts, the findings of the studies subjected to this
review could help in understanding the pathways of the metal
ion metabolism in living systems, and can be used in some
important processes, including phytoextraction, which is
based on the use of plants to remove heavy and toxic metals
from the environment.33
Prof. Miloš I. Djuran received his PhD in Chemistry at the University
of Kragujevac in 1985. He worked as a postdoc at the Leiden
University with Professor Jan Reedijk (1989–1990), and at the
University of London with Professor Peter J. Sadler (1992–1994). In
1985 he was appointed as an Assistant Professor at the University of
Kragujevac, where he subsequently became an Associate Professor
(1995) and a Full Professor (2001). He was elected as a member of
the Serbian Academy of Sciences and Arts in 2015. His research
focuses on the syntheses, structural characterization, and biological
evaluation of transition metal complexes.
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Dalton Transactions
Metal complexes with pyocyanin
To the best of our knowledge, pyocyanin is not used as a
ligand for the synthesis of biologically active metal complexes.
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However, the complexation reactions of pyocyanine with
different metal ions (Mn(II), Fe(II), Ni(II), Cu(II), and Zn(II)) as a
model for metal–flavin interactions have been studied by spec-
trophotometric and electrochemical techniques,34 and also
with alkali, alkaline earth and Zn(II) ions by electrospray mass
spectrometry.35
The results obtained for the reactions with different M(II)
ions (M(II) = Mn, Fe, Ni, Cu and Zn) in DMSO in the presence
of 0.1 M tetrapropylammonium perchlorate have shown that
pyocyanine is a useful model for riboflavin, based on both
redox considerations and the ability to form similar com-
plexes.34 The data obtained from UV-Vis measurements
suggested that the metal complexes formed in the studied
reactions contain one M(II) ion and one pyocyanin, and that
their relative stability constants are in the following order:
Ni(II) > Fe(II) > Zn(II) ≈ Cu(II) > Mn(II). However, the stability
constants of the complexes formed with the pyocyanin anion
radical determined from CV measurements are considerably
greater and are as follows: Zn(II) > Ni(II) > Mn(II) > Fe(II). This
method could not be used for the evaluation of the copper(II)
complex with the pyocyanin anion radical, due to the
reduction of the Cu(II) to the Cu(I) ion. It is worth mentioning
that metal complexation of the pyocyanin anion radical may
account for the well-known antimicrobial activity of
pyocyanin.34
From the results of mass spectrometry for the reactions of
pyocyanin with alkali, alkaline earth and Zn(II) ions, it can be
concluded that equimolar aqueous solutions of the metal ion
and pyocyanin yielded [M( pyocyanin)n]+ (M = alkali metal; n =
1–3), [M( pyocyanin)n]2+ (M = Mg(II), Ca(II), n = 1–4; M = Ba(II), n
= 1–3) and [Zn( pyocyanin)n]2+ (n = 2–3) complex species.35
Competition experiments have shown that the radius of the
studied metal ion has an influence on the complexation reac-
tion; pyocyanin is more strongly bound to the smaller metal
ion in each group. Moreover, the electrospray spectra of the
aqueous solutions containing equimolar amounts of the
corresponding metal cation, pyocyanin and 2,2′-bipyridine are
in accordance with the formation of both single- and mixed-
Dr Biljana Đ. Glišić was born in 1985 in Kragujevac (Serbia). She
finished her PhD studies in 2012 under the supervision of
Professor Miloš Djuran at the University of Kragujevac. She has
been a postdoctoral researcher at Heidelberg University, Adam
Mickiewicz University and University of Fribourg. Since 2022,
Dr Glišić has been an Associate Professor at the Faculty of Science,
University of Kragujevac. Her research has been focused on the
synthesis and structural characterization of transition metal com-
plexes, as well as evaluation of their antimicrobial and cytotoxic
activities. For extraordinary success in science, she was awarded
from the Serbian Chemical Society in 2017.
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Perspective
ligand complexes, with a preponderance of the 2,2′-bipyridine
containing ions. However, for the complexes containing one or
both the abovementioned ligands per metal ion, the collision
activated dissociation spectra showed that the preferred
decomposition pathway includes the loss of 2,2′-bipyridine
rather than the release of pyocyanin. In this experiment, the
Zn(II) ion was the only exception, for which a release of pyocya-
nin was favored.35
Metal complexes with pyochelin
Iron(III)
The experiments performed by adding iron(III) to the pyoche-
lin-containing solution showed that this biomolecule coordi-
nates to the Fe(III) ion with a stoichiometry of 2 : 1, respectively,
whereby the formed complex species has a remarkably low
stability constant of ca. 105 M−1.36 On the other hand, X-ray
diffraction analysis on the pyochelin–iron(III) complex has
revealed that the metal-to-ligand ratio is 1 : 1 and that only the
pyochelin I stereoisomer is present in the crystalline form of
the complex (Fig. 2).37 The preference of only one of the two
pyochelin stereoisomers to chelate the Fe(III) ion and the for-
mation of an iron(III) complex with the metal-to-ligand ratio of
1 : 1 in solution have also been proved by high-performance
liquid chromatography/electrospray ionization mass
spectrometry.38
Based on the single-crystal X-ray analysis, it was found that
the pyochelin–iron(III) complex has an octahedral geometry,
whereby four coordination sites of the Fe(III) ion are occupied
by the phenolate and the carboxylate oxygen atoms and by the
two nitrogen atoms of pyochelin I.37 Two equivalent pyoche-
lin–Fe(III) units are held together by a hydroxy group and an
acetate unit, satisfying the remaining two coordination sites of
Fe(III) (Fig. 2). The cavities between the pyochelin–Fe(III) moi-
eties, which contain disordered solvent molecules, are present
in the crystal of this complex.
Fig. 2 ORTEP representation of the iron(III) complex with pyochelin.37
Thermal ellipsoids are drawn at the 50% probability level. CSD refcode
YELJOP (CCDC number 299297).39 Reproduced with permission from
ref. 37. Copyright (2006) Verlag der Zeitschrift für Naturforschung.
Dalton Trans., 2023, 52, 4276–4289 | 4279

Perspective
Abdallah et al. reported the synthesis of the mixed iron(III)
complex with cepabactin, a siderophore from Burkholderia
cepacia,40 and pyochelin.41 Firstly, the authors performed the
total synthesis of cepabactin and the corresponding iron(III)
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complex, which contains three cepabactin molecules bident-
ately coordinated to the Fe(III) ion (Fig. 3a), and compared its
structure with that obtained from B. cepacia culture. For this
purpose, two B. cepacia strains (ATCC 17754 and ATCC 25416;
ATCC is American Type Culture Collection) were grown in a
succinate containing medium under iron-depleted conditions,
and when the supernatant was supplemented with iron(III)
chloride, along with pyochelin, two different iron(III) com-
plexes, an orange and a purple one, were isolated and purified
by chromatography.41 Based on the UV-Vis and mass spec-
trometry measurements, it was concluded that the orange
compound was an iron(III) complex with cepabactin, which was
identical to that obtained in the synthesis. On the other hand,
the spectroscopic results suggested that the purple colored
compound contains Fe(III), cepabactin and pyochelin constitu-
ents in a 1 : 1 : 1 molar ratio (Fig. 3b). In this complex, four
coordination sites of the Fe(III) ion are occupied by pyochelin
donor atoms, while the remaining two sites belong to the che-
lated cepabactin molecule. The results obtained from mass
spectrometry measurements also suggested that the iron(III)
complex with pyochelin has low stability in solution, and that
the most stable species is the iron(III) complex with cepabactin
or that with cepabactin and pyochelin.41
The high resolution crystal structure (2.00 Å) of the pyoche-
lin outer membrane receptor FptA bound to the pyochelin–
iron(III) complex isolated from P. aeruginosa has been reported
(Fig. 4).42 It was found that the FptA receptor can bind in vivo
the apo-form of its cognate siderophore and can also be puri-
fied as a stable iron-free FptA–pyochelin complex from
Pseudomonas strains, which were grown under iron-depleted
conditions.43 The FptA–pyochelin complex is less stable than
the co-purified FpvA–pyoverdine one44 and can also be loaded
with iron in vitro in the presence of iron(III) complexes with
pyochelin, citrate and ferrichrome.43
In the FptA–pyochelin–iron(III) species, pyochelin is tetra-
dentately coordinated to the Fe(III) ion, while the remaining
two coordination sites of the octahedral complex are occupied
Fig. 3 Structural representation of iron(III) complexes with cepabactin
(a) and cepabactin/pyochelin (b).41
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Dalton Transactions
Fig. 4 The crystal structure of FptA–pyochelin–iron(III) (RCSB Protein
Data Bank,45 the accession number 1XKW). The structure is reproduced
with permission from ref. 42. Copyright (2005) Elsevier Ltd.
by the oxygen atoms of the ethylene glycol, due to its high con-
centration in the crystallization solution. Although, ethylene
glycol does not represent a physiological molecule, it has the
appropriate position of the hydroxyl donor groups to achieve a
bidentate coordination of the Fe(III) ion. Based on these
results, it was proposed that only one pyochelin is necessary
for the recognition of the siderophore by FptA and its trans-
port into the periplasm. The structure presented in Fig. 4 is
most likely the physiological state of the complex, which
shows that pyochelin does not need to be the second chelating
ligand that would stabilize the Fe(III) ion and that it could be
water molecules, various ionic species or cepabactin as in the
case of FptA isolated from Burkholderia strains.42
The coordination properties of pyochelin and its two ana-
logues, 3″-nor-NH-pyochelin and the corresponding methyl
hydroxamate, 2-(2′-hydroxyphenyl)-4,5-dihydrothiazol-4-car-
boxylic acid methoxymethyl amide, (Fig. 5) to the Fe(III) ion in
methanol or buffered ( pH ∼ 2) (v/v 1 : 1) methanol–water mix-
tures were studied by different spectroscopic methods, includ-
ing UV-vis absorption, extended X-ray absorption fine struc-
tural analysis and electron paramagnetic resonance.46 It was
shown that all three investigated compounds form the ligand–
iron(III) complexes with 1 : 1 and 2 : 1 molar ratios, respectively.
The higher values of conditional equilibrium constants (Kcomp)
for the comproportionation of the 2 : 1 complex into two 1 : 1
species in the presence of Fe(III) were obtained for pyochelin
and 3″-nor-NH-pyochelin (Kcomp = 29.4 ± 16.7 and 10.3 ± 7.0,
Fig. 5 Structural formulas of pyochelin analogues, 3’’-nor-NH-pyoche-
lin and the corresponding methyl hydroxamate, used for the complexa-
tion of the Fe(III) ion.46 The atom assignment is the same for pyochelin
and 3’’-nor-NH-pyochelin.
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Dalton Transactions
respectively) relative to methyl hydroxamate (Kcomp = 1.16 ±
0.95). Based on this, it was concluded that the reaction
between the first two ligands and Fe(III) occurred with the pre-
dominant formation of 1 : 1 complexes, except for when the
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corresponding ligands are present in a large excess. This
further implies that pyochelin- and 3″-nor-NH-pyochelin–Fe(III)
systems exist principally in the extracellular medium, in the
form of a complex with the metal-to-ligand molar ratio 1 : 1,
whereby two remaining Fe(III) coordination sites are occupied
by hydroxides or water molecules. The same is true in the pres-
ence of methanol, where alkoxide ions serve as additional
ligands. These findings are in accordance with the abovemen-
tioned three-dimensional structure of the FptA receptor loaded
with the pyochelin–iron(III) complex (Fig. 4).42
In the complexes with ligand-to-metal molar ratio 2 : 1, one
pyochelin and a 3″-nor-NH-pyochelin molecule coordinate to
the Fe(III) ion in a tetradentate fashion via O1, N1, N2 and O3
donor atoms (see Fig. 5 for atom assignments), while the
second molecule behaves as a bidentate ligand and engages its
two donor atoms (O1, N1 or N2, O3), leading to the formation
of asymmetrical octahedral iron(III) complexes. On the other
hand, the corresponding methyl hydroxamate acts exclusively
as a tridentate ligand. It is proposed that two methyl hydroxa-
mate ligands offer the same set of donor atoms for coordi-
nation to the Fe(III) ion and form the symmetrical iron(III)
complex.46
In contrast to the abovementioned results for the low stabi-
lity constant of the pyochelin–iron(III) complex,36 a detailed
physico-chemical study of the reaction of pyochelin with Fe(III)
by potentiometry, spectrophotometry (absorption and emis-
sion) and electrospray ionization mass spectrometry showed
that this biomolecule has a significantly higher binding
affinity for Fe(III) ( pFe = 16.0 at pH = 7.4).20 Kinetic studies
revealed that the formation of the pyochelin–iron(III) com-
plexes proceeds through an Eigen–Wilkins dissociative ligand
interchange mechanism, which involves two protonated pyo-
chelin molecules and Fe(OH)2+ species, while no reactivity of
the dihydroxylated Fe(OH)2+ and dimeric Fe2(OH)24+ species
was observed. Based on the calculated constants (Table 1), it
can be concluded that pyochelin is a moderate iron(III) chela-
Table 1 pM values of transition metal complexes containing different
biologically relevant ligands at pH = 7.4 and 25 °C in water (M = Fe(III),
Zn(II) and Cu(II))
Biologically relevant ligands pFe pZn pCu
a,20
Pyochelin 16.0 11.8 14.9
Pyoverdineb,19 27.0
Chrysobactinb,53 17.3
Enterobactinc,47 35.5
Desferrioxamine Bd,48 26.5 6.6 11.2
La a,54 16.0
Ferrichromee,50,51 25.2
Desferrithiocine,52 20.4 7.9 12.9
Dihydroaeruginoic acide,52 21.1 6.5 12.1
a
Solvent: CH3OH/H2O (80/20 by weight); I = 0.1 M ((C2H5)4NClO4). b I =
0.1 M (NaClO4). c I = 0.1 M (KCl). d I = 0.2 M (KCl). e I = 0.1 M (KNO3).
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tor, which is not competitive compared to different hexaden-
tate siderophores, including enterobactin (Escherichia coli),47
ferrioxamine B (Streptomyces pilosus),48,49 and ferrichrome
(Ustilago sphaerogena),50,51 and some tridentate biochelators,
such as desferrithiocin (Streptomyces antibioticus),52 and dihy-
droaeruginoic acid (P. fluorescens).52 Moreover, pyochelin is
not able to extract Fe(III) from human serum transferrin at pH
= 7.4, which is consistent with the biochemical studies by Cox
et al.16 On the other hand, the pFe value of the pyochelin–iron
(III) complex is in agreement with those of chrysobactin
(Erwinia chrysanthemi)53 and a synthetic bidentate chelator
containing a phenol-oxazoline backbone (La).54
Importantly, pyoverdine, the other siderophore produced by
P. aeruginosa, with pFe = 27.0 (Table 1), is a much more
efficient iron(III) chelator and is considered to be the primary
siderophore of this bacterium, playing the key role in Fe(III)
uptake trafficking, while pyochelin is a secondary iron(III)
carrier ( pFe = 16.0), which can provide a sufficient amount of
iron to the bacteria under certain environmental or physiologi-
cal conditions.20 Nevertheless, both siderophores not only are
included in iron(III) shuttling but also play some other specific
biological roles, in subtle metal homeostasis in
microorganisms.20
Gallium(III)
The gallium(III) ion was found to inhibit P. aeruginosa growth
in both planktonic and biofilm forms and to cause significant
protection against infection with this bacterium in animal
models.55,56 The mechanism by which Ga(III) shows anti-
microbial properties originates from its chemical similarities
to Fe(III), which endow Ga(III) with iron mimetic properties,
such as a replacement of Fe(III) in iron-containing metalloen-
zymes. However, in contrast to the Fe(III) ion, Ga(III) is redox
inactive, and its incorporation in iron-containing enzymes
leads to the general disruption of iron metabolism.57 The
gallium(III) complexes with suitable carriers (synthetic chela-
tors or siderophores), which act as “Trojan horses”, were inves-
tigated with the aim of promoting the introduction of Ga(III)
ions into the bacterial cell, thus improving their antibacterial
activity.58 Thus, it was found that the complexation of Ga(III)
with pyochelin results in increased antibacterial activity
against P. aeruginosa, suggesting that pyochelin-based carriers
could be used in the design of Ga(III)-delivery systems.58
Synchrotron radiation induced X-ray photoelectron spec-
troscopy (SR-XPS) and X-ray absorption spectroscopy (XAS)
were used for the investigation of the electronic nature and
coordination chemistry of Fe(III) and Ga(III) ions in their com-
plexes with pyochelin.59 To provide complementary infor-
mation about the valence state, the coordination geometry and
next-neighbour atomic shells at Fe(III) and Ga(III) ions, XAS
experiments were carried out in the near-edge (XANES) and
extended (EXAFS) regions. The obtained results have shown
that both iron and gallium are in the +3 oxidation state and
have the same octahedral geometry, which involves six ligands
in the nearest neighbour shells, and a similar next-neighbour
coordination up to ca. 4.5 Å.59 These findings were in good
Dalton Trans., 2023, 52, 4276–4289 | 4281

Perspective
agreement with those of XPS measurements performed on
iron(III) and gallium(III) complexes with pyochelin.59
Zinc(II) and copper(II)
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In addition to Fe(III), pyochelin can bind Zn(II) and Cu(II) ions
( pZn = 11.8 and pCu = 14.9; Table 1), leading to the predomi-
nant formation of the complexes with metal-to-ligand molar
ratio 1 : 2, respectively, under physiological conditions.20
Furthermore, compared to the other biologically relevant
ligands presented in Table 1, both pZn and pCu values deter-
mined for pyochelin are the highest for these two metal ions.
Based on the presented data, it can be concluded that pyoche-
lin has a weak binding selectivity toward Fe(III) and a high
affinity to bind for Zn(II) and Cu(II) ions because of its tetraden-
tate behavior and the presence of nitrogen donor atoms.
Moreover, a dimeric zinc(II) complex with pyochelin (metal-to-
ligand ratio 2 : 2) was obtained in microcrystalline form and in
a relatively substantial yield during the biosynthesis of the
antibiotic pyoluteorin from a culture of Pseudomonas fluores-
cens.60 This complex was formed during chromatographic pro-
cedures, as a result of the extraction of Zn(II) from impurities,
which were present in the silica column. The NMR (1H and
13
C) spectroscopic data for the two pyochelin moieties of this
complex differ significantly from each other and from those of
uncoordinated pyochelin. The isolated pyochelin–zinc(II)
complex has unusual solubility and stability and can be easily
converted into highly pure pyochelin.60
Fast atom bombardment (FAB) mass spectrometry was also
used for the characterization of the zinc(II) complex with pyo-
chelin and the obtained results were compared with those for
the corresponding iron(III) complex.61 Under the investigated
conditions, the Fe(III) ion cannot be stabilized by pyochelin,
and only Fe(II) species were observed in the FAB mass spec-
trum. However, the zinc(II) complex with pyochelin formed the
[M + H]+ and [2M + H]+ ions in the presence of acetic acid in
the glycerol matrix. In the linked-scan studies of the pyoche-
lin–zinc(II) complex, it was always found that the Zn(II) ion is
bound to the deprotonated oxygen atom of the phenolic
group, with the molar ratio n(Zn2+) : n( pyochelin) being 1 : 1.
Moreover, the formation of a zinc(II) complex with the pyoche-
lin phenolate ion was confirmed by the spectrophotometric
study of Sodeau et al.62 Accordingly, the biosynthetic suppres-
sion of pyochelin from P. aeruginosa by the Zn(II) ion is
expected to be ineffective at physiological pH, as only a small
amount of the phenolate anion is formed under these circum-
stances. Indeed, it has been shown that the Zn(II) ion is a rela-
tively ineffective metal ion, which led to the ca. 25% repression
of pyochelin biosynthesis from P. aeruginosa at a concentration
of 100 μM.63 This is in contrast with the behavior of Co(II),
which displayed a Fe(III) mimicking effect and completely
reduced the production of pyochelin at concentrations of 5 to
10 μM.
Other metal ions
To confirm that pyochelin possesses a sequestering activity
towards the essential transition metal ions other than iron(III),
4282 | Dalton Trans., 2023, 52, 4276–4289
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Dalton Transactions
the effects of metal ion addition on the growth of P. aeruginosa
PAO1 in a metal-poor medium, on the biosynthesis of pyoche-
lin, and on the expression of outer membrane proteins
included in the siderophore-mediated iron uptake were investi-
gated.63 The obtained results have shown that all these pro-
cesses can also be modulated by transition metal ions other
than Fe(III) and have confirmed a considerable affinity of pyo-
chelin for them, suggesting that pyochelin can be involved in
the transport and delivery of different transition metals. The
appreciable affinity of pyochelin for binding Cu(II), Co(II),
Mo(VI), and Ni(II) ions has been confirmed by titration experi-
ments. In addition to this, as was mentioned above, Fe(III) and
Co(II) ions at 10 μM and Mo(VI), Ni(II), and Cu(II) at 100 μM
have inhibited the pyochelin biosynthesis and reduced the
iron-regulated outer membrane protein expression. All of the
studied metal ions, with the exception of Mo(VI), have a signifi-
cant influence on the growth of P. aeruginosa in a metal-poor
medium. It is important to note that Mo(VI), Ni(II) and Co(II)
were more efficient as inhibitors of P. aeruginosa growth in the
form of a complex with pyochelin than in the free form.63
Metal complexes with pyoverdine
Iron(III)
Investigation of the coordination properties of pyoverdines is
of great importance for the understanding of iron transport by
these siderophores. Taking this into account, pyoverdine PaA
and the corresponding iron(III) complex have been studied by
electrochemistry, potentiometry and spectrophotometry.19 A
single electronic spectrum with two charge transfer bands at
460 and 540 nm can be observed during the spectrophoto-
metric titration of the pyoverdine–iron(III) complex in water in
the pH range of 3.0–9.0. The results obtained from spectropho-
tometric and potentiometric measurements in the pH range
between 1.6 and 4.0, where spectrophotometric variations
could be observed, were used for the calculation of the thermo-
dynamic constant βLH2Fe(III), which is related to a diprotonated
iron(III) complex (log βLH2Fe(III) = 47.8 ± 0.2). On the other hand,
from the potentiometric results for both pyoverdine PaA and
its iron(III) complex for the exterior protonation sites (arginine
and succinic acid), the stability constants of the monoproto-
nated pyoverdine complex LHFe(III) and deprotonated LFe(III)−
were calculated to be log βLHFe(III) = 43.0 ± 0.3 and βLFe(III) =
30.8 ± 0.3, respectively. From the distribution curves for the
known and equal concentrations of pyoverdine PaA and
iron(III) at various pH values, it was concluded that the neutral
complex LHFe(III) is the only species formed under physiologi-
cal conditions.19
In order to compare the coordination properties of pyover-
dine PaA toward Fe(III) with some other natural siderophores,
the corresponding pM values, which include the equilibrium
concentration of Fe(III) at pH = 7.4 and at the studied ligand
and metal concentrations, are presented in Table 2. As can be
seen, although pyoverdine PaA contains a catechol moiety, its
pM value of 27.0 is similar to those determined for trihydroxa-
This journal is © The Royal Society of Chemistry 2023

Dalton Transactions
Table 2 pFe valuea for pyoverdine PaA in comparison with those for
different natural siderophores
Siderophores
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Tricatecholate siderophores
Enterobactin66
Trihydroxamate siderophores
Ferrichrome65
Ferrioxamine B65
Ferrioxamine E65
Coprogen64
Dihydroxamate siderophores
Rhodotorulic acid68
Mixed siderophores
Two hydroxamates and one hydroxyacid
Aerobactin67
Two hydroxamates and one catecholate-type
Pyoverdine PaA19
−6 −5
a
pM = −log[Fe ] when [Fe ] = 10
3+ 3+
M, [L] = 10 M, and pH = 7.4.
mate siderophores, such as coprogen ( pM = 27.5)64 and fer-
rioxamine B ( pM = 26.6).65 On the other hand, enterobactin,
which is a tricatecholate siderophore, has a significantly
higher pM value ( pM = 35.5)66 than pyoverdine PaA, while
aerobactin, which contains two hydroxamates and one hydro-
xycarboxylic acid, has a lower affinity to Fe(III) than pyoverdine
PaA with a pM value of 23.3.67
The reduction potential of the pyoverdine–Fe(III) complex
was determined by cyclic voltammetry and is found to be
−510 mV per NHE.19 This potential is 240 mV less negative
than that of enterobactin (−750 mV per NHE)66 and slightly
more negative than those of the trihydroxamate siderophores,
ferrichrome A (−448 mV per NHE), ferrioxamine B (−468 mV
per NHE)50 and coprogen (−447 mV per NHE).64 Furthermore,
the stability constant of the pyoverdine–Fe(II) complex was
found to be very similar to those determined for coprogen64
and ferrioxamine B.50 In general, pyoverdine PaA has a very
similar coordination mode to the Fe(II/III) ion as the other tri-
hydroxamate siderophores and accordingly, the release of the
Fe(III) ion from all these siderophores is the same and is fol-
lowed by its reduction to Fe(II) by physiologically available
reductants.19
Pyoverdine can compete directly with transferrin for iron,
which was confirmed by its fast removal from [59Fe]ferritrans-
ferrin by pyoverdine in vitro.13 This further suggests that pyo-
verdine can be considered as a primary essential virulence-
associated factor in P. aeruginosa.
As mentioned above, the pyoverdine–Fe(III) complex is trans-
ported into the periplasm by its cognate receptor, FpvA, which
belongs to the outer membrane transducer family. The crystal
structure for the full-length FpvA bound to pyoverdine–Fe(III) at
2.7 Å resolution was determined by Cobessi et al. and is pre-
sented in Fig. 6.69 Only one pyoverdine–Fe(III) conformer
bound to FpvA was observed, which reflects the conformer
selectivity of the binding site. The binding of pyoverdine–Fe(III)
to its receptor caused large conformational changes in the
N-terminal region of the plug, which increased its flexibility.
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pFe
35.5
25.2
26.6
27.7
27.5
21.76
23.3
27.0
Fig. 6 The crystal structure of FpvA–pyoverdine–iron(III) (RCSB Protein
Data Bank,45 the accession number 2IAH). The structure is reproduced
with permission from ref. 69. Copyright (2007) Elsevier Ltd.
This process had as a consequence the increased possibility of
the interaction between FpvA–pyoverdine–Fe(III) and TonB.
The photoreactivity of a mixture containing pyoverdine–
iron(III) complexes was studied under simulated cloud con-
ditions.70 A mixture of pyoverdines was synthesized from
Pseudomonas fluorescens 36b5, a bacterium isolated from the
atmospheric water, purified, and characterized by spectro-
scopic analyses, which were performed in water at pH = 4.0
and 6.0, as the typical pH values of cloud water.71 As expected,
pyoverdines coordinate Fe(III) ions, which are naturally present
in cloud water, and consequently modify their availability and
photoreactivity. Using the Job’s method in phosphate buffer
solution at pH = 4.0, it was found that the metal-to-ligand
molar ratio in the iron(III) complex with pyoverdine is 1 : 1,
which is consistent with the other results for the pyoverdine–
iron(III) complexes.19,72 The complex formation can also be
confirmed by the loss of fluorescence, since the pyoverdine–
iron(III) complex was found to be non-fluorescent. The stability
constants of the pyoverdine–iron(III) complex were found to be
20–27 times higher than those determined for carboxylate–
iron(III) complexes, indicating that pyoverdines and carboxylic
acids, both naturally present in cloud water, can compete for
iron(III) complexation, whereby the formation of the pyover-
dine–iron(III) complex is considered to be more favorable.70
The photoreactivity of the pyoverdine–iron(III) complex was
investigated at the pH values mentioned above under polychro-
matic irradiation.70 It was found that under the investigated
conditions this complex underwent a ligand-to-metal charge
transfer, which led to the formation of Fe(II) and organic
ligand radicals. The rates of Fe(II) formation are found to be
6.98 × 10−9 and 3.96 × 10−9 M s−1 at pH = 4.0 and 6.0, respect-
ively. This difference in rates could be explained by taking into
account the spontaneous oxidation of Fe(II) to Fe(III) by oxygen
Dalton Trans., 2023, 52, 4276–4289 | 4283

Perspective
in an aqueous solution at pH > 4.0.73 Besides this, the gener-
ated Fe(II) can participate in the Fenton reaction, thus provid-
ing an important source of hydroxyl radicals in the cloud
water.
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Gallium(III)
The ionic radius of Ga(III) is almost the same as that of Fe(III)
(62 and 65 pm, respectively), and it also forms complexes of
octahedral geometries, but opposite to Fe(III), Ga(III) is a dia-
magnetic ion. Considering this, gallium(III) complexes have
been used for NMR investigation as substitutes for the iron(III)
analogues. Cryosolvents (H2O/D2O (90 : 10) for measurements
at 1 °C, H2O/CD3OD/D2O (70 : 20 : 10) for measurements at
−9 °C and CD3CN/H2O/CD3OD/D2O (50 : 28 : 17 : 5) for
measurements at −20 °C) were used for the investigation of
the pyoverdine PvdI–Ga(III) complex at temperatures as low as
−20 °C.74 The structural formula of pyoverdine PvdI is shown
in Fig. 7a. Under the investigated conditions, two sets of
signals were observed in the NMR spectrum, and triple-reso-
nance experiments (13C, 15N and 1H) were performed to assign
both conformers of the pyoverdine PvdI–Ga(III) complex at −6
and −20 °C. Moreover, the three-dimensional structures of the
two PvdI–Ga(III) conformers in solution were determined,
whereas it was found that the observed conformational
exchange involves the stereoisomerization of the metal
binding coordination, accompanied by a change in the sidero-
phore global shape.75 However, as mentioned above, only one
of these two conformers was observed in the crystal structure
of the pyoverdine PvdI–Fe(III) complex with its specific outer
Fig. 7 The structural formulas of pyoverdine PvdI (a)74,75 and pyover-
dine G4R and G4RA (b).76,77
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Dalton Transactions
membrane receptor FpvA, which further suggests that the side-
rophore-receptor interaction is a stereoselective process.69
Pseudomonas putida is a nitrogen-fixing bacterium that
plays a role in the transformation of nitrogen into ammonia.
Two main pyoverdines, pyoverdine G4RA and pyoverdine G4R,
were obtained from the cultures of this bacterium, which were
grown under iron-depleted conditions, and their primary struc-
ture (Fig. 7b) was determined by FAB-MS and two-dimensional
NMR on unlabeled and 15N-labeled molecules.76 As can be
seen in Fig. 7b, these two pyoverdines contain the heptapep-
tide L-Asp-L-Orn-D-β-threo-OHAsp-L-Dab-Gly-L-Ser-L-cyclo-
OHOrn, which contains an amino acid resulting from the con-
densation of D-β-threo-hydroxyaspartic acid with L-2,4-diamino-
butyric acid. The gallium(III) complex with pyoverdine G4R was
characterized by NMR, fluorescence and UV-vis spectroscopy,
mass spectrometry and circular dichroism (CD), and its three-
dimensional structure was compared with those of other pyo-
verdines and siderophores.77 In the UV-Vis spectrum, this
complex has shown an absorption maximum at 408 nm at pH
= 5.0. After excitation of the complex solution, it manifests a
blue fluorescence at 364 nm, in contrast with the iron(III) ana-
logue, which is non-fluorescent. In the studied complex, the
metal-to-ligand molar ratio is 1 : 1, and was confirmed by the
magnetic electrospray mass spectrometric measurement on
the 15N-labeled pyoverdine G4R–Ga(III) complex.
CD spectroscopy was used to determine the absolute con-
figuration of the studied gallium(III) complex.77 By comparison
of its CD spectrum with those of pseudobactin B10–Fe(III)27
and pyoverdine GMII–Ga(III),78 it can be proposed that the pyo-
verdine G4R–Ga(III) complex predominantly has the Δ absolute
configuration.77
The three-dimensional structure of the pyoverdine G4R–
gallium(III) complex in solution was determined from the 1H
NMR spectra measured at 10 and 30 °C.77 It was found that
the site of complexation of the Ga(III) ion is located on the
surface of pyoverdine, indicating that Ga(III) can be released
without significant conformational changes, while the polar
groups of the peptide chain, which are responsible for the
receptor binding, are on the opposite side of the overall shape.
In the peptide chain, OHAsp3 and OHOrn7 are involved in
Ga(III) coordination, Dab4 and Gly5 are engaged in reversing
the direction of the peptide chain, while Asp1, Orn2, and Ser6
are possible residues for receptor recognition. However, it is
proposed that a surface formed by the ring of OHOrn7 and
parts of the side chains of Orn2, OHAsp3, and Ser6, i.e. the
fold around the octahedrally coordinated Ga(III) ion, is prefer-
ably recognized.77
Manganese(III)
The interaction of the Mn(III) ion with a pyoverdine-type sidero-
phore (PVDMnB1), produced by the Mn(II)-oxidizing bacterium
Pseudomonas putida strain MnB1, was investigated at pH = 8.0
and in a spent culture fluid.72 It was found that the PVDMnB1–
Mn(III) complex has a metal-to-ligand molar ratio of 1 : 1 and
that it has shown a fluorescence quenching and an absorption
spectrum different from those of the PVDMnB1–Fe(III) complex
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or PVDMnB1 itself. Moreover, Mn(III) can successfully compete
with the Fe(III) ion for binding to PVDMnB1; this can be seen
from the stability constant values for HPVDMnB1–Mn(III) and
HPVDMnB1–Fe(III) of 1047.5±0.5 and 1044.6±0.5 M−1, respectively,
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to the electronic effects resulting from the coordination. The
order of the protonation constants log KL′HM: Cd (9.7) > Cu
(5.5) > Fe (<1) is in accordance with the stability of the corres-
ponding metal complexes (log KL′M): Cd (8.2) < Cu (20.1) < Fe

determined at 4 °C under an infinite dilution reference state. (30.8).82

The finding that the stability constant KHPVDMnB1–Mn(III) is
greater than KHPVDMnB1–Fe(III) is in accordance with the affinity
of Mn(III) and Fe(III) cations for hydroxamate-containing sidero-
phores, and is correlated with the first hydrolysis constant of
the metal ion.79 The Mn(III) ion can also be strongly bound by
desferrioxamine B, which is a trihydroxamate siderophore.80
The stability of the PVDMnB1–Mn(III) complex was investi-
gated in the presence of different reduction agents or PVDMnB1
competing ligands, including ascorbate, which can reduce
Mn(III) to Mn(II), EDTA (ethylenediamine-N,N,N′,N′-tetraace-
tate), which is both a competing ligand and reductant because
its Mn(III) complex is susceptible to rapid intramolecular elec-
tron transfer, pyrophosphate (mainly HP2O73− and P2O74− at
pH = 8.0), a high-affinity ligand towards Mn(III) that does not
undergo an electron transfer process, and citrate, which can
be oxidized by Mn(III) at low pH but is relatively stable at pH =
8.0.72 The Mn(III) ion can only be partially released from the
PVDMnB1–Mn(III) complex in its reaction with an 830-fold
excess of pyrophosphate, indicating that this complex can act
as a reservoir of soluble Mn(III) in a natural environment.
Furthermore, the PVDMnB1–Mn(III) complex can participate in
environmentally important redox reactions, as it can oxidize
EDTA and ascorbate. The formation of the PVDMnB1–Mn(III)
complex has three major implications, including the effects of
pyoverdine on the microbial oxidation of Mn(II) to Mn(IV) via a
Mn(III) intermediate, the stabilization of Mn(III) in natural
waters and competition between Mn(III) and Fe(III) ions for pyo-
verdine binding that has an effect on iron bioavailability.72
The coordination properties of pyoverdine towards Cd(II)
and Cu(II) ions with some other chelate ligands, such as side-
rophores, non-humic organic acids and exogenous chelator
EDTA were compared on the basis of the pM values ( pM =
−log[Mz+]free) at pH = 5.0 and 8.0 with [L]tot = 10−5 M and
[Mz+]tot = 10−6 M (Table 3). At higher pH = 8.0, pyoverdine is
one of the most powerful chelators of Cu(II) and is competitive
with EDTA, which is used in chelate-assisted phytoextraction.83
The coordination properties of pyoverdine towards Cu(II) are
highly dependent on pH, since the pCu value is significantly
lower at pH = 5.0. In contrast to this, pyoverdine is not a strong
chelator for Cd(II) and cannot compete with EDTA, even at
higher investigated pH values (Table 3).
The mobility of Cd, Cu and Fe in soil was also investigated
by CaCl2 extraction.82 As expected, pyoverdine supply pro-
moted the mobility of Fe and Cu. On the other hand, the
amount of extracted Cd was low and remained almost the
same after the addition of pyoverdine, being in accordance
with the previously discussed coordination properties of this
biomolecule. The observed effect of pyoverdine on the mobility
of Fe and Cu is a consequence of the stability of its complexes
with these two metal ions at the pH predominant in the soil.
The diffusive gradient in thin films (DGT) is a very useful
technique for the characterization of the metal resupply from
the solid phase in environmental matrices89 and is used for
the investigation of the effect of addition of pyoverdine on the
fluxes of Cu and Cd resupply.82 The obtained results have
shown that pyoverdine induced an increase in the flux of Cu

Copper(II), nickel(II) and cadmium(II)

Fluorescence quenching experiments showed that the rate con-
stant of the reaction of Cu(II) and Fe(III) ions with pyoverdines
of Pseudomonas fluorescens 2-79, Pf-A, Pf-B, and Pf-C was much Table 3 pM values (M = Cd(II) and Cu(II)) for pyoverdine and environ-
mentally relevant ligands at 25 °C in aqueous solution. pH values 5.0
higher for Cu(II) (104–105 M−1 s−1) than that for the Fe(III) ion
and 8.0 are representative of acidic and calcareous soil, respectively
(102 M−1 s−1).81 However, the pyoverdine–Cu(II) complexes
were not stable in the presence of 0.1 mM EDTA and their pH = 5.0 pH = 8.0
complete dissociation occurred. On the other hand, the level
of pyoverdine–Fe(III) complex dissociation was relatively much Ligands pCd pCu pCd pCu
lower, at the same concentration of EDTA. Siderophores
The coordination properties of pyoverdine towards Cd(II) Pyochelina,20 — 11.46 — 14.90
Pyoverdineb,82 6.00 8.88 7.49 17.67
and Cu(II) ions were also investigated and the effect of its Desferrioxamine Bc,d,48 6.00 6.72 6.17 12.46
supply was assessed on the mobility, the phytoavailability and
the phytoextraction of these two metals in calcareous soil.82 Non-humic organic acids
Citric acid84,85 6.00b 6.23e 6.02b 6.24e
From spectrophotometric measurements, it was found that the Oxalic acid86 6.00b 6.23b,e 6.00b 6.24b,e
stability constant of the pyoverdine–Cu(II) complex (KL′Cu = Tartaric acid87 6.00 f 6.00 f 6.00 f 6.00 f
1020.1, L′ represents the pyoverdine system in which the ioniz-
Exogenous ligands
able sites of the arginine and succinate residues were omitted) EDTAg,88 10.20 13.23 14.47 17.50
is much higher than that of the pyoverdine–Cd(II) complex (KL′
a
8.2 Solvent: CH3OH/H2O (80/20 w/w); I = 0.1 M ((C2H5)4NClO4); solvent:
Cd = 10 ). The values of the protonation constants of the pyo-
H2O. b I = 0.1 M (NaClO4). c I = 0.2 M (KCl). d I = 0.15 M (KNO3), t =
verdine–Cu(II)/Cd(II) complexes (log KL′HCu = 5.5 and log KL′HCd 37 °C. e I = 0.1 M (KNO3 or NaNO3). f I = 1.0 M (NaClO4). g I = 0.1 M
= 9.7) are lower than that of the pyoverdine (log K2 = 10.8) due (KClO4).

This journal is © The Royal Society of Chemistry 2023 Dalton Trans., 2023, 52, 4276–4289 | 4285

Perspective
resupply and additionally confirmed its selective effect
towards copper.
The impact of pyoverdine addition on metal phytoextraction
by a monocotyledonous (strategy II) and a dicotyledonous
Published on 14 March 2023. Downloaded by Universidad Autonoma Metropolitana-Iztapalapa on 10/9/2023 11:55:50 PM.
(strategy I) plant was also studied.82 No significant influence of
pyoverdine on the plant biomass was observed at harvest, nor
on its toxicity. Surprisingly, the acquisition of Fe by the two
plants was not promoted after the addition of pyoverdine and
this was in contrast with previously reported results, which
have shown an effect of pyoverdine addition on the Fe status
of both types of plants.90 As expected, the amount of Cd in
plant tissues was also not significantly influenced by pyover-
dine supply, and the amounts of Zn, Ni, Mn and Ca. On the
other hand, the Cu content taken up by the two investigated
plants was significantly higher after the addition of pyover-
dine, suggesting the direct role of a bacterial siderophore in
the Cu phytoextraction. However, compared to EDTA, the effect
of pyoverdine on the Cu phytoextraction was weak.
The specificity of pyoverdine to Cu would help the Cu phy-
toextraction above neutral pH when Ca can compete for
binding with Cu91 and in metal-polycontaminated matrices.82
However, the use of bacteria that are the producers of pyover-
dine will be limited to the phytoremediation of Cu-contami-
nated areas and to the alkaline matrices.
The ability of pyoverdine to coordinate Ni(II) ion and the
effect of its supply on Cd and Ni phytoavailability in hydropo-
nics have also been studied.92 The succinate and arginine resi-
dues of pyoverdine do not participate in the coordination of
the Ni(II) ion and, accordingly, their protonation constants
were similar to those of pyoverdine itself. The stability con-
stant of the pyoverdine–Ni(II) complex (KL′Ni, L′ represents the
pyoverdine system in which the ionizable sites of the arginine
and succinate residues were omitted) was found to be 1010.9.
This value is higher than that of the pyoverdine–Cd(II) complex
(KL′Cd = 108.2),82 but is significantly lower than those calculated
for Cu(II) (KL′Cd = 1020.1)82 and Fe(III) ions (KL′Fe = 1030.8).19 The
mobilization of Ni from smectite was promoted after the
addition of 50 μM pyoverdine, while no change in the equili-
brium Cd concentration was observed, which is in accordance
with the higher stability constant of the pyoverdine–Ni(II)
complex. Both the affinity of pyoverdine for the metal ion and
the sorption of the pyoverdine–metal complex onto the solid
phase have the influence of pyoverdine supply on the metal
mobility. The phytoavailability of Cd and Ni was also studied
by determining the amounts of Cd and Ni in plant roots,92
while it was found that pyoverdine itself did not influence the
plant growth. A decrease in phytoavailability for Cd and Ni was
observed after the addition of 50 μM pyoverdine, in the
absence of smectite. The addition of pyoverdine has a more
pronounced effect on the phytoavailability of Ni than that of
Cd, with a decline of 90% and 57%, respectively, as a conse-
quence of its higher affinity for Ni, and the different uptake
kinetics of these two metals by plant roots. The influence of
inoculation with a pyoverdine-producing bacterium, such as
P. aeruginosa PAO1, on Ni phytoavailability was also investi-
gated and the obtained results were compared with those for
4286 | Dalton Trans., 2023, 52, 4276–4289
View Article Online
Dalton Transactions
the direct addition of pyoverdine.92 Inoculation with
P. aeruginosa was found to have a similar effect on Ni phytoa-
vailability to the direct addition of pyoverdine.
Conclusions
This article presents an overview of the results achieved in the
complex formation between three natural products derived
from P. aeruginosa, namely pyocyanin, pyochelin and pyover-
dine(s), and different transition metal ions.
The investigation of the pyocyanin complexation reactions
with different metal(II) ions has shown that this biomolecule
forms stable complexes whose stability constants decreased in
the following order: Ni(II) > Fe(II) > Zn(II) ≈ Cu(II) > Mn(II).
However, the stability constants for the complexes of these
metal(II) ions with the pyocyanin anion radical are consider-
ably greater and decrease as follows: Zn(II) > Ni(II) > Mn(II) >
Fe(II). The stability constants of the copper(II) complex with the
pyocyanin anion radical could not be determined due to the
fast reduction of Cu(II) to Cu(I) ions. The binding of pyocyanin
to alkali and alkaline earth ions proceeds with the formation
of complexes with different numbers of coordinated pyocyanin
and is strongly dependent on the radius of the studied metal
ion.
The single-crystal X-ray diffraction analysis of the pyoche-
lin–Fe(III) complex showed that only the pyochelin I stereoi-
somer is coordinated to the Fe(III) ion. In this octahedral
complex, four coordination sites of Fe(III) are occupied by the
phenolate and the carboxylate oxygen atoms and by the two
nitrogen atoms of pyochelin I. Two pyochelin–Fe(III) units are
connected by a hydroxy and an acetate groups, thus satisfying
the remaining two coordination sites of the Fe(III) ion. The
crystal structure of the pyochelin outer membrane receptor
FptA bound to the pyochelin–Fe(III) complex, isolated from
P. aeruginosa, showed that pyochelin is tetradentately co-
ordinated to the Fe(III) ion, while the remaining two coordi-
nation sites of the octahedral complex are occupied by the
ethylene glycol oxygen atoms. Two octahedral complexes, one
with three cepabactin molecules, a siderophore from
Burkholderia cepacia, bidentately coordinated to the Fe(III) ion,
and the second containing Fe(III), cepabactin and pyochelin in
a 1 : 1 : 1 molar ratio, have also been reported. The coordi-
nation properties of pyochelin to Fe(III) were compared to
those of its two analogues, 3″-nor-NH-pyochelin and the
corresponding methyl hydroxamate. From the obtained
results, it can be concluded that pyochelin is a moderate Fe(III)
chelator, which is not competitive in comparison with
different hexadentate siderophores, such as enterobactin,
ferrioxamine B, ferrichrome, desferrithiocin, and dihydro-
aeruginoic acid. Furthermore, no extraction of the Fe(III) ion
from human serum transferrin by pyochelin is observed at
pH = 7.4.
Considering the fact that the ionic radius of Ga(III) is almost
the same as that of Fe(III), a review of the literature data indi-
cates that its complexes with pyochelin have the same octa-
This journal is © The Royal Society of Chemistry 2023

Dalton Transactions
hedral geometry. In addition to Fe(III) and Ga(III), pyochelin
can bind Zn(II) and Cu(II) ions, leading to the predominant for-
mation of complexes with a metal-to-ligand molar ratio of 1 : 2,
respectively, under physiological conditions.
Published on 14 March 2023. Downloaded by Universidad Autonoma Metropolitana-Iztapalapa on 10/9/2023 11:55:50 PM.
Investigation of the coordination ability of pyoverdines for
Fe(III) is of great importance considering the fact that these
siderophores play an important role in the transport of this
metal ion. Although pyoverdine PaA contains a catechol
moiety, its coordination properties toward Fe(III) are similar to
those of trihydroxamate siderophores (coprogen and ferrioxa-
mine B). On the other hand, enterobactin, a tricatecholate
siderophore, has a significantly higher pM value, while aero-
bactin, containing two hydroxamates and one hydroxycar-
boxylic acid, has a lower affinity to Fe(III) than pyoverdine PaA.
The stability constants of the pyoverdine–Fe(III) complexes
were higher than those determined for the carboxylate–iron(III)
complexes, indicating that pyoverdines and carboxylic acids
can compete for Fe(III) complexation, whereby the formation of
the pyoverdine–Fe(III) complex is considered to be more favor-
able. The crystal structure for the full-length FpvA bound to
pyoverdine–Fe(III) has shown that the binding of pyoverdine–
Fe(III) to its receptor caused large conformational changes in
the N-terminal region of the plug, which increased its
flexibility.
NMR spectroscopy has been successfully applied for the
structural determination of the pyoverdine–Ga(III) complexes,
used as models for the corresponding siderophore–Fe(III) com-
pounds. The three-dimensional structure of the two pyover-
dine PvdI–Ga(III) conformers in solution was determined,
whereas it was found that the observed conformational
exchange involves the stereoisomerization of the metal
binding coordination, accompanied by a change in the sidero-
phore global shape. However, only one of these two confor-
mers was observed in the crystal structure of the complex of
pyoverdine PvdI–Fe(III) with its specific outer membrane recep-
tor FpvA. Furthermore, the three-dimensional structure of the
gallium(III) complex with pyoverdine G4R, obtained from the
cultures of Pseudomonas putida, was confirmed, and it was
found that in this complex the site of Ga(III) complexation is
located on the surface of the pyoverdine, indicating that this
metal ion can be released without significant conformational
changes.
The complexation reaction of the pyoverdine-type sidero-
phore (PVDMnB1) with Mn(III) ions led to the formation of the
PVDMnB1–Mn(III) complex, which has a metal-to-ligand molar
ratio of 1 : 1. Based on the difference in the stability constant
values between the Mn(III) and Fe(III) complexes, it can be con-
cluded that Mn(III) can successfully compete with the Fe(III) ion
for binding to this siderophore. Moreover, the competitive
experiments for the reactions of Cu(II) and Fe(III) ions with pyo-
verdines have shown that the rate constant for the formation
of the pyoverdine–Cu(II) complexes was much higher than that
for the corresponding Fe(III) complexes. However, the copper(II)
complexes were less stable in comparison with the corres-
ponding Fe(III) complexes, but much more stable than the
Cd(II) and Ni(II) analogues.
This journal is © The Royal Society of Chemistry 2023
View Article Online
Perspective
The content of this review article could direct future devel-
opments of new bacterially derived natural product-metal com-
plexes, which can serve as a model for the study of metal ion
metabolism in living systems, and might also be considered as
novel therapeutic agents for potential use in medicine, as an
exciting area of research that should be encouraged
worldwide.
Author contributions
Conceptualization, V. M., M. I. Dj. and B. Đ. G.; investigation,
T. P. A., D. P. A., B. P. and N. Lj. S.; writing – original draft
preparation, T. P. A., D. P. A., B. P. and N. Lj. S.; writing –
review and editing, V. M., M. I. Dj. and B. Đ. G; visualization,
T. P. A., D. P. A., B. P., N. Lj. S. and V. M.; supervision, M. I. Dj.
and B. Đ. G. All authors read and approved the final
manuscript.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
This research was supported by the Science Fund of the
Republic of Serbia, Grant No. 7730810, Value-added biologics
through eco-sustainable routes – BioECOLogics. This research
has also received funding from the Ministry of Science,
Technological Development and Innovation of the Republic of
Serbia (Agreements No. 451-03-47/2023-01/200122 and 451-03-
47/2023-01/200378) and the Serbian Academy of Sciences and
Arts under project No. F128.
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