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Siderophores in cloud waters and potential impact on atmospheric chemistry: Photoreactivity of iron complexes under sun-simulated conditions Monica Passananti, Virginie Vinatier, Anne-Marie Delort, Gilles Mailhot, and Marcello Brigante Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02338 • Publication Date (Web): 03 Aug 2016 Downloaded from http://pubs.acs.org on August 10, 2016
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Siderophores in cloud waters and potential impact on atmospheric chemistry: Photoreactivity of iron complexes under sun-simulated conditions
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Monica Passanantia,b, Virginie Vinatiera,b, Anne-Marie Delorta,b, Gilles Mailhota,b, Marcello Brigantea,b*
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a
7
F-63000 CLERMONT-FERRAND, FRANCE
8
b
Université Clermont Auvergne, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448,
CNRS, UMR 6296, ICCF, F-63171 AUBIERE, FRANCE
9 10 11
* Corresponding author MB: University Blaise Pascal, Institute of Chemistry of Clermont-Ferrand, avenue des Landais 63171 Aubière, France; Phone +33 0473405514 e-mail:
[email protected] 12 13
Submitted to Environmental Science and Technology
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Abstract
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In the present work, the photoreactivity of a mixture of iron(III)-pyoverdin (Fe(III)-Pyo) complexes was
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investigated under simulated cloud conditions. Pyoverdins are expected to complex ferric ions naturally
21
present in cloud water, thus modifying their availability and photoreactivity. The spectroscopic properties
22
and photoreactivity of Fe(III)-Pyo were investigated, with particular attention to their fate under solar
23
irradiation, also studied through simulations. The photolysis of the Fe(III)-Pyo complex leads to the
24
generation of Fe(II), with rates of formation ( RFe( II ) ) of 6.98 and 3.96 × 10-9 M s-1 at pH 4.0 and 6.0,
25
respectively. Interestingly, acetate formation was observed during the iron-complex photolysis, suggesting
26
that fragmentation can occur after the ligand-to-metal charge transfer (LMCT) via a complex reaction
27
mechanism. Moreover, photogenerated Fe(II) represent an important source of hydroxyl radical via the
28
Fenton reaction in cloud water. This reactivity might be relevant for the estimation of the rates of
29
formation and steady-state concentrations of the hydroxyl radical by cloud chemistry models and for
30
organic matter speciation in the cloud aqueous phase. In fact, the conventional models, which describe the
31
iron photoreactivity in terms of iron-aqua and oxalate complexes, are not in accordance with our results.
f
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Introduction
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Clouds play a key role in the atmospheric system and may indeed influence the troposphere composition,
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the oxidizing capacity of the atmosphere and radiation phenomena (such as light diffusion). Therefore,
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clouds have a strong impact on the climate. It is well known that the aqueous phase in clouds is an
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important medium for chemical reactions, in which the fate of organic and inorganic constituents is
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strongly dependent on the oxidative capacity and cloud water composition.1, 2 The determination of cloud
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composition has become a popular subject of research in recent years.3, 4 While the inorganic composition
41
of clouds has been well elucidated,5,
42
characterized due to its complexity.7, 8
43
Only 25-30% of the organic components of clouds have been characterized, while the remaining 70-75% is
44
unidentified.9-11 The identified compounds are mainly acetic, formic and oxalic acids and other small chain
45
acids10, 12 or aldehydes.13 A recent study reported the molecular weight distribution for the organic fraction
46
of cloud samples and showed that the main compounds have molecular weights between 50 and 2500
47
Da.14 The small chain carboxylic acids are derived from anthropogenic and biological emission and from
48
chemical transformation in the atmosphere and in clouds. The higher molecular weight material (> 500 Da)
49
could be humic-like substances (also called HULIS), macromolecules and biogenic nanoscale material
50
(BONM)
51
water.14, 17, 18 Indeed the presence of microorganisms such as bacteria and fungi has been reported mainly
52
in the last decade.19-21 Bauer and coworkers reported that microorganisms can contribute to the organic
53
carbon (OC) content of cloud water: in particular bacteria and fungi account for 1.7% of the OC (in mgC/L).9
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Living microorganisms can have a direct effect on the chemical composition of cloud water via
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biocompound production and/or by the degradation of species present in atmospheric water including
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carboxylic acids, aldehydes, sugars and H2O2.22-24 The chemical species produced by microorganisms can
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also interact with the species present in atmospheric water. In a companion paper it has been shown that a
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large number of bacteria and yeasts isolated from cloud waters are able to produce siderophores which are
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strong complexing agents that uptake iron from the extracellular environment. These molecules have been
15, 16
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the composition of the organic fraction has not been fully
and this organic fraction is an important portion of the organic matter present in cloud
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shown to modify iron speciation and photostability in oceanic systems,
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occur in atmospheric waters. Barbeau and co-workers investigated the photostability of different Fe(III)-
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siderophores and suggested that their photostability depends strongly on the chemical structure of the iron
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binding groups.28 In fact, the presence of specific groups such as catechols and α-hydroxy carboxylates can
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possibly lead to the photooxidation of the iron complexes and reduction of Fe(III) into Fe(II) in aquatic
65
media.
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Iron (Fe(II) and Fe(III)) that is naturally present in the atmosphere has been considered as one of the most
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important sources of the hydroxyl radical (HO•) in cloud water, via the Fenton and Fe(III) photolysis
68
processes (R1 and R2).29, 30
69
Fe( II ) + H2O2 → Fe( III ) + HO− + HO•
(R1)
70
hν Fe( III ) + H2O → Fe( II ) + H + + HO•
(R2)
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HO• is a strong and non-selective photooxidant and reacts with a wide variety of compounds at nearly
72
diffusion-controlled rates leading to the oxidation of organic compounds present in natural water.31 The
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photoinduced generation of hydroxyl radicals plays a key role in the oxidative capacity of the cloud aqueous
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phase. Therefore, its formation mechanism needs to be investigated.
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Fenton and photo-Fenton reactions refer to free iron and have been extensively studied in atmospheric
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waters.32,
77
models represent iron as free ions, aquo-complexes or Fe-oxalate complexes.34 As recently shown,
78
atmospheric aqueous models tend to overestimate hydroxyl radical formation from iron species, probably
79
due to an incorrect modeling of iron (free or Fe-oxalate complexes).35
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The photoreactivity and impact on the oxidative budget of iron complexes with the most abundant
81
carboxylic acids found in cloud water (oxalate, formate, etc.) have been extensively studied.34, 36 However,
82
to the best of our knowledge, the fate (under environmental conditions) of iron complexes in the presence
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of large and functionalized molecules (i.e. polycarboxylic acids, siderophores, etc.) is not yet understood.
33
and the same could thus
To evaluate the contribution of iron to hydroxyl radical formation, atmospheric chemistry
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The photoreacctivity of iron-siderophore complexes in the aqueous phase of clouds could be substantially
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different from that of Fe-oxalate or Fe-aqua complexes and should therefore be evaluated to assess the
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contribution of the source to the generation of Fe(II) and the hydroxyl radical in cloud water.
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In the companion paper of this work, 450 microbial strains isolated from cloud waters were screened for
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their production of siderophores. Of these strains, 43% are able to produce siderophores and among them
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36% belong to the genus Pseudomonas. This genus is the major group present in cloud waters
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known to produce pyoverdins, which are siderophores containing both hydroxamate and catechol-
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chelating functions.37 These molecules are strong iron complexants containing a chromophore and,
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consequently, may impact photoreactivity of iron in clouds. In this paper, we examined a pyoverdin
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mixture produced by Pseudomonas fluorescens 36b5, a bacterial strain isolated from the aqueous phase of
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clouds. Microorganisms are metabolically active and are also able to grow at low temperatures in clouds as
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recently demonstrated using in situ measurements performed directly in cloud water and in lab cultures.24,
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38, 39
97
The aforementioned pyoverdin mixture (Pyo) was purified and characterized by spectroscopic tools, and
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the photochemical behaviour of iron(III)-pyoverdin complexes (Fe(III)-Pyo) was studied under simulated
99
solar irradiation. In particular, the rates of formation of Fe(II) and the hydroxyl radical were measured to
100
21
and is
assess the impact of the Fe(III)-Pyo complex on the oxidative capacity of the aqueous phase of clouds.
101 102
Materials and methods
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Pyoverdin purification
104
Pyoverdins were produced by the bacterial strain Pseudomonas fluorescens 36b5 (JF706586) isolated from
105
cloud water collected at the puy de Dôme station.21 The bacteria were grown at 25°C under shaking for 24
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h in a 1 L of distilled water with 6.0 g of K2HPO4, 3.0 g of KH2PO4, 1.0 g of (NH4)2SO4, 0.2 g of MgSO4•7H2O
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and 4.0 g of succinic acid. The pH was adjusted to 7.0 with NaOH before sterilisation.
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After centrifugation the supernatant was filtered on a 0.2 µm Polyethersulfone (PES) membrane and loaded
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on a 120 g Puriflash IR-C18 (50 µm) cartridge (Interchim). The cartridge was placed on a Spot II (Armen)
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flash chromatography system and rinsed 5 times with of water to remove mineral compounds. The
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pyoverdin mixture was then eluted with H2O/methanol with a linear gradient from 5 to 25% methanol over
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10 minutes. After HPLC analysis, the fractions containing pyoverdins were evaporated. Further purification
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was carried out with the same gradient on a higher-resolution cartridge with smaller particles
114
(InterchimPuriflash C18-HQ-15 µm-35 g). Pure fractions were freeze, dried and stored under argon in a
115
polypropylene
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No additive was added to the mobile phase during purification to avoid eventual artefacts. Plasticware was
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used to avoid complexation with iron, and pyoverdins were obtained in the free form.
centrifuge
tube.
118 119
Determination and analysis of chemical and physicochemical parameters
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All chemicals were used without further purification: iron(III) perchlorate (Fe(ClO4)3) (98%), nitrobenzene (>
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99 %), potassium oxalate (> 99%) (Sigma-Aldrich) and acetonitrile (chromasolv) were obtained from Sigma
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Aldrich. GPR Rectapur methanol was purchased from VWR. All solutions were prepared in water purified by
123
a Millipore milli-q device (Millipore αQ, resistivity 18 MΩ cm, DOC < 0.1 mg L-1). The pH was modified by the
124
addition of HClO4 or NaOH.
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Fluorescence measurements were performed using a Varian Cary Eclipse fluorescence spectrofluorimeter,
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adopting a 5 nm bandpass on both excitation and emission. The fluorescence excitation–emission matrix
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(EEM) was obtained from 250 to 460 nm for excitation and from 250 to 600 nm for emission. UV-vis spectra
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were acquired with a Varian Cary 300 UV–vis spectrophotometer.
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Mass spectrometry was performed by mixing pyoverdin samples (1:1) with R-cyano-4-hydroxycinnamic acid
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(7 mg mL-1 in 30% acetonitrile/0.1% trifluoroacetic acid) and spotting them on a MTP384 Ground Steel
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plate. MALDI-TOF-TOF analyses were performed on an Autoflex Speed (Bruker) in the positive reflectron
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mode. The LIFT technique was used for MS/MS.
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A Perkin Elmer series 200 HPLC system equipped with a DAD detector was used for pyoverdin
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quantification and to check purity. Samples were injected on a Varian Polaris amide C18 column (150 × 4.6
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mm; 5 µm). Solvent A contained 1 mM ethylenediaminetetraacetic acid in 0.1% formic acid. Solution B was
136
acetonitrile. Elution was performed at 1 mL min-1 with a linear gradient from 0 to 60 % B starting 5 minutes
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after injection.
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Iron (II) was quantified using a colorimetric complexant (ferrozine,3-(2-pyridyl)-5,6-bis(4-phenylsulfonic
139
acid)-1,2,4-triazine) coupled with UV/vis spectrophotometric detection at λmax = 562 nm 40. The well-known
140
molar absorption coefficient of the complex between ferrozine and Fe(II) (ε562nm = 27900 M-1 cm-1) was used
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to calculate the Fe(II) concentration.
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Ion chromatography analyses were performed for the quantification of carboxylic acids (material: Dionex
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DX320, column AS11 for anions, eluent KOH). The elution method has been previously described.2 The
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method adopted to calculate the Fe(III)-Pyo transformation ( RFe(III )−Pyo ) and Fe(II) ( RFe( II ) ) formation rates
145
as well as quantum yields are described in the Supplementary Materials section.
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The stoichiometry of the Fe(III)-Pyo complex was investigated using Job’s method in a buffer solution
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(phosphate buffer) at pH 4.0.41 The complex formation was monitored by spectroscopic analysis following
148
the absorption peak at 480 nm characteristic of the Fe(III)-Pyo complex. At pH 4.0, complex absorbance
149
shows a maximum at XFe(III) (Fe(III) molar fraction) of 0.5, corresponding to a Fe(III):Pyo stoichiometry of 1:1.
d
f
150 151
Irradiation experiments
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Aqueous solutions were irradiated in a 40 mL cylindrical reactor at a constant temperature (278 ± 2
153
K) controlled by water circulation. The reactor was located at the focal point of the lamp to ensure a
154
constant irradiation of the whole sample, and was equipped at the top with a Pyrex filter to remove
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wavelengths lower than ~290 nm. Samples were continuously stirred with a magnetic stirrer and a
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The emission spectrum of the Xenon lamp reported in Figure 1 was recorded using an optical fiber coupled
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with a CCD spectrophotometer (Ocean Optics USD 2000+UV-VIS). A reference lamp (DH-2000-CAL, Ocean
159
Optics) was used for calibration. The energy was normalized to the actinometry results obtained using a
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paranitroanisole (PNA)/pyridine actinometer.42 A total flux of 34 W m-2 was measured over the wavelength
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range 290-400 nm. As shown in Figure 1, the intensities are similar to those measured in cloudy conditions
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at the top of the puy de Dôme mountain in autumn 2013.
163 164
Detection of hydroxyl radicals
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Hydroxyl radicals (HO•) were quantified using nitrobenzene (NB) as a trapping molecule. Nitrobenzene,
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which reacts directly with photogenerated hydroxyl radicals to produce different nitrophenols and 1,3-
167
dinitrobenzene,43 was measured by HPLC. Further details about the detection method are reported in the
168
Supporting Information (Text S1).The kinetic approach to estimate the rate of hydroxyl radical formation is
169
obtained from the following equation (eq.1)
170
d f RNB = RHO − •
k NB ,HO• [NB]
(eq. 1)
k NB ,HO• [NB] + ∑ k i [Si ] i
d
171
f where RNB is the rate of degradation of nitrobenzene (M s-1), RHO • is the formation rate of the hydroxyl
172
radical (M s-1), k NB, HO • the second order rate constant for the reaction between nitrobenzene and the
173
hydroxyl radical (3.9 × 109 M-1 s-1)
44
and
∑k [S ] i
i
is the pseudo-first-order rate constant (s-l) for HO•
i
174
scavenging by all constituents (i.e., Fe-complexes) of the solution except for the probe NB.
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In the presence of Fe(III)-Pyo a competition to react with HO• between iron-complex and NB was
176
f considered to determine RHO k NB,HO• [NB] and k NB,HO• [NB] • . Under adopted conditions,
177
estimated to be 1.17 × 106 s-1 and 1.0 × 106 s-1 respectively indicating that 54 % of photogenerated hydroxyl
178
radicals react with NB probe (see Text S1 and figures S1-2 for details).
can be
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Results and discussion
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Production, isolation and structural characterization of pyoverdins
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Pseudomonas fluorescens 36b5 was grown in a succinate minimal medium for 24 h. Separation of the
183
supernatant components by reverse phase chromatography showed the presence of two major and two
184
minor products with spectra characteristic of pyoverdins. Longer incubations increased the yield, but
185
degradation products appeared as the pH of the medium was increased.
186
Pyoverdin was extracted and pre-purified from the supernatant in a single step by ultraperformance flash
187
chromatography on a C18 cartridge. Further purification was performed on a high quality column with a
188
smaller particle size (15 µm). The global yield of the culture medium was 50 mg L-1. HPLC analyses showed
189
that the major components were present in identical proportions and represented 91% of the total
190
pyoverdin (Figure S3).
191
Experiments were performed on a mixture of pyoverdins to take into account the diversity of
192
pyoverdins in the environment. Exact molecular masses of various pyoverdins were determined by
193
MALDI-TOF. The two major compounds had molecular weights of 1142.6 and 1160.6 Da. Minor
194
compounds had molecular masses of 1171.6 and 1188.6 Da. As the masses of the major compounds
195
differ by 18u, they probably share the same peptide chain in a linear and a cyclic form. Indeed,
196
cyclisation has already been described in terms of ester bond formation between a carboxylic acid
197
and the alcohol function of serine 45 with a loss of H2O.
198
Structural investigation of the major linear form was performed by fragmentation of the [M-H2O+H]+ ion
199
(m/z = 1143) by MALDI-TOF-TOF. The major ion corresponds to the A1 fragment (m/z = 417) with a loss of
200
H2O (m/z = 399) which indicates that serine is the first amino acid linked to the chromophore (Figures S4, S5
201
and Table S1) associated with a succinic acid side chain.46 Retro-Diels-Alder opening of the chromophore
202
gives characteristic fragments (m/z = 286 and 204). Loss of 74u due to the elimination of COOH-CHO by a
203
Mc Lafferty rearrangement indicates the presence of a hydroxyaspartic acid residue. The presence of an N9 ACS Paragon Plus Environment
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acetyl-N-hydroxyornithine residue is suggested by the loss of 73u (-NOH-COCH3). A hypothetical structure
205
matching the exact mass and the fragmentation pattern can then be proposed as follows: Chr-Ser-
206
AcOHOrn-Pro-Val OHAsp-Ala-Lys. (Figure 2).
207 208
Spectroscopic analyses of pyoverdin and Fe(III)-pyoverdin complex
209
Pyoverdins are strong Fe(III) complexing agents. Therefore, it is highly probable that in atmospheric water
210
they may exist in the form (Fe(III)-Pyo). A spectroscopic analysis of pyoverdin and the Fe(III)-pyoverdin
211
complex (Fe(III)-Pyo) was carried out in water at pH 4.0 and pH 6.0 (with addition of HClO4), considered as
212
the typical pH range values of cloud water.12 The pyoverdin mixture solution (~100 µM, without iron) was
213
found to be stable in water in the dark for more than 72 h. The natural pH of the solution was 8.3 and the
214
UV-vis spectrum in Figure S6 shows an absorption band at 230 nm [ε230 = (2.00 ± 0.02) × 104 M-1 cm-1] with a
215
shoulder at 280 nm and a band centred at 408 nm [ε408 = (9.18 ± 0.09) × 103 M-1 cm-1]. The UV-vis spectrum
216
was observed to change with pH. In particular, a hypochromic shift was observed at more acidic pH values.
217
The pyoverdin solution at pH 4.0 was not stable, and the UV-vis spectrum changed during 72 h (in the dark),
218
as reported in Figure S7. When the solution was returned to its natural pH (~ 8.0), a bathochromic shift in
219
the UV-vis spectrum was observed. In aqueous solution, pyoverdin acts as a buffer, and its pH slowly
220
changes towards the natural pH (~ 8.3). Pyoverdins are fluorescent molecules containing a chromophores,
221
generally a catechol, that is responsible for colour and fluorescence.47 A fluorescence excitation−emission
222
matrix (EEM) of pyoverdin was recorded at natural pH (Figure 3). Pyoverdin excited at λex= 408 nm emits at
223
a maximum (λem) centred at 460 nm. The measurement of fluorescence is a sensitive method for detecting
224
pyoverdin (detection limit estimated to be approximately 0.3 µM), and a linear correlation between
225
fluorescence intensity and concentration was observed up to 40 µM (Figure S8). After this concentration
226
corresponding to Abs > 0.2, fluorescence quenching was observed.48 The 1:1 Fe(III):Pyo stoichiometry of the
227
Fe(III)-Pyo complex was confirmed using Job’s method in buffer solution (phosphate buffer) at pH 4.0
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(Figure S9). Pyoverdins have three bidentate chelating groups (a catechol and two hydroxamate functions)
229
and form strong 1:1 complexes with iron (III), in agreement with the results reported in the literature.49, 50
230
A spectroscopic analysis of Fe(III)-pyoverdin shows that the complex formation can be confirmed by
231
fluorescence loss. Indeed, the Fe(III)-Pyo complexwas found not to be fluorescent, and in the EEM of the
232
complex solution, only Rayleigh and Raman scattering were observed (Figure S10).51
233
The UV-vis spectrum of the complex at pH 4.0 shows a shoulder at 273 nm and a band centred at 386 nm
234
[ε3869 = (1.52 ± 0.09) × 103 M-1 cm-1] with a tail extending to 700 nm (Figure 1). The UV-vis spectrum at pH
235
6.0 has an absorption band at 230 nm [ε230 = (4.04 ± 0.10) × 103 M-1 cm-1], a shoulder at 273 nm and the
236
same absorption band at 386 nm as that observed for the complex at pH 4 [ε386 = (1.46 ± 0.11) × 103 M-1 cm-
237
1
238
observed after 2 h.
]. The stability of the complexes was investigated in the dark, and no change in the UV-vis spectra were
239 240
Photochemical behaviour of pyoverdin and the Fe(III)-pyoverdin complex
241
The photoreactivity of Fe(III)-Pyo was investigated at pH 4.0 and 6.0 under polychromatic irradiation with,
242
particular attention to the generation of Fe(II) and small carboxylic acids. Fe(III)-Pyo under simulated solar
243
irradiation undergoes a ligand-to-metal-charge-transfer (LMCT) leading to the formation of Fe(II) and
244
organic ligand radicals.52 The quantity of Fe(II) produced during the irradiation is directly proportional to
245
the Fe(III)-Pyo concentration (see the Supporting Information for the method adopted to measure the
246
formation/loss rates).
247
at pH 6.0 (3.96 ×10-9 M s-1) (Table 1), as shown in Figure 4A. The evaluated quantum yields are 1.9 × 10-4
248
and 5.5 × 10-5 respectively for pH 4.0 and 6.0. The difference of
249
considering the spontaneous oxidation of Fe(II) to Fe(III) by oxygen in waters at pH > 4, as previously
250
reported by Morgan and Lahav.53
RFef ( II ) at pH 4.0 is 6.98 ×10-9 M s-1 and is approximately two times the value observed
RFef ( II ) at pH 4 and 6 can be explained by
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Photolysis was performed on Fe(C2O4)2- and on a mixture of Fe(C2O4)2-/Fe(C2O4)33- (50/50) to compare the
252
formation rates of Fe(II) with those reported for Fe(III)-Pyo. Iron-oxalate complexes were prepared as
253
reported in the Supporting Information (Text S4 and Figures S11-12 ), and the pH was fixed at 2.5 and 3.8 to
254
obtain the maximum concentration of the desired stoichiometry of the complex or mixture. RFe( II ) were
255
estimated to be 5.35 ± 0.60 ×10-7 M s-1 for Fe(C2O4)2- and 1.88 ± 0.29 ×10-7 M s-1 for the Fe(C2O4)2–/
256
Fe(C2O4)33– mixture under polychromatic irradiation. These values are approximately two orders of
257
magnitude higher than the RFe( II ) found for the Fe(III)-Pyo complexes. This difference could explain the
258
overestimation of iron reactivity by cloud chemistry models. Indeed, it has been observed that cloud
259
chemistry models tend to overestimate the contribution of iron to the oxidative capacity of clouds,
260
probably due to the incorrect description of iron in this complex medium.35 The iron is generally described
261
as iron oxalate and iron aquo-complexes, whereas strong iron-organic complexes (like Fe(III)-Pyo) are not
262
taken into account in the models. In presence of Fe(III)-Pyo complexes in cloud waters the Fe(III) photo-
263
reduction rate and associated free radical production decrease compared to the iron-oxalate and iron
264
aqua-complexes.
265
The photolysis of Fe(III)-organic complexes produces radical compounds that might generate reactive
266
oxygenated species such as HO2•/O2•–, H2O2 and HO•.54-56 In the case of iron aqua complexes, the photolysis
267
directly leads to the formation of Fe(II) and the hydroxyl radical, while in the case of Fe(III)-oxalate
268
complexes ([Fe(C2O4)]+, [Fe(C2O4)2]– and [Fe(C2O4)3]3–), the so-called LMCT involving nonbonding p-orbitals
269
of the coordinating O-atom and empty d-orbitals of the metal ion leads to the generation of an oxalate
270
radical followed by its reaction with molecular oxygen (expected to have a reaction rate of ~2 × 109 M-1 s-1)
271
57
272
more complicated due to the chemical structure of the organic ligand. In the Fe(III)-Pyo complex the LMCT
273
mechanism can occur by two different reaction pathways, A) and B) (see Figure 5). According to A), photo-
274
dissociation of the complex from the carbonyl binding group can lead to the formation of ferrous ions
275
(Fe(II)) and a carbonyl radical (1). The carbonyl radical undergoes a decarboxylation resulting in the
f
f
and the formation of CO2 and O2•–. In the case of Fe(III)-Pyo the photolysis mechanism is found to be
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formation of an unstable carbon-centred radical (2) that reacts with molecular oxygen to generate a
277
peroxide radical (3). The intermediate 3 can decompose into the hydroperoxide radical (HO2●) and a
278
derivative (4). Such a mechanism has been previously suggested for other Fe(III)-polycarboxylated
279
complexes in water under irradiation.58 Finally, hydroperoxide radical disproportionation leads to the
280
formation of hydrogen peroxide, which can react with ferrous ions to generate hydroxyl radical in water.
281
In the second pathway (B), the LMCT reaction can occur, leading to the oxidation of the N-hydroxylamine
282
functional group. In this way, the radical cation (5) leads to the protonated nitroxide radical (6a) via an
283
intramolecular hydrogen shift. Nitroxide radicals are stable. Thus, their formation is favoured, and they are
284
often used in organic synthesis and for their redox properties.59, 60 Radical 6a is in equilibrium with its basic
285
form 6b, with a pKa estimate of approximately 5.5 on the basis of reported values for dialkylnitroxide.61
286
Different pathways have been suggested for the nitroxide radical transformation: one possible mechanism
287
involves hydrogen transfer between the protonated (6a) and deprotonated (6b) forms of the nitroxide
288
radical, 62, 63 which yields hydroxylamine (7) and the N-oxoammonium cation (8). The latter can lead to the
289
formation of acetic acid (9) and pyoverdin product (10) via hydrolysis, acetic acid could be also produced by
290
reaction of 8 with hydroxyl radical that could lead to formation of 9 and a nitro derivative. Another possible
291
transformation pathway has been suggested by Goldestein and co-workers, who investigated the effect of
292
the hydroperoxide radical/superoxide radical anion couple (HO2•/O2•–, pKa = 4.8) on the stability of
293
nitroxides. These authors showed that the formation of the N-oxoammonium cation (8) is enhanced by the
294
presence of HO2•, while the back reaction (i.e., oxidation of oxoammonium cations to the nitroxide radical)
295
is promoted by O2•–.64 The suggested mechanism is also supported by experimental data, which show a
296
faster formation of acetic acid at pH 4.0 than at pH 6.0 (see Figure 4B). Acetate formation at pH 4.0 is found
297
to be approximately two times higher compared to that at pH 6.0 during the first 10 minutes.
298
The formation rate of the hydroxyl radical ( RHO• ) was measured to evaluate the impact of the photolysis of
299
Fe(III)-Pyo on the cloud chemistry oxidative budget. The
300
9
f
f RHO • value was on the order of magnitude of ~10
‒10-8 M s-1 under simulated solar irradiation of Fe(III)-Pyo (100 µM for both pH 4.0 and 6.0), showing that 13 ACS Paragon Plus Environment
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secondary species such as HO2•/O2•– and H2O2 can be generated during iron complex photolysis, leading to
302
the formation of the hydroxyl radical.
303
In Figure S13, Fe(III)-Pyo (100 µM at pH 6.0) was irradiated for 1 hour, and Fe(II) and acetate were
304
quantified. The data show that the Fe(III)-Pyo is photolysed faster at pH 4.0 (Figure 4A) compared to pH 6.0,
305
and in stronger acidic conditions, the formation rates of Fe(II) and acetate are approximately 2 times
306
greater than those at pH 6.0 (Figure 4B). Hydroxyl radical formation rates were measured to be 9.6 × 10-9
307
and 7.8 × 10-9 M s-1 at pH 4.0 and 6.0, respectively (see Supporting Information Text S1).
308 309
Atmospheric implications
310
This work reports the first investigation of the photochemical behaviour Fe(III)-pyoverdin complex, in which
311
the pyoverdin mixture produced by a bacterium isolated from the cloud aqueous phase is shown to
312
complex with ferric ions. Particular attention was paid to the generation of Fe(II) and HO• under aqueous
313
phase conditions of clouds. Additionally, the stability constants of Fe(III)-Pyo (log K) were estimated to be
314
20-27 times higher than those reported for other Fe(III)-carboxylate complexes. Hence, pyoverdins and
315
carboxylic acids, naturally present in the aqueous phase of clouds, can compete for Fe(III) complexation and
316
the formation of Fe(III)-Pyo can be considered as relevant.
317
Previous studies have not reported any data on the concentration of pyoverdins in cloud water, although
318
siderophores have been quantified in rain water. Cheize and coworkers have reported that the
319
concentrations of siderophores in rainwater are in the range of 0.104 to 0.366 µM equivalent of Fe(III), with
320
the dissolved Fe (dFe) concentrations being in the range of 0.073 to 0.988 µM.
321
dissolved iron concentrations in cloud samples are on the same order (µM) and much more concentrated
322
than those in sea waters (nM). Furthermore, concentrations of compounds found in rain water are
323
estimated to be between 2 and 23 times more diluted that the corresponding concentrations in cloud
324
water 66. Therefore, the siderophore concentration is expected to be in the same range as that of dissolved
325
iron in cloud water. Considering a reasonable concentration of Fe(III) 2 µM 35, 67 and Pyo ranging from 0.5 to 14 ACS Paragon Plus Environment
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Measurements of
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326
2 µM, we can estimate the competition between Pyo and oxalate (Ox) to complex Fe(III) at a pH of 4.0 (see
327
text S4). Due to the high complexation constant between Fe(III) and Pyo, the complex Fe(III)-Ox2 is expected
328
to be predominant for [Ox]/[Pyo] ratios greater than 200 (see figure S14). In Figure 6, the expected
329
concentrations of Fe(III)-Pyo and Fe(III)-Ox2 (Fe(C2O4)2-) complexes are determined as a function of oxalate
330
concentration in cloud water.2,12 As expected, all Pyo is complexed with iron and Fe(III)-Ox2 concentration
331
becomes relevant only when Pyo concentration is lower than iron concentration. In fact, in the presence of
332
0.5 µM of Pyo (and 2 µM of Fe(III)), Fe(III)-Ox2 concentration increases up to 1.5 µM, when reaches a
333
plateau due to the complete Fe(III) complexation. This estimation suggests that the presence of Fe(III)-Pyo
334
complex (and as consequence the relevance of this complex) depends on the relative Pyo/Fe(III)
335
concentrations ratio. However, our estimation is performed considering only one type of siderophores that
336
is expected to be produced by bacteria in cloud water. Therefore more siderophores are expected to be
337
present in cloud waters.
338
The presence of pyoverdins in the aqueous phase of clouds can impact the composition and oxidative
339
capacity of this medium via the following:
340
i) Iron cycle modification leading to the complexation of ferric ions and their increased availability 68 and
341
photochemistry.
342
ii) Formation of new ferric ion complexes from the pyoverdin oxidative products (after LMCT reaction), as
343
shown for marine siderophores.27, 69 These species can be attributed to the formation of new complexes
344
between Fe(III) (still present in solution under its soluble form) and degradation products of pyoverdin).
345 346
iii) Scavenging of the hydroxyl radical in cloud water (pyoverdins’ reactivity with the hydroxyl radical has been estimated to be approximately 1010 M-1 s-1).70
347
iv) Decrease the Fe(III) photo-reduction rate and consequently the associated free radical production.
348
v) Formation of low molecular weight organic compounds, such as carboxylic acids, under solar irradiation.
349
Finally, the iron chemistry and its photoreactivity in cloud water should be deeply investigated in the
350
future, and the related impact on the oxidative capacity of cloud water must be reconsidered by cloud 15 ACS Paragon Plus Environment
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351
chemistry models. In fact, cloud chemistry models are generally restricted to iron oxalate and iron aqua
352
complexes and tend to overestimate the contribution of iron to the oxidative capacity of this complex
353
medium. This work considers only one type of siderophores, the pyoverdins, but the real environment can
354
have many other biological or anthropogenic compounds with a strong affinity to iron (e.g.,
355
aminopolycarboxylic acids). It is likely that other Fe(III)-siderophore complexes may have a similar reactivity
356
to Fe(III)-Pyo, but this hypothesis should be confirmed through future studies. In summary, other studies
357
are needed to better investigate the photoreactivities of other strong Fe(III)-organic complexes.
358 359
Acknowledgements
360
Authors acknowledge financial support from the Regional Council of Auvergne and from the "Fédération de
361
Recherches en Environnement" through the CPER “Environnement” founded by the “Région Auvergne,” the
362
French government, FEDER from the European community, and the ANR BIOCAP (ANR-13-BS06-0004).
363
AMD and VV gratefully acknowledge Christophe Chambon and Didier Viala (INRA, Plateforme d'exploration
364
du métabolisme, F-63122 Saint-Genès Champanelle, France) for mass spectrometry analysis.
365 366
Associated content
367
Supporting information. Experimental setups details and formation rates calculations, Table S1 and Figures
368
S1-14.
This
information
is
available
free
of
charge
via
Internet
at
http://pubs.acs.org/
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Environmental Science & Technology
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68.Kraemer, S., Iron oxide dissolution and solubility in the presence of siderophores. Aquat. Sci. 2004, 66, (1), 3-18. 69.Vraspir, J. M.; Butler, A., Chemistry of marine ligands and siderophores. Ann. Rev. Mar. Sci. 2009, 1, (1), 43-63. 70.Hoe, S.; Rowley, D. A.; Halliwell, B., Reactions of ferrioxamine and desferrioxamine with the hydroxyl radical. Chem.-Biol. Interact. 1982, 41, (1), 75-81. 71.Ongena, M.; Jacques, P.; Delfosse, P.; Thonart, P., Unusual traits of the pyoverdin-mediated iron acquisition system in Pseudomonas putida strain BTP1. BioMetals 2002, 15, (1), 1-13. 72.Smith, R. M.; Martell, A. E., Critical Stability Constants. Springer US: 1977; Vol. 3-6.
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RFef ( II ) (M s-1)
φFe ( II )
Stability constant (log K)
Fe(III)-Pyo pH 4.0
6.98 ± 0.07 ×10-9
1.9 × 10-4
19.7-21.2 * (at pH 5)71
Fe(III)-Pyo pH 6.0
3.96 ± 0.04 ×10-9
5.5 × 10-5
25.5-27.1 * (at pH 7)71
Fe(III)-oxalate complexes [Fe(C2O4)2]–
5.35 ± 0.60 ×10-7
16.2 72
[Fe(C2O4)2]–/ [Fe(C2O4)3]3–
1.88 ± 0.29 ×10-7
16.2 / 26.2 72
50/50 551 f
552
Table 1. Fe(II) formation rate ( RFe( II ) ) and Fe(II) quantum yield formation ( φFe ( II ) ) of the 100 µM Fe(III)-Pyo
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complex at pH 4.0 and 6.0 and 100 µM Fe-oxalate complexes (di-oxalate : [Fe(C2O4)2]– and a mixture of di and tri-oxalate [Fe(C2O4)2]–/ [Fe(C2O4)3]3–) obtained in this work under simulated solar irradiation. Stability constants (log K) of [Fe(C2O4)2]– and [Fe(C2O4)3]3– are reported from the literature. * Data are taken from other iron(III)-pyoverdins.
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Figure Caption
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1)
2) 3) 4) 5) 6)
Emission spectrum of Xenon lamp (solar simulator), sun emission spectrum measured at the top of puy de Dôme mountain in autumn 2013 during cloudy conditions and molar absorption coefficients of the aqueous Fe(III)-Pyo complexes at pH 4.0 and 6.0. Proposed structure for the linear form of the major pyoverdin. A, B and Y indicate the identified fragmentations reported in Figure S3 Fluorescence excitation-emission matrix (EEM) of pyoverdin. Fe(II) (A) and acetate (B) formations from Fe(III)-Pyo (100µM) irradiation in water at pH 4.0 and 6.0 at 278 ± 2 K. The lines are the exponential rise fits of data. Proposed reaction mechanisms from the LMCT reaction of Fe(III)-Pyo under polychromatic irradiation The Fe(III)-Pyo and Fe(III)-Ox2 complexes concentration are reported as a function of oxalate concentration. The competition between Fe(III)-Pyo and Fe(III)-Ox2 complexes is calculated at pH 4.0, considering 2 µM of Fe(III) in the presence of Pyo 0.5µM (dashed lines) and 2 µM (continuous lines). The shaded region of the graph highlights the most significant concentration range of oxalate found in cloud waters.
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Figure 1
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Figure 2
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Figure 3
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Figure 6
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