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Aug 3, 2016 - ABSTRACT: In the present work, the photoreactivity of a mixture of iron(III)−pyoverdin (Fe(III)−Pyo) complexes was investigated unde...
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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

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F-63000 CLERMONT-FERRAND, FRANCE

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b

Université Clermont Auvergne, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448,

CNRS, UMR 6296, ICCF, F-63171 AUBIERE, FRANCE

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* 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]

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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

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present in cloud water, thus modifying their availability and photoreactivity. The spectroscopic properties

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and photoreactivity of Fe(III)-Pyo were investigated, with particular attention to their fate under solar

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irradiation, also studied through simulations. The photolysis of the Fe(III)-Pyo complex leads to the

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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,

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respectively. Interestingly, acetate formation was observed during the iron-complex photolysis, suggesting

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that fragmentation can occur after the ligand-to-metal charge transfer (LMCT) via a complex reaction

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mechanism. Moreover, photogenerated Fe(II) represent an important source of hydroxyl radical via the

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Fenton reaction in cloud water. This reactivity might be relevant for the estimation of the rates of

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formation and steady-state concentrations of the hydroxyl radical by cloud chemistry models and for

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organic matter speciation in the cloud aqueous phase. In fact, the conventional models, which describe the

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iron photoreactivity in terms of iron-aqua and oxalate complexes, are not in accordance with our results.

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TOC/Abstract

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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

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of clouds has been well elucidated,5,

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characterized due to its complexity.7, 8

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Only 25-30% of the organic components of clouds have been characterized, while the remaining 70-75% is

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unidentified.9-11 The identified compounds are mainly acetic, formic and oxalic acids and other small chain

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acids10, 12 or aldehydes.13 A recent study reported the molecular weight distribution for the organic fraction

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of cloud samples and showed that the main compounds have molecular weights between 50 and 2500

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Da.14 The small chain carboxylic acids are derived from anthropogenic and biological emission and from

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chemical transformation in the atmosphere and in clouds. The higher molecular weight material (> 500 Da)

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could be humic-like substances (also called HULIS), macromolecules and biogenic nanoscale material

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(BONM)

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water.14, 17, 18 Indeed the presence of microorganisms such as bacteria and fungi has been reported mainly

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in the last decade.19-21 Bauer and coworkers reported that microorganisms can contribute to the organic

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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

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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

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processes (R1 and R2).29, 30

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Fe( II ) + H2O2 → Fe( III ) + HO− + HO•

(R1)

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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

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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,

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models represent iron as free ions, aquo-complexes or Fe-oxalate complexes.34 As recently shown,

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atmospheric aqueous models tend to overestimate hydroxyl radical formation from iron species, probably

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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

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carboxylic acids found in cloud water (oxalate, formate, etc.) have been extensively studied.34, 36 However,

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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.

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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

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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

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solar irradiation. In particular, the rates of formation of Fe(II) and the hydroxyl radical were measured to

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21

and is

assess the impact of the Fe(III)-Pyo complex on the oxidative capacity of the aqueous phase of clouds.

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Materials and methods

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Pyoverdin purification

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Pyoverdins were produced by the bacterial strain Pseudomonas fluorescens 36b5 (JF706586) isolated from

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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

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(InterchimPuriflash C18-HQ-15 µm-35 g). Pure fractions were freeze, dried and stored under argon in a

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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.

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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

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a Millipore milli-q device (Millipore αQ, resistivity 18 MΩ cm, DOC < 0.1 mg L-1). The pH was modified by the

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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

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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

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acid)-1,2,4-triazine) coupled with UV/vis spectrophotometric detection at λmax = 562 nm 40. The well-known

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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

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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

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the absorption peak at 480 nm characteristic of the Fe(III)-Pyo complex. At pH 4.0, complex absorbance

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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

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Irradiation experiments

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Aqueous solutions were irradiated in a 40 mL cylindrical reactor at a constant temperature (278 ± 2

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K) controlled by water circulation. The reactor was located at the focal point of the lamp to ensure a

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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

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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.

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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-

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dinitrobenzene,43 was measured by HPLC. Further details about the detection method are reported in the

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Supporting Information (Text S1).The kinetic approach to estimate the rate of hydroxyl radical formation is

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obtained from the following equation (eq.1)

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d f RNB = RHO − •

k NB ,HO• [NB]

(eq. 1)

k NB ,HO• [NB] + ∑ k i [Si ] i

d

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f where RNB is the rate of degradation of nitrobenzene (M s-1), RHO • is the formation rate of the hydroxyl

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radical (M s-1), k NB, HO • the second order rate constant for the reaction between nitrobenzene and the

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hydroxyl radical (3.9 × 109 M-1 s-1)

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and

∑k [S ] i

i

is the pseudo-first-order rate constant (s-l) for HO•

i

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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

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f considered to determine RHO k NB,HO• [NB] and k NB,HO• [NB] • . Under adopted conditions,

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estimated to be 1.17 × 106 s-1 and 1.0 × 106 s-1 respectively indicating that 54 % of photogenerated hydroxyl

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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

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supernatant components by reverse phase chromatography showed the presence of two major and two

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minor products with spectra characteristic of pyoverdins. Longer incubations increased the yield, but

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degradation products appeared as the pH of the medium was increased.

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Pyoverdin was extracted and pre-purified from the supernatant in a single step by ultraperformance flash

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chromatography on a C18 cartridge. Further purification was performed on a high quality column with a

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smaller particle size (15 µm). The global yield of the culture medium was 50 mg L-1. HPLC analyses showed

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that the major components were present in identical proportions and represented 91% of the total

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pyoverdin (Figure S3).

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Experiments were performed on a mixture of pyoverdins to take into account the diversity of

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pyoverdins in the environment. Exact molecular masses of various pyoverdins were determined by

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MALDI-TOF. The two major compounds had molecular weights of 1142.6 and 1160.6 Da. Minor

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compounds had molecular masses of 1171.6 and 1188.6 Da. As the masses of the major compounds

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differ by 18u, they probably share the same peptide chain in a linear and a cyclic form. Indeed,

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cyclisation has already been described in terms of ester bond formation between a carboxylic acid

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and the alcohol function of serine 45 with a loss of H2O.

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Structural investigation of the major linear form was performed by fragmentation of the [M-H2O+H]+ ion

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(m/z = 1143) by MALDI-TOF-TOF. The major ion corresponds to the A1 fragment (m/z = 417) with a loss of

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H2O (m/z = 399) which indicates that serine is the first amino acid linked to the chromophore (Figures S4, S5

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and Table S1) associated with a succinic acid side chain.46 Retro-Diels-Alder opening of the chromophore

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gives characteristic fragments (m/z = 286 and 204). Loss of 74u due to the elimination of COOH-CHO by a

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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

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matching the exact mass and the fragmentation pattern can then be proposed as follows: Chr-Ser-

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AcOHOrn-Pro-Val OHAsp-Ala-Lys. (Figure 2).

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Spectroscopic analyses of pyoverdin and Fe(III)-pyoverdin complex

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Pyoverdins are strong Fe(III) complexing agents. Therefore, it is highly probable that in atmospheric water

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they may exist in the form (Fe(III)-Pyo). A spectroscopic analysis of pyoverdin and the Fe(III)-pyoverdin

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complex (Fe(III)-Pyo) was carried out in water at pH 4.0 and pH 6.0 (with addition of HClO4), considered as

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the typical pH range values of cloud water.12 The pyoverdin mixture solution (~100 µM, without iron) was

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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

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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

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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

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was observed to change with pH. In particular, a hypochromic shift was observed at more acidic pH values.

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The pyoverdin solution at pH 4.0 was not stable, and the UV-vis spectrum changed during 72 h (in the dark),

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as reported in Figure S7. When the solution was returned to its natural pH (~ 8.0), a bathochromic shift in

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the UV-vis spectrum was observed. In aqueous solution, pyoverdin acts as a buffer, and its pH slowly

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changes towards the natural pH (~ 8.3). Pyoverdins are fluorescent molecules containing a chromophores,

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generally a catechol, that is responsible for colour and fluorescence.47 A fluorescence excitation−emission

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matrix (EEM) of pyoverdin was recorded at natural pH (Figure 3). Pyoverdin excited at λex= 408 nm emits at

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a maximum (λem) centred at 460 nm. The measurement of fluorescence is a sensitive method for detecting

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pyoverdin (detection limit estimated to be approximately 0.3 µM), and a linear correlation between

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fluorescence intensity and concentration was observed up to 40 µM (Figure S8). After this concentration

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corresponding to Abs > 0.2, fluorescence quenching was observed.48 The 1:1 Fe(III):Pyo stoichiometry of the

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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)

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and form strong 1:1 complexes with iron (III), in agreement with the results reported in the literature.49, 50

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A spectroscopic analysis of Fe(III)-pyoverdin shows that the complex formation can be confirmed by

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fluorescence loss. Indeed, the Fe(III)-Pyo complexwas found not to be fluorescent, and in the EEM of the

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complex solution, only Rayleigh and Raman scattering were observed (Figure S10).51

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The UV-vis spectrum of the complex at pH 4.0 shows a shoulder at 273 nm and a band centred at 386 nm

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[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

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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

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same absorption band at 386 nm as that observed for the complex at pH 4 [ε386 = (1.46 ± 0.11) × 103 M-1 cm-

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1

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observed after 2 h.

]. The stability of the complexes was investigated in the dark, and no change in the UV-vis spectra were

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Photochemical behaviour of pyoverdin and the Fe(III)-pyoverdin complex

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The photoreactivity of Fe(III)-Pyo was investigated at pH 4.0 and 6.0 under polychromatic irradiation with,

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particular attention to the generation of Fe(II) and small carboxylic acids. Fe(III)-Pyo under simulated solar

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irradiation undergoes a ligand-to-metal-charge-transfer (LMCT) leading to the formation of Fe(II) and

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organic ligand radicals.52 The quantity of Fe(II) produced during the irradiation is directly proportional to

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the Fe(III)-Pyo concentration (see the Supporting Information for the method adopted to measure the

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formation/loss rates).

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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

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and 5.5 × 10-5 respectively for pH 4.0 and 6.0. The difference of

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considering the spontaneous oxidation of Fe(II) to Fe(III) by oxygen in waters at pH > 4, as previously

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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

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formation rates of Fe(II) with those reported for Fe(III)-Pyo. Iron-oxalate complexes were prepared as

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reported in the Supporting Information (Text S4 and Figures S11-12 ), and the pH was fixed at 2.5 and 3.8 to

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obtain the maximum concentration of the desired stoichiometry of the complex or mixture. RFe( II ) were

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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–/

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Fe(C2O4)33– mixture under polychromatic irradiation. These values are approximately two orders of

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magnitude higher than the RFe( II ) found for the Fe(III)-Pyo complexes. This difference could explain the

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overestimation of iron reactivity by cloud chemistry models. Indeed, it has been observed that cloud

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chemistry models tend to overestimate the contribution of iron to the oxidative capacity of clouds,

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probably due to the incorrect description of iron in this complex medium.35 The iron is generally described

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as iron oxalate and iron aquo-complexes, whereas strong iron-organic complexes (like Fe(III)-Pyo) are not

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taken into account in the models. In presence of Fe(III)-Pyo complexes in cloud waters the Fe(III) photo-

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reduction rate and associated free radical production decrease compared to the iron-oxalate and iron

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aqua-complexes.

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The photolysis of Fe(III)-organic complexes produces radical compounds that might generate reactive

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oxygenated species such as HO2•/O2•–, H2O2 and HO•.54-56 In the case of iron aqua complexes, the photolysis

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directly leads to the formation of Fe(II) and the hydroxyl radical, while in the case of Fe(III)-oxalate

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complexes ([Fe(C2O4)]+, [Fe(C2O4)2]– and [Fe(C2O4)3]3–), the so-called LMCT involving nonbonding p-orbitals

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of the coordinating O-atom and empty d-orbitals of the metal ion leads to the generation of an oxalate

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radical followed by its reaction with molecular oxygen (expected to have a reaction rate of ~2 × 109 M-1 s-1)

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57

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more complicated due to the chemical structure of the organic ligand. In the Fe(III)-Pyo complex the LMCT

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mechanism can occur by two different reaction pathways, A) and B) (see Figure 5). According to A), photo-

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dissociation of the complex from the carbonyl binding group can lead to the formation of ferrous ions

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(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

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peroxide radical (3). The intermediate 3 can decompose into the hydroperoxide radical (HO2●) and a

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derivative (4). Such a mechanism has been previously suggested for other Fe(III)-polycarboxylated

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complexes in water under irradiation.58 Finally, hydroperoxide radical disproportionation leads to the

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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

553 554 555 556

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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