Photochemical and Nonphotochemical Transformations of Cysteine

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Photochemical and non-photochemical transformations of cysteine with dissolved organic matter Chiheng Chu, Paul R. Erickson, Rachel A. Lundeen, Dimitrios Stamatelatos , Peter J. Alaimo, Douglas E. Latch, and Kristopher McNeill Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01291 • Publication Date (Web): 12 May 2016 Downloaded from http://pubs.acs.org on May 22, 2016

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Environmental Science & Technology

Manuscript

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Photochemical and non-photochemical transformations of cysteine with dissolved organic

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matter

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Chiheng Chu †, Paul R. Erickson †, Rachel A. Lundeen †, Dimitrios Stamatelatos †, Peter J. Alaimo ‡, Douglas E. Latch ‡, and Kristopher McNeill †*

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† Institute of Biogeochemistry and Pollutant Dynamics (IBP), Department of Environmental Systems Science, ETH Zurich, 8092 Zurich, Switzerland

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‡ Department of Chemistry, Seattle University, Seattle, WA 98122, USA

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*Corresponding authors

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Kristopher McNeill

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Tel. +41 (0)44 6324755; Fax. +41 (0)44 6321438

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Email: [email protected]

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Number of Figures:

3

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Number of Tables:

2

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Total word count:

6083

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1 ACS Paragon Plus Environment


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Abstract

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Cysteine (Cys) plays numerous key roles in the biogeochemical processes in natural waters.

23

Despite its importance, a full assessment of Cys abiotic transformation kinetics, products and

24

pathways under environmental conditions has not been conducted. This study is a mechanistic

25

evaluation of the photochemical and non-photochemical (dark) transformations of Cys in

26

solutions containing chromophoric dissolved organic matter (CDOM). The results show that Cys

27

underwent abiotic transformations under both dark and irradiated conditions. Under dark

28

conditions, the transformation rates of Cys were moderate and highly pH- and temperature-

29

dependent. Under UVA or natural sunlight irradiations, Cys transformation rates were enhanced

30

by up to two orders of magnitude compared to rates under dark conditions. Product analysis

31

indicated cystine and cysteine sulfinic acid were the major photooxidation products. In addition,

32

this study provides an assessment of the contributions of singlet oxygen, hydroxyl radical,

33

hydrogen peroxide, and triplet dissolved organic matter in Cys photochemical oxidation

34

reactions.

35

photochemical loss of Cys, which will require further study to identify.

The results suggest that another unknown pathway was dominant in the


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INTRODUCTION

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Thiols (RSH) are of great importance in both biological and geochemical systems in natural

38

waters. Thiol-containing compounds, such as cysteine (Cys) and Cys-containing biomolecules,

39

are abundant in microbes and involved in numerous biochemical processes, such as binding

40

metals, stabilizing protein structure through disulfide linkages, and acting as redox-active

41

nucleophiles in enzymes.1-4 When released into the extracellular environment, Cys and Cys-

42

containing biomolecules play key roles in environmental nitrogen, sulfur, and metal cycling. For

43

instance, Cys is known to be important in the speciation, transport, reactivity, and bioavailability

44

of metals (e.g., Hg, Cu, Zn, Cd, and Pb) in aquatic systems.5-8 Recent studies have shown that

45

complexation of Hg by Cys significantly affects both the Hg methylation by microbes9-12 and

46

methylmercury photoreactivity13,14 in natural waters. From a nutrient bioavailability viewpoint,

47

Cys is not only a bioavailable source of organic nitrogen (along with other free amino acids), but

48

also a rare source of reduced organic sulfur in natural waters. Additionally, Cys in surface waters

49

has been hypothesized to be the direct precursor to carbon disulfide and carbonyl sulfide in

50

marine systems, and thus plays a key role in the global sulfur cycle.1,15 Despite the important

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roles that Cys plays in the environment, there is little known about its transformation processes

52

in natural waters. These transformation processes regulate the aqueous steady-state

53

concentrations of Cys (~ 10-10 - 10-11 M in surface waters, including both free Cys and oxidized

54

Cys disulfide dimer)3 and subsequently affect environmental processes involving Cys.

55

Previous studies have suggested that photochemical transformation is an important sink for

56

thiols in surface waters.16,17 While direct phototransformation of Cys is not possible due to its

57

negligible absorbance in the solar spectrum (Figure 1), photochemical oxidation of Cys may

58

occur indirectly by reaction with various photochemically produced reactive intermediates



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(PPRI).18-22 In natural waters, chromophoric dissolved organic matter (CDOM) acts as the major

60

sensitizer of PPRI.23 Previous photochemical studies on free amino acids have demonstrated that

61

the relative importance of each PPRI-mediated photooxidation pathway may vary greatly among

62

different amino acid species during CDOM-sensitized photolysis.24-27 To date, the susceptibility

63

of Cys to phototransformation in sunlit surface waters and the relative importance of PPRI on

64

Cys phototransformation remain unclear.

65

In addition to photochemical transformation, abiotic non-photochemical transformation (i.e.,

66

under dark conditions) may also play an important role in the fate of Cys in environmental

67

systems. Recent work shows that hydrogen sulfide (H2S), the simplest sulfhydryl-containing

68

molecule present in surface waters (~ 10-9 - 10-11 M),28 can be oxidized non-photochemically in

69

CDOM solutions with a half-life of days and numerous oxidation products are formed upon H2S

70

dark oxidation.29 Analogously, the free thiol group of Cys may undergo similar or even faster

71

dark oxidation when in solution with CDOM, considering the lower proton coupled electron

72

transfer potential for Cys compared to H2S.30 The abiotic transformation of Cys, either through

73

the photochemical or non-photochemical transformation pathways, may be dependent on

74

solution pH because of the higher electron density of the deprotonated anionic thiol moiety (Cys-)

75

compared to the protonated neutral species (Cys0).

76

While there is considerable prior work indicating that photochemical and non-photochemical

77

transformations of free Cys should occur in natural waters, an evaluation of Cys transformation

78

in aquatic systems has not yet been conducted. The lack of in-depth assessment of the free Cys

79

has lagged behind that of the other canonical free amino acids, which have been readily studied

80

in natural waters,24 largely due to two reasons. First, free Cys is extremely reactive, especially

81

under oxic conditions when trace metals are present. Metal-catalyzed oxidation of thiols can


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easily occur on the benchtop,31 for instance, due to trace amounts of metals in glassware, water

83

or reagents used in experiments, leading to artifacts in reactions rates of Cys oxidation. Secondly,

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some methods to analyze and quantify free amino acids, such as ortho-phthaldialdehyde-

85

derivatization,24,32 are not compatible with Cys. These analytical drawbacks related to Cys

86

analysis are evident from both laboratory studies24 and many environmental studies,33-35 which

87

have analyzed all of the other 19 proteinogenic amino acids but omitted Cys from their studies.

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The goal of this study was to evaluate the abiotic photochemical and non-photochemical

89

transformations of free Cys in natural waters. We assessed the transformation rates of Cys under

90

UVA irradiation and natural sunlight conditions, as well as dark conditions, in CDOM solutions

91

at pH 5.7-9.9. In addition, the reaction rate constants of Cys with environmentally relevant PPRI

92

(i.e., singlet oxygen (1O2), triplet state excited sensitizers (3Sens*), and hydrogen peroxide

93

(H2O2)) were established over a range of solution pH values. Utilizing PPRI-specific rate

94

constants, the relative contributions of each PPRI-mediated transformation pathways to the

95

transformation of Cys during CDOM-sensitized photolysis were assessed. These assessments

96

were conducted following trace metal clean techniques to avoid unwanted metal-catalyzed

97

oxidation reactions. In addition, a selective thiol derivatization agent, monobromobimane

98

(mBBr), was used as fluorescence-based probe for Cys and also as a protecting group to prevent

99

the oxidation of Cys before analysis.36

100 101

MATERIALS AND METHODS

102

Materials and sample preparation. Detailed information on sources and preparation

103

methods of chemicals is provided in the supporting information (SI, Section S1). All

104

experiments conducted in H2O solutions were pH-buffered using acetate (pH 4.0-6.0), phosphate 5 ACS Paragon Plus Environment


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(pH 6.0-7.5), tris (pH 7.5-9.5), carbonate (above pH 9.5) all at 5 mM. To prevent contamination

106

with trace metals, which might catalyze the oxidation of Cys, all glassware was acid-washed

107

prior to use.

108

Suwannee River Natural Organic Matter (SRNOM, Lot Nr. 1R101N) and Pahokee Peat

109

Humic Acid Standard (PPHA, Lot Nr. 15103H) were purchased from the International Humic

110

Substances Society (IHSS) and were used as received. SRNOM and PPHA were chosen as

111

model CDOM because of their representative origins of decomposing vegetation and agricultural

112

peat soil, respectively. CDOM solutions were prepared following previously published

113

methods.25 All CDOM solutions used in this study had a concentration of 11.4 mgC/L.

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Cys reaction with singlet oxygen. The photochemical transformation rate constants of Cys

115

with 1O2 were assessed either through steady-state photolysis in the presence of a model 1O2

116

sensitizer or through time-resolved 1O2 phosphorescence measurements.

117

Steady-state photolysis. Steady-state photolysis experiments using perinaphthenone as the 1O2

118

sensitizer were conducted at pH 5.0 and 10.0, where Cys was in the neutral form (Cys0) or

119

anionic form (Cys-), respectively (n.b., the pKa1 of the thiol group is 8.42; see below). During the

120

steady-state photolysis, Cys oxidation was mediated by a combination of 1O2 and other PPRI.

121

The involvement of 1O2 on Cys oxidation was probed by using D2O as a solvent in place of H2O

122

due to the well-known solvent isotope effect.37 D2O solutions were pD-adjusted with NaOD and

123

DCl where pD = pH* + 0.4 (pH* corresponds to the uncorrected pH meter reading). All

124

solutions during the steady-state photolysis experiments contained Cys (initial concentration of

125

40 µM), furfuryl alcohol (FFA, initial concentration of 100 µM), a pH buffer species (5 mM),

126

and a 1O2 sensitizer (i.e., perinaphthenone). At pH 5.0, any secondary kinetic isotope effect on


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the Cys transformation rate due to the conversion of S-H bonds to S-D in D2O solution was

128

assumed to be negligible and ignored. The observed pseudo-first-order phototransformation rate

129

HO constants (units of s–1) of Cys0 and Cys- in H2O ( kCys and ,obs

130

DO and ( kCys ,obs

131

Cys transformation rate constants could be described by Equations 1-4.

2

0

2

0

HO kCys ,obs , respectively) and D2O 2

−

DO kCys ,obs, respectively) were assessed at solution pH 5.0 and pH 10.0. The observed 2

−

1

H2O

O2

1

H2 O

Other

kCys ,obs = kCys ,rxn [ O2 ] ss,pH5 +kCys 0

0

1

D2O

O2

(૚)

0

1

D2O

Other

kCys ,obs = kCys ,rxn [ O2 ] ss,pH5 +kCys 0

0

1

H2O

O2

(૛)

0

H2O

1

Other

kCys ,obs = kCys ,rxn [ O2 ] ss,pH10 +kCys −

−

1

D2O

O2

(૜)

−

1

D2 O

Other

kCys ,obs = kCys ,rxn [ O2 ] ss,pH10 +kCys −

−

(૝)

−

132

O O O O where [ 1O2 ]Hss,pH5 ， [ 1O2 ]Hss,pH10 ， [ 1O2 ]Dss,pH5 ， and [ 1O2 ]Dss,pH10 were the steady-state concentrations

133

of 1O2 at pH 5.0 and 10.0 in H2O and D2O;

134

constants (units of s–1) of Cys0 and Cys- with other PPRI, respectively. The 1O2-mediated

135

bimolecular reaction rate constants (units of M–1s–1) of Cys0 and Cys- ( k

136

were thus obtained (Equation 5 and 6).

2

2

2

2

k

=

k

−

D 2O

H 2O

0

0

1

D 2O

1

H 2O

1

O2 0

Cys ,rxn

1

and

O2

k Cys ,rxn ) −

(૞)

[ O 2 ] ss,pH5 −[ O 2 ] ss,pH5 =

D 2O

H 2O

−

−

k Cys ,obs − k Cys ,obs

1

O2 Cys − ,rxn

0

k Cys ,obs − k Cys ,obs

1

O2 Cys 0 ,rxn

Other Other kCys and kCys were the first order reaction rate

1

D 2O

1

H 2O

(૟)

[ O 2 ] ss,pH10 − [ O 2 ] ss,pH10



1

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O2

137

The 1O2 reaction rate constants of Cys ( kCys,rxn ) between pH 5.0 and 10.0 were modeled

138

O (Equation 7) accounting for the respective reaction rate constants of Cys0 and Cys- ( kCys and ,rxn

139

kCys ,rxn ) and their respective fractions ( fCys

1

2 0

1

O2

and

0

−

1

1

O2

fCys ) to total Cys, −

1

O2

O2

k Cys,rxn = k Cys ,rxn f Cys + k Cys ,rxn f Cys 0

0

−

−

(ૠ)

140

The fractions of Cys0 and Cys- were calculated based on the pKa of thiol sidechain (pKa1) and

141

the solution pH (Equation 8 and 9). The pKa1 values of thiol sidechain in H2O and D2O

142

measured by UV-vis spectroscopic titration were 8.42 and 8.37, respectively (Figure S2, pKa1 in

143

D2O was obtained as uncorrected pH meter reading).

fCys = 0

1 + 1+Ka1 /[H ]

(ૡ)

+

fCys

−

Ka1 /[H ] = + 1+Ka1 /[H ]

(ૢ)

144

Time-resolved phosphorescence. Time-resolved 1O2 phosphorescence measurements were

145

conducted at pH 10.2 with perinaphthenone as the 1O2 sensitizer using a laser flash photolysis

146

setup, where the excitation wavelength was 350 nm and 1O2 phosphorescence was monitored at

147

1270±5 nm. Cys was added at concentrations ranging from 0.06 mM to 20 mM to quench the

148

1

149

decrease in the 1O2 lifetime upon Cys addition to Equation 10:

O2 phosphorescence. The bimolecular rate constant

1/ τ

1

O2

1

1

O2

kCys ,rxn −

1

O2

= kCys ,rxn [Cys]+1/ τ 0

O2

−

1

1

150

where [Cys] was the concentration of Cys and

151

with or without Cys in solution, respectively.

τO

2

and

τ 0O

2

was determined by fitting the

(૚૙)

were the measured 1O2 lifetime


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152

Cys and 2,4,6-trimethylphenol (TMP) reactions with triplet state ketone sensitizers. The

153

photochemical transformation rate constants of Cys with model triplet state sensitizers (3Sens*)

154

were assessed at pH 4.4 and 10.2 using transient absorption spectroscopy following previously

155

published methods.38 As triplet ketones are believed to be among the key oxidants formed upon

156

the photolysis of CDOM mixtures,39 a series of ketone sensitizers (i.e., perinaphthenone,

157

lumichrome, 2-acetonaphthone, 3’-methoxyacetophenone, and benzophenone-4-carboxylate,

158

herein defined as 3Sens*, see Section S2 for details) were used to represent the range of ketone

159

triplet oxidants in CDOM with varying excited triplet state redox potentials. 3

160

Sens* The bimolecular reaction rate constant of Cys with 3Sens* ( kCys,rxn , units of M–1s–1) was

161

determined by fitting the decay of 3Sens* lifetime as a function of increasing Cys concentration

162

(ranging from 0.25 mM to 30 mM) to Equation 11:

1/ τ 3

τ

Sens*

3

and

τ 0 Sens*

3

Sens*

3

3

Sens*

= kCys,rxn [Cys]+1/ τ 0

Sens*

(૚૚)

were the measured 3Sens* lifetime with or without Cys. The

163

where

164

bimolecular reaction rate constant of TMP with 3Sens* was assessed using the same approach.

165

Cys reaction with hydrogen peroxide. H2O2-mediated Cys oxidation was carried out in 2

166

mL protein LoBind eppendorf tubes in the dark at different solution pH values ranging from 5.0

167

to 10.4. The solutions contained Cys (initial concentrations of 20 µM), a buffer species (5 mM),

168

and H2O2 (500 µM). Aliquots were removed at various time points for kinetic analysis of Cys.

169

The bimolecular reaction rate constant,

HO (units of M–1s–1), of Cys with H2O2 was kCys,rxn 2

2

170

HO calculated from the observed pseudo-first-order rate constant of Cys transformation ( kCys,obs ) and

171

the concentration of H2O2 ([H2O2]) (Equation 12).

2

2



HO kCys,obs = [H2O2 ] 2

H2O2 Cys,rxn

k 172

The experimental

Page 10 of 37

2

(૚૛)

H2O2

kCys,rxn at pH values between 5.0 and 10.4 was fit to a model (Equation 13) H2O2

H2O2 − ,rxn

173

considering the respective reaction rate constants of Cys0, Cys- and Cys2- ( kCys ,rxn , kCys

174

kCys

0

H2O2 2−

,rxn ),

and their respective fractions (

H2O2

fCys , fCys 0

H2O2

−

and

fCys

2−

H2O2

) to total Cys,

H2O2

kCys,rxn = kCys ,rxn fCys + kCys ,rxn fCys + kCys 0

175 176

0

−

and

−

2−

,rxn

fCys

2−

(૚૜)

The fractions of Cys species were calculated based on the thiol pKa1 (pKa1 = 8.42)，Nterminal amine pKa2 (pKa2 = 10.28),40 and the solution pH (Equation 14-16). +

Ka1 /[H ] = + + 1+Ka1 /[H ]+[H ]/Ka2

(૚૝)

−

1 + + 1+Ka1 /[H ]+[H ]/Ka2

(૚૞)

2−

[H ]/Ka2 = + + 1+Ka1 /[H ]+[H ]/Ka2

fCys

0

fCys =

+

fCys

(૚૟)

177

Cys transformation in CDOM solutions. Phototransformation. The photolysis experiments

178

of Cys in CDOM solutions were conducted at different solution pH ranging from 5.7 to 9.9. The

179

solutions contained Cys (initial concentrations of 40 µM), a buffer species (5 mM), and CDOM

180

(i.e., either SRNOM or PPHA, 11.4 mgC/L). In order to probe the 1O2, •OH, and H2O2

181

production in CDOM solutions, identical photolysis solutions were prepared containing a PPRI

182

probe and CDOM, as described in the SI (Section S5). Briefly, FFA was used as a probe to

183

determine the steady-state 1O2 concentration in the bulk aqueous phase ([1O2]ss). Potassium

184

terephthalic acid (TPA) was used as a probe to determine the steady-state •OH concentration in


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185

the bulk aqueous phase ([•OH]ss).41,42 Ampliflu Red was used to probe H2O2 formation rate

186

( RH2O2 , unit of M/s) by the formation of fluorescent resorufin in the presence of horseradish

187

peroxidase.43 light

188

CDOM ) was The first-order transformation rate of Cys in CDOM-containing solutions ( kCys,obs

189

obtained in UVA- or sunlight-irradiated experiments. The relative importance of each PPRI-

190

mediated Cys transformation pathway in irradiated CDOM solutions was estimated by

191

comparing the estimated pseudo-first-order reaction rate constant of each PPRI-mediated Cys

192

PPRI CDOM phototransformation ( kCys ) with kCys,obs .24,44-46 For 1O2 and •OH, the pseudo-first-order reaction

193

O rate constants ( kCys and

194

O constants for the reaction between Cys and 1O2 or •OH ( kCys,rxn and

195

steady-state 1O2 or •OH concentrations ([1O2]ss and [•OH]ss) in CDOM solutions, respectively

196

(Equation 17 and 18).

light

1

2

•OH ) were estimated by multiplying the bimolecular reaction rate kCys 1

2

1

1

O2

1

O2

O2

•OH

kCys,rxn ) with the measured

1

kCys = (kCys ,rxn fCys + kCys ,rxn fCys )[ O2 ]ss 0

0

•OH

−

(૚ૠ)

−

(૚ૡ)

•OH

kCys = kCys,rxn [•OH]ss 197

Unlike other PPRI, H2O2 was relatively long-lived upon formation.47 H2O2-mediated pseudo-

198

HO first-order reaction rate constant of Cys ( kCys ) increased linearly with irradiation time (t)

199

HO (Equation 19). An averaged kCys for a typical sampling period (i.e., 60 min) was adapted to

200

assess the contribution of H2O2-mediated transformation.

2

2

H2O2

2

2

H2O2

H2O2

H2O2

kCys = (kCys ,rxn fCys + kCys ,rxn fCys + kCys 0

0

−

−

2−

,rxn

fCys )RH O t 2−

2

2

(૚ૢ)



Page 12 of 37

201

Dark transformation. Dark transformation experiments were carried out using the identical

202

solutions from the above Cys phototransformation experiments: Cys (40 uM), buffer species

203

(5mM, pH 5.7-9.9), and either SRNOM or PPHA. Identical dark controls were also prepared

204

without CDOM (referred to herein as CDOM-free controls). Aliquots of the solutions were

205

transferred to 2 mL protein LoBind eppendorf tubes and stored in a refrigerator (set at 15 °C) or

206

oven (set at 32 °C). Aliquots from the solution were removed at various time points for kinetic

207

analysis of Cys.

208

Steady-state photolysis setups. The steady-state photolysis experiments were carried out in

209

separate experimental setups. The light intensity of UVA and sunlight was monitored through

210

combined use of p-nitroanisole/ pyridine actinometry and radiometer measurements (Ocean

211

Optics Jaz, see SI for more information, Section S6).

212

UVA photolysis. Acid-washed borosilicate test tubes, which were transparent to the

213

wavelengths used in this study, were employed for all photolysis experiments. Sensitized Cys

214

photolyses were conducted in a photochemical reactor (Rayonet) equipped with two (for

215

solutions containing perinaphthenone) or twelve (for solutions containing CDOM) 365 nm bulbs

216

(Southern New England Ultraviolet Co., RPR-3500 Å). The photolysis experiments were

217

conducted in a temperature-controlled room set at 10 °C. At designated time points, aliquots

218

from

219

hydroxyterephthalate (hTPA), p-nitroanisole or resorufin.

the

photolysis

solution

were

removed

for

kinetic

analysis

of

Cys,

FFA,

220

Sunlight photolysis. Sunlight photolysis experiments were conducted during midday (10 am

221

to 3 pm) on clear days on the roof of the CHN building at ETH Zurich (47°22’45’’N,

222

8°32’55’’E). Solutions in acid-washed test tubes were aligned at a 45° angle to the ground in a


Page 13 of 37


223

home-built rack. The rack was turned periodically to keep the tubes facing the sun. Aliquots

224

were removed at regular time points for analysis of Cys, FFA, hTPA, or p-nitroanisole.

225

Quantification of FFA, p-nitroanisole, resorufin, hTPA, Cys and Cys transformation

226

products. FFA and p-nitroanisole concentrations were quantified using a Waters ACQUITY

227

ultra high-pressure liquid chromatography (UPLC) coupled to a photodiode array detector. Cys

228

was derivatized with mBBr and subsequently analyzed by UPLC with fluorescence detection.

229

Resorufin and hTPA concentrations were directly analyzed by UPLC with fluorescence

230

detection. Cys transformation products were analyzed on a Waters ACQUITY nanoUPLC

231

coupled to an Orbitrap high resolution mass spectrometer (HRMS, Thermo Exactive) equipped

232

with electrospray ionization (ESI). Previously published nanoUPLC-ESI-HRMS methods48 were

233

followed. Detailed information on sample preparation, UPLC separation and detection

234

parameters are provided in the SI (Section S4).

235

RESULTS AND DISCUSSION

236

General observations. Cys was found to spontaneously oxidize in solution upon standing in

237

air at room temperature, which added an experimental challenge. While taking care to work with

238

acid-cleaned glassware to remove metals that catalyzed the oxidation, the spontaneous oxidation

239

of Cys was limited. We conducted a number of control experiments to assess the Cys stability in

240

CDOM-free solutions, prior to evaluating the abiotic transformations of Cys in CDOM-

241

containing solutions. Results from CDOM-free controls show that less than 10% of Cys was

242

transformed under UVA irradiation and less than 5% of Cys was transformed under dark

243

conditions over the course of 8 hours. In addition, we performed control experiments of Cys

244

photolysis in the presence of the metal chelating agent EDTA to test the possibility of trace



Page 14 of 37

245

metal contamination in CDOM isolates. The result shows that the transformation rates of Cys

246

remained unchanged upon addition of EDTA, indicating that Cys transformation was not likely

247

affected by trace metal from CDOM isolates (Figure S10). These results established that

248

unwanted metal-catalyzed oxidation of Cys was negligible compared to Cys transformation with

249

CDOM under all study conditions within the timeframe of the experiments.

250

When Cys was allowed to react with CDOM in the dark, slow transformation of Cys was

251

observed with a half-life on the timescale of days. This observation is consistent with two

252

distinct transformation pathways: (i) CDOM is redox active and capable of accepting

253

electrons,49-51 and thus may oxidize Cys; and, (ii) CDOM can act as an electrophile, and reacts

254

with S-based nucleophiles.29,52,53 The balance between these two reaction modes is not known in

255

this case. Under sunlight or UVA irradiation in the presence of CDOM, Cys was found to

256

degrade rapidly with a half-life on the timescale of minutes, indicating the fast indirect

257

phototransformation of Cys in CDOM solutions. Evaluations of the kinetics, pathways and

258

transformation products from the abiotic transformations of Cys are provided in the subsequent

259

sections.

260

Cys transformation in CDOM solutions. The non-photochemical and photochemical

261

transformation reactions of Cys in CDOM solutions all followed pseudo-first-order degradation

262

kinetics and the rates were found to be highly pH-dependent (Figure 2a, showing Cys

263

transformation in UVA-irradiated SRNOM solutions as an example). Under UVA irradiation,

264

the solution temperatures gradually increased from 10 °C to 15 °C. Under sunlight irradiation,

265

the solution temperature increased from 25 °C to around 32 °C within 30 min and then remained

266

CDOM stable during irradiation. Accordingly, Cys dark transformation rate constants ( kCys,obs ) obtained

dark


Page 15 of 37


light

267

CDOM at 15 °C and 32 °C were compared with kCys,obs

268

respectively.

under UVA and sunlight irradiations,

dark

269

CDOM Cys non-photochemical transformation. In both SRNOM and PPHA dark solutions, kCys,obs

270

values increased from pH 5.7 to pH 8.7, suggesting higher transformation rates of Cys- than Cys0.

271

CDOM slightly decreased with increasing solution pH. In both SRNOM and Above pH 8.7, kCys,obs

272

PPHA solutions, the Cys reactivity was sensitive to solution temperature with two-fold higher

273

rates at 32 °C than at 15 °C (Figure 2b). Despite different origins of the DOM isolates, the Cys

274

oxidation rates in PPHA solutions were quite similar to those in SRNOM under dark conditions

275

(Figure 2b).

dark

light

276

CDOM Cys photochemical transformation. Generally, kCys,obs values of Cys in both SRNOM and

277

PPHA irradiated solutions increased from pH 5.7 to 9.9, with slight decrease in PPHA solutions

278

under sunlight irradiation from pH 8.7 to pH 9.9 (Figure 2c). The increased indirect

279

phototransformation rates with increasing solution pH suggest higher photoreactivity of Cys-

280

than Cys0. The indirect transformation rates of Cys were higher under sunlight irradiation than

281

UVA irradiation both in SRNOM and PPHA solutions. In addition, Cys underwent faster

282

indirect phototransformation in solutions containing PPHA than SRNOM (Figure 2c). The

283

differences in Cys photoreactivity between SRNOM and PPHA are most likely due simply to the

284

fact that PPHA has a higher absorbance (Figure 1).

285

photoreactivity in PPHA and SRNOM solutions matches their absorbance ratio.

The magnitude of the ratio in Cys

286

Cys phototransformation products in CDOM solutions. The transformation products of

287

Cys during the irradiation were assessed. Previous studies report that Cys is prone to oxidation at



Page 16 of 37

288

the thiol functional group (RSH) that forms disulfide (RSSR, cystine), sulfenic acid (RSOH),

289

sulfinic acid (RSO2H), and sulfonic acid (cysteic acid, RSO3H) under various conditions.54-60 In

290

this study, the formation of these transformation products in UVA-irradiated CDOM solution

291

was investigated at pH 5.7 and pH 9.9, where the thiol and thiolate forms were the dominant

292

species, respectively. The results showed that RSSR and RSO2H were the two major products of

293

Cys oxidation in irradiated CDOM solutions with an initial Cys concentration of 40 µM, and

294

these products together contribute around 30% of Cys transformation products (Figure S4).

295

Notably, the concentrations of Cys found in surface waters are considerably lower than Cys

296

concentrations used in this study.3 While this is not expected to impact the disappearance

297

kinetics, as they were cleanly first-order, this may affect the product distribution, especially the

298

formation of RSSR. We did not detect the formation of RSO3H, which was likely due to the

299

short photolysis period. In addition to the HRMS-identified transformation products, we predict

300

that unidentified RSH-CDOM conjugates may also form. This could be due to the conjugation

301

capability of thiols to quinones (Figure 3), for example.29,52,53 No effort was made to quantify

302

RSH-CDOM conjugate formation in this study.

303

The photochemical reactivity of RSSR and RSO2H was further tested in UVA-irradiated

304

CDOM solutions. RSSR transformed quite slowly over the course of 6 hours irradiation. The

305

product analysis indicates formation of small amounts of Cys upon phototransformation of

306

RSSR (Figure S5). This result agrees with the previous study that reported the photochemical

307

formation of free thiol by cleavage of disulphide linkages.55 When RSO2H was subjected to

308

photochemical reaction in the presence of SRNOM or PPHA, RSO3H was identified as the

309

major transformation product (Figure S6). This transformation pathway in CDOM solutions was

310

in accordance with the observed oxidation of RSO2H into RSO3H in cellular systems.61,62 The


Page 17 of 37


311

transformation rates of RSO2H were much higher at pH 5.7 than at pH 9.9 in both SRNOM and

312

PPHA solutions. The reason for the higher reactivity of RSO2H at lower pH is not known.

313

Reaction rate constants of Cys with PPRI. The prior sections focused on kinetics and

314

products of Cys abiotic transformation in CDOM solutions. Below we examine the intrinsic

315

reaction rate constants of Cys with the well-defined PPRI in CDOM solutions, to provide a basis

316

for assessing the contribution of each PPRI-mediated pathway (Table 1).

317

Reaction rate constants of Cys with singlet oxygen. The photochemical transformation rate

318

constants of Cys with 1O2 were assessed through either steady-state photolysis or time-resolved

319

phosphorescence. The steady-state phototransformation of Cys with 1O2 followed pseudo-first-

320

order kinetics at pH 10.0. The 1O2 steady-state concentration in D2O was 10-fold higher than in

321

O was H2O, which was consistent with the solvent isotope effect.37 The rate constant kCys ,rxn

322

determined to be 2.3 × 108 M–1s–1. At pH 5.0, no Cys transformation was observed through

323

O O photolysis (Table 1). The lower kCys than kCys is in agreement with the higher 1O2 reactivity ,rxn ,rxn

324

of the electron-rich thiolate form.20,21 With the respective 1O2 reaction rate constants for different

325

protonation states of Cys,

1

2 −

1

1

2 0

2 −

1

O was modeled as a function of solution pH (Figure S3). kCys,rxn 2

326

In addition to steady-state photolysis, the reaction rate constant of Cys with 1O2 was further

327

investigated by time-resolved 1O2 phosphorescence. At pH 10.2, the quenching rate constant of

328

1

329

O study.20 Nevertheless, the kCys value measured by 1O2 phosphorescence quenching experiment ,rxn

330

was lower than the value of

O O2 by Cys- ( kCys ) was determined to be 1.5 × 108 M–1s–1, which is consistent with a previous ,rxn 1

2 −

1

2 −

1

8

O2

kCys ,rxn measured by steady-state photolysis (i.e., 2.3 × 10 −

M–1s–1).



1

O2

kCys ,rxn values

Page 18 of 37

331

We tentatively attribute the different

332

time-scales. The time-resolved photolysis experiment, which followed the loss of 1O2, allowed

333

for the direct observation of the elementary reaction step between one Cys and one 1O2 molecule.

334

In the steady-state experiment, in which the loss of Cys was followed, the observed decay was

335

the sum of all of the Cys loss processes. For example, if additional oxidants are formed in the

336

reaction, such as superoxide or H2O2, they would contribute to the observed Cys loss in the

337

steady-state experiments. The ratio of the steady-state and laser flash photolysis rate constants is

338

1.5, indicating an overall stoichiometry of approximately 1.5 cysteine molecules per 1O2. At pH

339

5.0, 1O2 phosphorescence was not depressed upon addition of Cys, which indicated no 1O2-

340

mediated reaction of Cys0 and was in accordance with the result of the steady-state experiments.

−

to different stoichiometry on the different

341

Reaction rate constant of Cys with hydroxyl radical. The previously reported diffusion-

342

controlled bimolecular reaction rate constant (1.8 × 1010 M–1s–1) was adapted for the reaction of

343

•OH with both Cys0 and Cys– in this study (Table 1).63

344

Reaction rate constants of Cys with hydrogen peroxide. The H2O2-mediated Cys HO increased from pH kCys,rxn

345

transformation rate constants were highly pH-dependent (Figure S3):

346

5.0 to pH 9.8, indicating higher reactivity of Cys- with H2O2 than Cys0. Above pH 9.8,

347

decreased with increasing solution pH, indicating that the reactivity may also be modulated by

348

the protonation state of N-terminal amine (pKa2 = 10.28). Previous work suggests that hydrogen

349

bonding between the protonated amine in Cys and H2O2 takes place, which may consequently

350

reduce the electron density in H2O2 and promote the reactivity of H2O2 as an electrophile

351

towards Cys.64 At high pH, deprotonation of the N-terminal amine suppresses hydrogen bond

352

formation and lowers the reaction rate of Cys2- with H2O2. The reaction rate constants of Cys0,

2

2

HO kCys,rxn 2

2


Page 19 of 37


353

Cys-, and Cys2- with H2O2 were obtained from the fit of experimental rate constants (Table 1,

354

Equation 13) and agreed with previous work.64

355

Reaction rate constants of Cys with triplet state sensitizers. The photochemical

356

transformation rate constants of Cys with model excited triplet state sensitizers (3Sens*) were

357

assessed by transient absorption spectroscopy (Figure S1). The quenching rate constants of

358

3

Sens* by Cys- varied from 5.0 × 108 to 1.4 × 109 M–1s–1, with varying redox potentials of

359

3

Sens* (Table S1).38,65-71 Comparatively, no clear quenching of 3Sens* by Cys0 was observed

360

(Table 1), indicating the rate constants were ≤ 2.2 × 106 M–1s–1.

361

Contribution of individual PPRI-mediated transformation pathway to Cys oxidation in

362

irradiated CDOM solutions. In the previous sections, the Cys transformation rate constants

363

with individual PPRI were established over a wide range of solution pH. Based on these results,

364

we investigated the formation of PPRI in irradiated CDOM solutions and sought to evaluate the

365

CDOM contribution of each transformation pathway (e.g., kCys,obs ,

366

in irradiated CDOM solutions (Equation 20). The processes that are not attributable to 1O2,

367

H2O2, •OH and dark reactions are combined into a single pseudo-first-order rate constant,

dark

CDOM light

CDOM dark

1

O2

•OH

1

•OH

2

H 2O 2

Other

kCys,obs = k Cys,obs + kCys + kCys + k Cys + k Cys 368

H2O2

kCysO , kCys , kCys

) to Cys oxidation

Other

kCys

.

(૛૙)

Singlet oxygen-mediated Cys phototransformation in CDOM solutions. The contribution of

369

1

370

because of the reactivity of Cys with 1O2 and 1O2 formation in CDOM solutions were dependent

371

on solution pH. UVA- and sunlight-irradiation of SRNOM yielded similar steady-state 1O2

372

concentrations, which decreased with increasing solution pH. Comparatively, the [1O2]ss in

O2 to the photooxidation of Cys during CDOM photolysis varied across experimental pH



Page 20 of 37

373

PPHA-sensitized experiments was highly dependent on the light source. The measured [1O2]ss in

374

PPHA-sensitized experiments was 2- to 4-fold higher than [1O2]ss in SRNOM-sensitized

375

O experiments (Table 2). In both CDOM solutions, kCys increased with solution pH, despite the

376

O lower [1O2]ss at high pH. In SRNOM solutions, kCys on average contributed around 4.3% and 3.3%

377

of the Cys oxidation under UVA and sunlight irradiations, respectively (Table 2). The

378

contribution increased as solution pH increased from 5.7 to 9.9. In PPHA solutions, the

379

contribution of 1O2 reactions were higher, which on average accounted for 7.8% and 11.0% of

380

Cys oxidation under UVA and sunlight irradiation, respectively.

1

2

1

2

381

The involvement of 1O2 in Cys oxidation in CDOM solutions was further probed through the

382

use of azide (N3-) as a 1O2 quencher under UVA irradiation at varying pH ranging from 5.7 to

383

9.9 (SI, Section S3). Approximately 65% of [1O2]ss was quenched upon addition of 1 mM N3-.

384

The Cys transformation rates decreased along with observed lower [1O2]ss, suggesting the

385

involvement of

386

transformation to Cys oxidation were estimated to be 35% and 33% on average during SRNOM-

387

and PPHA-sensitized photolysis, respectively (Table S3). Notably, the contributions of 1O2

388

predicted from 1O2 quenching experiments were significantly higher than the 1O2 contributions

389

obtained above (i.e., 4.3 % and 7.8 % in SRNOM and PPHA solutions, respectively). These

390

results indicate that in addition to 1O2, azide may also quench other PPRI (e.g., •OH), which are

391

involved in Cys photooxidation transformations during CDOM-sensitized experiments.

1

O2 in Cys oxidation (Table S2). The contributions of

1

O2-mediated

392

Hydroxyl radical-mediated Cys phototransformation in CDOM solutions. The •OH-

393

mediated Cys photooxidation rates varied little across measured solution pH. The steady-state

394

concentrations of •OH ([•OH]ss) were low relative to other PPRI concentrations (Table 2). In


Page 21 of 37


395

both CDOM solutions, sunlight irradiation produced higher [ • OH]ss than UVA irradiation.

396

Higher [•OH]ss were observed in PPHA solutions than in SRNOM solutions (Table 2). Despite

397

•OH the low steady-state concentrations, •OH-mediated phototransformation rates ( kCys ) were

398

significant due to the diffusion-controlled reactivity of •OH with Cys0 and Cys- (Figure S3).

399

Notably, •OH-mediated phototransformation was more important at low solution pH (i.e., at pH

400

•OH 5.7), where Cys0 reacted with other PPRI at relatively slow rates. The contribution of kCys

401

decreased with increasing solution pH. For instance, the •OH contribution decreased from 4.4%

402

at pH 5.7 to 1.6% at pH 9.9 in UVA irradiated SRNOM solutions (Table 2). Overall, •OH-

403

mediated phototransformation had minor importance on Cys oxidation in irradiated CDOM

404

solutions.

405

Hydrogen peroxide-mediated Cys phototransformation in CDOM solutions. The

406

contribution of H2O2-mediated Cys photooxidation during CDOM-sensitized photolysis was

407

strongly pH-dependent. The photochemical formation rate of H2O2 was investigated in only the

408

HO UVA irradiated system. The results show that kCys increased rapidly with increasing solution

409

HO pH, giving higher H2O2 formation rates at higher pH (Table 2). For instance, kCys increased by

410

HO over 2 orders of magnitude from pH 5.7 to pH 9.9 and hence, the contribution of kCys to Cys

411

HO oxidation increased from 0.15% to 7.1% in SRNOM solutions. At pH 7.3, kCys contributed to

412

0.58% and 1.2% of Cys oxidation in SRNOM and PPHA solutions, respectively. These results

413

suggest that H2O2-mediated phototransformation pathway had minor importance on Cys

414

oxidation in irradiated CDOM solutions.

2

2

2

2

2

2

2

2



Page 22 of 37

415

Cys phototransformation in CDOM solutions via other pathways. The contributions of 1O2,

416

•OH, and H2O2 to Cys photooxidation in CDOM-sensitized experiments from the discussions

417

above were estimated on the basis of measured rate constants and measured steady-state

418

concentrations of individual PPRI. This section addresses other processes, collectively referred

419

to as

420

varied depending on solution pH and light source. Yet the trends of

421

independent of light or CDOM, which generally increased with increasing solution pH (Table 2).

Other

kCys

that may contribute to Cys phototransformation (Equation 20). The values of

The contribution of 3CDOM*-mediated oxidation to

422

Other

kCys

Other

kCys

Other

kCys

with pH are

was assessed using multiple

423

approaches. Generally, the first-order kinetics were expected for 3CDOM*-mediated oxidation

424

because molecular oxygen was the major quencher for

425

concentration of 3CDOM* was not affected by addition of Cys. Based on this assumption, we

426

first estimated the correlation of

427

TMP as a reference compound. TMP has been previously employed as a reference compound for

428

3

CDOM* reactivity.72-76 These estimates were not used to directly calculate the contribution of

429

3

CDOM*-mediated oxidation but were rather used for assessing whether 3CDOM* is a major

430

contributor to

431

CDOM* 0.66) with the pseudo-first-order degradation rate constant of TMP ( kTMP,obs ), indicating that

432

3

Other

kCys

Other

kCys

3

CDOM* and the steady-state

with 3CDOM*-mediated Cys photooxidation rates using

. The results show that

Other

kCys

3

Other CDOM* had a correlation ( kCys / kTMP,obs =1.01, r2 = 3

CDOM*-mediated pathway in Cys phototransformation was plausible (Figure S7).

433

Nevertheless, uncertainty may exist using TMP as a reference compound because Cys has

434

lower reactivity towards 3CDOM* than TMP. We used triplet state model ketones (3Sens*) as

435

representative 3CDOM* and compared the reactivity of TMP and Cys with 3Sens*. The


Page 23 of 37


436

quenching rate constants of 3Sens* with TMP varied from 5.9 × 108 to 43 × 108 M–1s–1, which

437

were higher than the values of Cys (5.0 - 14.0 × 108 M-1 s-1, Table S1). More notably, the ratio

438

of the rate constants for TMP and Cys oxidation by the five model triplet ketones varied from

439

1.2 to 5.4, indicating that assuming a simple reactivity ratio between the two compounds is likely

440

not valid. Finally, while the model triplet ketones showed high reactivity with the thiolate form,

441

Cys-, they showed very slow kinetics with the thiol form, Cys0. This was not mirrored in the

442

kCys

Other

values, which had substantial contributions over the entire experimental pH range.

443

The reactivity of Cys and TMP towards 3CDOM* was further studied via monitoring 1O2

444

phosphorescence in SRNOM solutions. The concentration of 3CDOM* was greatly depressed by

445

addition of TMP, resulting in depressed 1O2 formation. Comparatively, we observed constant

446

1

447

by Cys (Figure S8). Additional evidence against significant contribution of 3CDOM*-mediated

448

transformation came from deceased Cys transformation rates in N2-sparged CDOM solutions,

449

where the steady-state concentration of 3CDOM* was enhanced (Figure S9). This result is

450

consistent with 3CDOM*-mediated transformation being only a minor contributor to Cys

451

photooxidation. It is however not conclusive evidence, since there are mechanisms initiated by

452

triplet sensitizers that are slowed by oxygen removal, such as oxygen-dependent radical chain

453

processes.

O2 formation upon Cys addition in SRNOM solution, indicating minor quenching of 3CDOM*

454

It thus appears that processes other than reaction with 1O2, •OH, and H2O2 are responsible for

455

the majority of the indirect phototransformation of Cys and that 3CDOM* is not the main

456

contributor to these other processes that are represented by

457

these other processes are reaction with an unidentified oxidant (e.g., peroxy radicals) and/or

Other . kCys

The leading candidates for



Page 24 of 37

458

photo-initiated reactions between Cys and CDOM. Identifying the major processes responsible

459

for CDOM-sensitized degradation of Cys remains a need for future work.

460

Environmental implications. This study establishes that abiotic transformation processes,

461

both photochemical and non-photochemical, are expected to be an important sink of Cys in

462

surface waters. We expect that the susceptibility of Cys to abiotic transformations also applies to

463

other Cys-containing biomolecules, such as glutathione, phytochelatins and enzymes, which act

464

as powerful antioxidants and chelators for microorganisms experiencing oxidative stress and

465

toxic metal stress.31,77-79 These Cys-containing biomolecules are not only intracellular, but also

466

are released into the extracellular environment and detected in the bulk solution of

467

environmental aquatic systems.1-4 While quantifying the transformations of these structurally

468

diverse Cys-containing biomolecules might be challenging, this mechanistic study on free Cys

469

lays an important foundation for that work. An assessment of Cys reactivity in structurally

470

higher order biomolecules is currently being addressed in ongoing work.

471

The rapid photochemical and non-photochemical transformation processes significantly

472

depress the steady-state concentrations of thiols, which provides an explanation for the observed

473

low concentrations of thiol compounds in the oxic water column.80 Compared to the slow biotic

474

removal rate constants (~10-5-10-6 s-1, phytochelatin as example),77 the rate constants of abiotic

475

non-photochemical transformation (~10-5 s-1) or photochemical transformation (~10-3 s-1) are

476

substantially higher in CDOM solutions. Further studies are needed for a full understanding of

477

concurrent abiotic and biotic processes of individual thiol species in natural waters.

478

The depressed thiol concentrations resulting from abiotic transformation have profound

479

impacts on numerous environmental processes, for instance, metal cycling. The speciation,


Page 25 of 37


480

transportation, transformation, and bioavailability of soft metals (e.g., Hg, Cu, Zn, Cd, and Pb)

481

in aquatic systems are largely dependent on complexation with thiols. Soft metals complexed

482

with either thiol or thiol oxidantion products (e.g, disulfide) vary greatly in their stability

483

constants81 and biogeochemical properties. Thus, organisms that use thiols to control free metal

484

concentrations in natural waters must cope with the higher reactivity and lower lifetime of these

485

thiols under photochemical conditions.

486



Page 26 of 37

487

Supporting Information Available

488

Supporting figures, tables, detailed experimental methods and additional experiments described

489

within the manuscript are provided. This material is available free of charge via the Internet at

490

http://pubs.acs.org.

491

Acknowledgements

492

This work was financially supported by grants from the Swiss National Science Foundation

493

(Project numbers 200021_138008 and 200020_159809). We thank Prof. Heileen Hsu-Kim

494

(Duke University) for helpful discussions regarding this study.

495


Page 27 of 37


496

0.8 Absorbance

2.0

UVA

1.5

PPHA

0.6 1.0 0.4

SRNOM sunlight 0.5

0.2 Cys- Cys0

0.0 250

497 498 499 500

300

350 400 450 Wavelength (nm)

0.0 500

Light intensity (10-6 Es L-1s-1)

1.0

Figure 1. (Left y-axis) Absorbance spectra of Cys0 (40 µM at pH 5.7), Cys- (40 µM at pH 9.9), PPHA (11.4 mgC/L at pH 7.3), and SRNOM (11.4 mgC/L at pH 7.3). (Right y-axis) Intensity of UVA and natural sunlight.

501



502 503 504 505 506 507 508 509 510 511 512 513

Page 28 of 37

Figure 2. (a) Photochemical and non-photochemical transformation kinetics of Cys in CDOM solutions at pH 5.7-9.9 (showing UV-A irradiated SRNOM solutions as example). The natural logarithm of the ratio of Cys concentration at time point t and initial concentration, ln([Cys]t/[Cys]0), was plotted versus photolysis time. (b) Dark transformation rates (units of s–1) of Cys in SRNOM solutions at 15 °C (red triangle), SRNOM solutions at 32 °C (green dot), PPHA solutions at 15 °C (cyan square), and PPHA solutions at 32 °C (blue diamond). (c) Phototransformation rate (units of s–1) of Cys in UVA-irradiated SRNOM solutions (red triangle), sunlight-irradiated SRNOM solutions (green dot), UVA-irradiated PPHA solutions (cyan square), and sunlight-irradiated PPHA solutions (blue diamond). The observed rates in panel (b) and (c) at different pH values are connected to show the tendency of rates over pH (solid lines). Note that the scale of the y-axis in panel c covers a 80-fold larger range than in panel b.

514


Page 29 of 37


515 516 517

Figure 3. Schematic overview of Cys phototransformation and product formations in irradiated CDOM solutions.

518



519

Page 30 of 37

Table 1. Reaction rate constants (units of M–1s–1) of Cys with PPRI. Rate constants (M-1s-1)

520

PPRI

1

521 522 523

O2 (steady-state)

Photochemical and Nonphotochemical Transformations of Cysteine

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