Study on the molecular interactions of hydroxylated polycyclic aromatic hydrocarbons with catalase using multi-spectral methods combined with molecular docking

Journal Pre-proofs Study on the molecular interactions of hydroxylated polycyclic aromatic hydrocarbons with catalase using multi-spectral methods combined with molecular docking Jing Zhang, Linfeng Chen, Yaxian Zhu, Yong Zhang PII: DOI: Reference:

S0308-8146(19)31874-6 https://doi.org/10.1016/j.foodchem.2019.125743 FOCH 125743

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Food Chemistry

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1 October 2018 11 September 2019 17 October 2019

Please cite this article as: Zhang, J., Chen, L., Zhu, Y., Zhang, Y., Study on the molecular interactions of hydroxylated polycyclic aromatic hydrocarbons with catalase using multi-spectral methods combined with molecular docking, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125743

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1

Study on the molecular interactions of hydroxylated polycyclic

2

aromatic hydrocarbons with catalase using multi-spectral

3

methods combined with molecular docking

4

Jing ZHANGa, Linfeng CHENb, Yaxian ZHUc, Yong ZHANGb*

5 6

a

Key Laboratory of Estuarine Ecological Security and Environmental Health, Tan

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Kah Kee College, Xiamen University, Zhangzhou, 363105, PR China,

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[email protected] (Jing ZHANG)

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b State

Key Laboratory of Marine Environmental Sciences of China (Xiamen

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University), College of Environment and Ecology, Xiamen University, Xiamen,

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361102, PR China, [email protected] (Linfeng CHEN),

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[email protected] (Yong ZHANG)

13 14

c Department

of Chemistry, College of Chemistry and Chemical Engineering, Xiamen

University, Xiamen, 361005, PR China, [email protected] (Yaxian ZHU)

15 16 17

*Corresponding

18

Address: State Key Laboratory of Marine Environmental Science of China (Xiamen

19

University), College of Environment and Ecology, Xiamen University, 361102 Xiamen,

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Fujian Province, China.

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Tel.: +86 592 2188685; Fax: +86 592 2888685

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E-mail address: [email protected] (Y. Zhang).

Author：

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Declarations of interest: none 1

25

Abstract

26

To reveal the potential effects of hydroxylated polycyclic aromatic hydrocarbons

27

(OH-PAHs) on catalase (CAT), the interactions of 1-hydroxynaphthalene (1-OHNap),

28

9-hydroxyphenanthrene (9-OHPhe) and 1-hydroxypyrene (1-OHPyr) with CAT were

29

investigated

30

Fluorescence analysis showed that 1-OHNap, 9-OHPhe and 1-OHPyr can form 1:1

31

complex with CAT, with the binding constant of 6.31×103, 1.03×104 and 2.96×105 L

32

mol-1 at 17 oC. Thermodynamic and docking parameters demonstrated that van der

33

Waals’ force, hydrogen bonds and hydrophobic interactions dominated the three

34

binding processes. Molecular docking also revealed the specific binding mode of OH-

35

PAHs with CAT. Synchronous fluorescence and circular dichroism spectral results

36

indicated that the three OH-PAHs induced varied structural changes of CAT.

37

Furthermore, CAT activity was promoted by 9-OHPhe, but inhibited by either 1-

38

OHNap or 1-OHPyr. Under the maximum experimental concentration of OH-PAHs,

39

the percent change of CAT activity induced by 1-OHNap, 9-OHPhe and 1-OHPyr were

40

8.42%, 4.26% and 13.21%.

using

multi-spectroscopic

and

molecular

docking

techniques.

41 42

Keywords: Hydroxylated polycyclic aromatic hydrocarbons; Catalase; Binding

43

interaction; Spectroscopic techniques; Molecular docking

44 45

Chemical compounds studied in this article

46

1-hydroxynaphthalene (PubChem CID: 7005); 9-hydroxyphenanthrene (PubChem CID:

47

10229); 1-hydroxypyrene (PubChem CID: 21387)

48 49

Abbreviated (running) title: Molecular interactions of OH-PAHs with catalase 2

50

1. Introduction

51

Polycyclic aromatic hydrocarbons (PAHs), as typical persistent organic pollutants,

52

can accumulate in human beings through food intake, as they may be formed during

53

the process of food preparation, such as barbecuing, smoking, or frying (Gysel et al.,

54

2018; Lao, Xie, Wu, Bao, Tao, & Zeng, 2018). After entering the human body, parent

55

PAHs can be primarily metabolized by cytochrome P450 enzymes and converted to

56

more active oxy-derivatives such as epoxides and hydroxyl compounds. These

57

metabolites have the potential ability to bind with biomacromolecules (e.g., DNA and

58

proteins), which may induce damage to the DNA or the proteins (Moorthy, Chu, &

59

Carlin, 2015). Therefore, the investigations on the molecular interactions between

60

biomacromolecules and the oxygenated metabolites of PAHs are of importance to

61

understand the toxic effects of PAHs. Hydroxylated polycyclic aromatic hydrocarbons

62

(OH-PAHs), a type of important metabolites of PAHs, are considered to be potentially

63

more toxic than their parent PAHs (Diamante et al., 2017), while they have not been

64

studied extensively as their parent PAHs. Hence, more attention should be paid to the

65

molecular interactions of OH-PAHs with functional biomacromolecules and the

66

corresponding effects.

67

Catalase (CAT, EC 1.11.1.6) is a heme containing metalloenzyme, which

68

catalyzes the degradation of hydrogen peroxide (H2O2) into one molecule of H2O and

69

a half molecule of O2 (Deisseroth & Dounce, 1970), thus playing an important role in

70

protecting cells from oxidative injure induced by reactive oxygen species (ROS). As is

71

well-known, ROS have been considered to be an important factor in aging, cancer,

72

diabetes, hypertension and inflammation, and thus the denaturation of CAT is

73

correlated with these pathological states (Glorieux & Calderon, 2017). Recently, more

74

and more studies have been performed on the potential toxicity of pollutants on CAT 3

75

in vivo and in vitro. Intake of exogenous environmental pollutants is suggested to cause

76

the conformational destruction of CAT and/or to affect the catalytic activity of CAT

77

(Chen, Zhang, Zhu, & Zhang, 2017; J. Wang, Wang, Xu, Liu, & Chen, 2016; Xu,

78

Cui, Zhao, Hu, Zong, & Liu, 2018). Even so, the toxicity mechanism of pollutants on

79

CAT is still far from being fully understood.

80

As the toxicity of contaminants is originated from their interactions with

81

functional biomacromolecules (Moorthy et al., 2015), the investigation on the

82

molecular interactions of OH-PAHs with CAT are crucial to reveal the toxicity

83

mechanism of OH-PAHs on the antioxidant enzyme system. In our previous work, the

84

interaction between CAT and 1-hydroxypyrene (1-OHPyr) were preliminary

85

investigated (Chen, Zhang, Zhu, & Zhang, 2015), and the strong binding affinity of

86

1-OHPyr to CAT and the adverse effect of 1-OHPyr on the conformation and function

87

of CAT were confirmed. However, minor changes in the structure of OH-PAHs may

88

induce their different interaction behaviors with biomacromolecules and govern their

89

toxicity in organisms (Sievers, Shanle, Bradfield, & Xu, 2013; L. R. Wang, Wang,

90

Chen, & Guo, 2009). Wu et al. (Wu, Wu, Guan, Su, & Cai, 2007) proved the

91

formation of the complex between hydroxynaphthalene and bovine serum albumin

92

(BSA), and revealed that the binding of 1-hydroxynaphthalene (1-OHNap) with BSA

93

was more cooperative than that of 2-hydroxynaphthalene. Wang, et al. (2009)

94

investigated the binding of 26 OH-PAHs with DNA, which suggested that steric effect

95

was vital in the interaction of DNA with differently substituted OH-PAH. Ohura,

96

Kurihara, and Hashimoto (2010) investigated the binding interactions between OH-

97

PAHs and aryl hydrocarbon receptor (AhR), and the results indicated that the number

98

and site of hydroxy-groups substituted on PAH skeleton obviously influenced the AhR-

99

ligand binding capacity of OH-PAHs, with the octanol-water partition coefficient (Kow) 4

100

value of OH-PAH significant related to the AhR activity. Li, et al. (2011) developed a

101

QSAR model to characterize the interactions of OH-PAHs with DNA, which suggested

102

that the electrostatic potential, molecular size and polarizability of OH-PAHs were

103

closely correlated with their binding capacity to DNA. Sievers, et al. (2013) reported

104

that 1-OHNap, 9-hydroxyphenanthrene (9-OHPhe) and 1-hydroxypyrene (1-OHPyr)

105

were able to competively bind ERβ, induce ERβ homodimers, and regulate ERβ target

106

genes, whereas, the relative activity of each compound is quite different. These studies

107

confirmed that the binding affinity/the adverse effects of OH-PAHs to/on

108

biomacromolecules varied as the structural difference of OH-PAHs. Specifically, the

109

logKow, charges and steric effect of OH-PAHs are important factors affecting the

110

binding interactions. Therefore, in order to fully understand the effects of the structural

111

difference of OH-PAHs on their interactions with CAT, further work on different OH-

112

PAHs with varied number of benzene rings should be conducted.

113

Herein, based on our previous work, the interactions of CAT with 2-4 ring

114

monohydroxy PAHs (1-OHNap, 9-OHPhe and 1-OHPyr) were further investigated in

115

the present study. Briefly, this work is undertaken based on the following hypothesis:

116

1) OH-PAHs with different numbers of aromatic rings may have varied binding affinity

117

to CAT, and the binding ability of CAT with OH-PAHs may be associated with the

118

hydrophobicity of OH-PAHs; 2) OH-PAHs with different numbers of aromatic rings

119

can induce different structural and functional changes of CAT, due to the varied

120

molecular interaction of OH-PAHs with CAT. To verify these hypotheses, firstly,

121

multi-spectroscopy, in combination with molecular docking method, was employed to

122

study the binding information of OH-PAHs with CAT. Then, synchronous fluorescence

123

and circular dichroism (CD) spectroscopy methods were employed to clarify the effects

124

of the binding interactions on the structure of CAT. Enzyme activity measurements 5

125

were further utilized to interpret the response of CAT activity after OH-PAHs exposure.

126

Meanwhile, the differences of the interactions of CAT with OH-PAHs with different

127

numbers of aromatic rings were discussed throughout the study. These investigations

128

aim to help clarify the molecular mechanism of the interactions between CAT and OH-

129

PAHs, and provide an important basis for further understanding the toxicity of OH-

130

PAHs on antioxidant enzyme systems in vivo and further warn the health risk of the

131

PAHs via dietary intake.

132 133

2. Materials and methods

134

2.1 Materials

135

Three OH-PAHs (1-OHNap, 9-OHPhe, and 1-OHPyr) with purity > 99% and CAT

136

from bovine liver were purchased from Sigma-Aldrich Company, USA. 30% H2O2 was

137

from Xilong Chemical Company, Ltd, China. Stock solutions of 5.0×10-5 mol L-1 CAT

138

were prepared in Tris-HCl buffer. 1.0×10-2 mol L-1 1-OHNap, 2.0×10-3 mol L-1 9-

139

OHPhe and 2.0×10-3 mol L-11-OHPyr were all prepared in ethanol and stored at 4 oC in

140

the dark. All chemicals were of analytical grade, and ultrapure water (> 18.2 MΩ cm-1)

141

was used throughout the experiments.

142

2.2 Methods

143

All of the samples were prepared in a series of 10 mL colorimetric tubes and were

144

diluted with 0.05 mol L-1 Tris–HCl buffer (containing 0.10 mol L-1 NaCl, pH = 7.40) to

145

volume, and mixed. Exceptionally, the samples used for CD measurements were diluted

146

with ultrapure water. Each sample should contain no more than 5‰ (v/v) ethanol.

147

Besides, the sample balance placing time was 20 min (unless explicitly stated). All

148

measurements were conducted at 17 °C and performed in triplicate (unless explicitly

149

stated). 6

150

2.2.1 Fluorescence spectra measurements

151

The fluorescence quenching experiments were performed at three different

152

temperatures (17, 27 and 35 oC), after maintained the samples in a thermostatic water

153

bath for 1 h. The steady-state fluorescence spectra of the samples were measured on a

154

Cary

155

excitation/emission slit widths of 5 /10 nm, respectively. The fluorescence emission

156

spectra were recorded in the range of 290-550 nm at the excitation wavelength of 280

157

nm. The synchronous fluorescence spectra were scanned at Δλ = 15 nm (excitation

158

wavelength from 270 to 310 nm), and Δλ = 60 nm (excitation wavelength from 250 to

159

310 nm).

160

2.2.2 CD spectra measurements

Eclipse

fluorescence

spectrophotometer

(Varian,

USA),

with

the

161

CD spectra of the CAT in different systems were measured from 200 to 250 nm in

162

1.0 nm intervals with a JASCO-810 spectrometer (Shimadzu, Japan), at the scanning

163

speed of 500 nm min-1. Each CD spectrum of the CAT was the average of three scans,

164

and was baseline corrected by subtracting the spectrum of the corresponding solutions

165

without CAT. The secondary structural contents of the CAT were calculated by CDpro

166

software combined with the CONTIN/ LL method.

167

2.2.3 Molecular docking process

168

AutoDock 4.2.6 software and Autodock Tool (ADT) (the Scripps Research

169

Institute, La Jolla, CA, USA) (Morris et al., 2010) were employed to perform the

170

molecular docking processes of the interactions of 1-OHNap, 9-OHPhe, or 1-OHPyr

171

with CAT. GaussView 5.0.8 program (the Gaussian Inc., Pittsburgh, PA, USA) was

172

utilized to draw the three-dimensional (3-D) structures of 1-OHNap, 9-OHPhe and 1-

173

OHPyr. The obtained structures were further optimized by Gaussian 09 software

174

package (Frisch et al., 2009) using the DFT/B3LYP method combined with the 67

175

31G(d) basis set. The native structure of CAT was obtained from the Protein Data Bank

176

(PDB ID: 1TGU). To seek the binding sites of each OH-PAH in CAT, molecular

177

docking was carried out as described in previous work (Chen, Zhang, Zhu, & Zhang,

178

2018). Afterwards, the conformation with the lowest binding free energy from docking

179

results was chosen for further analysis. Besides, the Protein–Ligand Interaction Profiler

180

(projects.biotec.tu-dresden.de/plip-web), a web service for fully automated detection of

181

relevant non-covalent protein–ligand contacts in 3-D structures (Salentin, Schreiber,

182

Haupt, Adasme, & Schroeder, 2015), was used to detect the interactions between each

183

OH-PAH and its surrounding amino acid residues of the CAT molecule. Finally,

184

PyMOL software (the PyMOL Molecular Graphics System, Version 1.3r1 Schrödinger,

185

LLC., NY, USA) was used to visualize and analyze the docking results (DeLano, 2002).

186

2.2.4 CAT activity measurements

187

Catalase activity was determined using the method described in previous work

188

(Chen, Zhang, Zhu, & Zhang, 2018). The activity measurements were performed at

189

17 °C. Each sample was tested for 6 times, and mean ± standard deviation was regarded

190

as the final result. Statistical differences among various groups were analyzed using

191

one-way analysis of variance (ANOVA) followed by Student’s t-test (Chen et al.,

192

2017). All data analyses were performed using SPSS 16.0 statistical software program

193

for Windows (SPSS, Inc., Chicago, IL, USA). Values were considered statistically

194

significant when P< 0.05.

195 196

3. Results and Discussion

197

3.1 Effects of 1-OHNap, 9-OHPhe or 1-OHPyr on the fluorescence spectra of CAT

198

The intrinsic fluorescence of CAT is mainly attributed to its tryptophan (Trp) and

199

tyrosine (Tyr) residues, and there are 6 Trp and 20 Tyr residues in each subunit of CAT 8

200

(Schroeder et al., 1982). The fluorescence spectra of CAT with various concentrations

201

of 1-OHNap, 9-OHPhe or 1-OHPyr were shown in Fig. 1. As shown in Fig. 1, pure

202

CAT displays a strong fluorescence emission peak at 350 nm upon excitation at 280

203

nm. However, the fluorescence emission intensity of 1-OHNap, 9-OHPhe and 1-OHPyr

204

can be ignored around 350 nm with identical excitation as shown in Fig. S1. The

205

fluorescence intensity of CAT at the emission wavelength of 350 nm was obviously

206

quenched with the addition of 1-OHNap, 9-OHPhe or 1-OHPyr, which indicated that

207

an interaction had occurred between CAT and each OH-PAH. Moreover, isosbestic

208

points were observed at 398 nm for 1-OHNap, 361 nm for 9-OHPhe and 376 nm for 1-

209

OHPyr. This suggested the existence of the interaction equilibrium of each OH-PAHs-

210

CAT system (Ghosh, Mitra, Pal, Basu, & Saha, 2017; Song et al., 2000).

211

Interestingly, comparing the spectra of Fig. 1 and Fig. S1, there are inversions in the

212

fluorescence intensity of 1-OHNap and 9-OHPhe in their maximum concentration in

213

the presence and absence of CAT. It was because that CAT increased the fluorescence

214

intensity of 1-OHNap, while decreased the intensity of 9-OHPhe. The relevant

215

mechanism may be related to the varied interaction mechanism between the two

216

compounds with CAT, which needs further investigation.

217

3.2 Fluorescence quenching mechanisms of CAT by 1-OHNap, 9-OHPhe or 1-

218

OHPyr

219

The mechanisms of fluorescence quenching are usually classified as either

220

dynamics quenching (collisional encounters) or static quenching (complex formation)

221

(J. Zhang et al., 2016). In order to reveal the interaction mechanism of CAT with 1-

222

OHNap, 9-OHPhe or 1-OHPyr, the fluorescence quenching spectra of CAT in the

223

presence of different concentrations of individual OH-PAHs were measured under three

224

different temperatures (17, 27 and 35 oC), and the fluorescence quenching data were 9

225

analyzed according to the Stern–Volmer equation (Eq. S1) (Liu, Xiao, Li, Geng, Ma,

226

& Liang, 2017).

227

The Stern–Volmer plots for the quenching of CAT by OH-PAHs at three different

228

temperatures were shown in Fig. S2. The values of the Stern–Volmer quenching

229

constant (Ksv) and the quenching rate constant (Kq) were calculated and shown in Table

230

S1. As can be seen from Fig. S2 and Table S1, the KSV values of 9-OHPhe-CAT and

231

1-OHPyr-CAT decreased with the increment of the temperature. Moreover, the Kq

232

values of 9-OHPhe-CAT and 1-OHPyr-CAT were both much larger than the maximum

233

dynamic quenching constant of the various quenchers (2.0×1010 L mol−1 s−1 (Liu et al.,

234

2017)). These results indicated that the fluorescence quenching of CAT induced by 9-

235

OHPhe or 1-OHPyr was a static process. While for 1-OHNap, the KSV values of 1-

236

OHNap-CAT increased with the increment of the temperature, which indicated a

237

dynamic process was involved. What's more, the Kq values of 1-OHNap-CAT at three

238

temperatures were higher than 2.0×1010 L mol−1 s−1, indicating a static process also

239

occurred. That is to say, a combined quenching process (both dynamic and static

240

quenching) existed in the system of CAT and 1-OHNap (Zhang et al., 2016) .

241

3.3 Binding parameters of 1-OHNap, 9-OHPhe, and 1-OHPyr with CAT

242

In order to get a better understanding of the static quenching process of CAT

243

induced by the three OH-PAHs, investigation on the binding parameters (e.g., binding

244

constant, number of binding sites, thermodynamic parameters, and binding distance)

245

between CAT and OH-PAHs is necessary. Thus, a further research focused on the OH-

246

PAHs-CAT binding interactions was performed.

247

3.3.1 Binding constant (Kb) and the number of binding sites (n) of each OH-PAH with

248

CAT

249

When OH-PAHs molecules are bound independently to a set of equivalent sites 10

250

on CAT macromolecule, the Kb and n values can be obtained by the double logarithm

251

equation (Eq. S2) (Jahanban-Esfahlan & Panahi-Azar, 2016). The values of Kb and

252

n at three different temperatures were shown in Table 1. As can be seen in Table 1, the

253

Kb values of the 1-OHNap–CAT, 9-OHPhe–CAT and 1-OHPyr–CAT complexes were

254

6.31×103, 1.03×104 and 2.96×105 L mol-1 at 17oC, indicating that the binding capacity

255

of the three OH-PAHs with CAT increased with the increment of the number of

256

benzene rings under this experimental condition. Moreover, the n values were all

257

approximately equal to 1 for the three OH-PAHs-CAT systems at 17oC, which indicated

258

that there was one binding site for each OH-PAH in CAT at this temperature. The values

259

of Kb and n of the three systems all decreased at higher temperatures, which

260

demonstrated that higher temperature hindered the formation of the OH-PAH-CAT

261

complex.

262

Moreover, the binding constants of the three OH-PAHs with CAT were in the

263

order of 1-OHNap ＜ 9-OHPhe ＜ 1-OHPyr at 17 and 27oC. Meanwhile, the

264

octanol/water partition coefficients (logKow) of 1-OHNap, 9-OHPhe and 1-OHPyr are

265

2.689, 3.865 and 4.453, which is in the same order as the binding constants at the two

266

temperatures. This suggested that the hydrophobicity of OH-PAHs could affect their

267

affinities to CAT, which was also revealed by previous study that focused on the

268

binding of organic ligands with protein (Zhang, Zhang, Tong, Chen, Zhuang, & Liu,

269

2013). While at 35 oC, the order of the binding constants turned into 9-OHPhe ＜ 1-

270

OHNap ＜ 1-OHPyr. This interesting phenomenon can be well explained by the

271

thermodynamic parameters presented in section 3.3.2. As ∆H<0, the binding

272

interactions of the three OH-PAHs with CAT were exothermic. Thus, the binding

273

affinity of OH-PAHs with CAT decreased with the increment of temperature for each

274

system. As |∆H| of 9-OHPhe with CAT was larger than that of 1-OHNap, the binding 11

275

of 9-OHPhe with CAT was more susceptible to temperature than 1-OHNap. Therefore,

276

the binding affinity of 9-OHPhe with CAT decreased to a value which was lower than

277

that of 1-OHNap at 35 ºC, which was different from the order at 17 and 27 ºC.

278

3.3.2 Thermodynamic parameters in the binding of the OH-PAHs with CAT

279

The binding forces between biomolecules and small molecular ligands mainly

280

include hydrophobic interactions, hydrogen bonds, van der Waals interactions, and

281

electrostatic forces. The type of binding forces dominated in the binding process can be

282

determined based on enthalpic change (∆H) and entropic change (∆S) of the interactions

283

(Ross & Subramanian, 1981). Because the temperature range was not too wide (from

284

17 oC to 35 oC), the ∆H could be regarded as a constant. Therefore, the thermodynamic

285

parameters ∆H, ∆S, and ∆G (the free energy change) could be calculated using the

286

Van’t Hoff equation (Fq. S3) and thermodynamic equation (Fq. S4). The ∆H and ∆S

287

values were calculated from the slope and intercept values of the plot of lnKb versus

288

1/T, respectively. The calculated thermodynamic parameters of the binding of CAT

289

with each OH-PAH were listed in Table S2. As can be seen, ∆G<0 revealed that the

290

binding interactions were all spontaneous; ∆H<0 indicated that the binding interaction

291

in each system was an exothermic process. As ∆G<0, ∆H<0 and -T∆S>0, it suggested

292

that the binding process was enthalpy-driven (Pan, Jiang, Liu, Wang, & Shi, 2017).

293

All in all, the binding interactions between the three OH-PAHs and CAT were

294

spontaneous, exothermic and enthalpy-driven. Moreover, the negative ∆H and negative

295

∆S indicated that van der Waals and hydrogen bonds forces played major roles in the

296

binding process of all the three OH-PAHs with CAT (Mu et al., 2019) .

297

3.3.3 Binding distances of the OH-PAHs with CAT

298

Fluorescence resonance energy transfer (FRET) is a useful technique to determine

299

the distance between a donor (fluorophore) and an acceptor. As the absorption spectrum 12

300

of each OH-PAH and the fluorescence emission spectrum of CAT overlap enough (Fig.

301

S3), the energy transfer from CAT to OH-PAHs may happen with a high probability

302

(Förster, 1965). Herein, the average distances between the TRP residues of CAT and

303

each OH-PAH were investigated using FRET. After fitting data to Eqs. (S5) – (S7), all

304

of the calculated parameters were shown in Table S3.

305

As shown in Table S3, the energy transfer efficiency (E) of CAT to 1-OHNap, 9-

306

OHPhe and 1-OHPyr was 3.83%, 4.05% and 11.83%, respectively. The average

307

distances from the TRP residue of CAT to 1-OHNap, 9-OHPhe and 1-OHPyr (r) were

308

3.28, 4.06 and 3.48 nm, which were all less than 7 nm. This confirmed that there is a

309

high probability of energy transfer from CAT to each OH-PAH. Moreover, it

310

demonstrated again that the static quenching process occurred in the interactions

311

between OH-PAHs and CAT.

312

3.4 Molecular docking of the OH-PAHs with CAT

313

To further locate the specific binding site of each OH-PAH in CAT and obtain

314

more detailed binding information at the molecular level, blind molecular docking

315

method was utilized to reveal the optimal binding mode of each OH-PAH with CAT.

316

During the docking simulations, the lowest energy-ranked binding mode was chosen

317

among 25 conformers of docking results in each OH-PAH-CAT system. The obtained

318

optimal locations of each OH-PAH on CAT and the specific interactions between OH-

319

PAHs and its surrounding amino acid residues were shown in Fig. 2. The relevant

320

information was listed in Table S4–S7.

321

As illustrated in Fig. 2A, C and E, the three OH-PAHs were all located near the

322

active center (Heme) of CAT, which indicated that the three OH-PAHs might make

323

impact on the activity of CAT. Fig. 2B, D and F illustrates the neighboring amino acid

324

residues around each OH-PAH, and also the varied non-covalent interactions between 13

325

these amino acid residues and each OH-PAH. As can be seen in Fig. 2B and Table S4,

326

1-OHNap formed hydrophobic interactions with ARG169, PRO171, PHE325 and

327

ARG387; three hydrogen bonds were formed between 1-OHNap and ASN323,

328

PHE325, ASN396, with the bond length of 1.87 Å, 1.74 Å, and 3.68 Å, respectively.

329

9-OHPhe had hydrophobic contacts with TYR369, LEU370, and GLN386; there were

330

four hydrogen bonds formed between 9-OHPhe and CAT, one between 9-OHPhe and

331

ASN368 (2.10 Å), one between 9-OHPhe and LEU370 (3.05 Å), and two between 9-

332

OHPhe and GLN 371 (3.11 Å and 2.45 Å) (Fig. 2D and Table S5). As for 1-OHPyr

333

(Fig. 2F and Table S6), hydrophobic interactions were formed between 1-OHPyr and

334

LYS168, ARG169, PRO171, ARG387, ASN402; three hydrogen bonds were observed

335

between 1-OHPyr and ASN323, PHE325, ASN396, with the bond length of 1.82 Å,

336

1.98 Å, and 3.58 Å, respectively; moreover, a benzene ring of 1-OHPyr and the

337

heterocyclic ring of HIS165 were closely packed in parallel and had the π-

338

Stacking interactions which could enhance the binding of 1-OHPyr to CAT. Combined

339

the results in thermomechanical analysis and theoretical studies, it suggested that van

340

der Waals interactions, hydrogen bonds and hydrophobic interactions played major

341

roles in the binding of three OH-PAHs with CAT.

342

Moreover, for the selected conformations, the Kb values calculated from the

343

docking studies of the OH-PAHs-CAT systems were ranked in 1-OHNap< 9-OHPhe<

344

1-OHPyr (as shown in Table S7). The results were consistent with the experimental

345

results by fluorescence analysis, indicating that molecular docking technique was

346

reliable to study the binding interactions between CAT and OH-PAHs. In addition, for

347

the three OH-PAHs-CAT systems, the discrepancies between the estimated Kb values

348

and experimental Kb values may be due to the fact that the X-ray structure of the CAT

349

crystals used in the molecular docking study was different from that of the aqueous 14

350

system reported previously (Sharma, Anandakumar, & Ilanchelian, 2014).

351

3.5 Effects of the OH-PAHs on the conformation of CAT

352

It had been confirmed that the interactions of each OH-PAHs with CAT could

353

cause the fluorescence characteristics change of the CAT. However, it was still unclear

354

whether the binding interactions could influence the microenvironment and

355

conformation of the CAT, and whether the effects varied among the three OH-PAHs.

356

In order to address these issues, the synchronous fluorescence and CD spectroscopy

357

were employed to investigate the structural changes of the CAT when binding with OH-

358

PAHs in the following sections.

359

3.5.1 Effects of OH-PAHs on the synchronous fluorescence spectra of CAT

360

Synchronous fluorescence spectroscopy can be used to characterize the

361

microenvironmental changes around chromosphere molecules like Tyr or Trp residues

362

in CAT (Chen, Zhang, Zhu, & Zhang, 2018). The synchronous fluorescence spectra

363

of CAT in the presence of different concentrations of OH-PAHs were shown in Fig. 3.

364

With the addition of each OH-PAH, the synchronous fluorescence intensity of Trp (Δλ

365

= 60 nm) and Tyr (Δλ = 15 nm) residues both decreased. Meanwhile, the maximum

366

emission wavelength of Trp and Tyr residues had no obvious shift with the addition of

367

1-OHNap and 9-OHPhe. It indicated that neither 1-OHNap nor 9-OHPhe did perturb

368

the polarity of microenvironment around Trp and Tyr residues significantly. However,

369

the maximum emission wavelength of Trp residues had a distinct red shift (from 282 to

370

286 nm) with the addition of 1-OHPyr (Fig. 3C), which indicated that the Trp residues

371

were placed in a less hydrophobic environment (Xu et al., 2018). Moreover, the Ksv

372

values for Tyr and Trp residues in each OH-PAHs-CAT system were calculated and

373

shown in Table S8. The results suggested that 1-OHNap and 9-OHPhe induced greater

374

perturbation of the microenvironment around Tyr than Trp residues, while 1-OHPyr 15

375

showed a greater effect on Trp than Tyr residues.

376

3.5.2 Effects of the OH-PAHs on the circular dichroism (CD) spectra of CAT

377

In order to quantitatively study the effects of the OH-PAHs on the secondary

378

structure of CAT, CD spectroscopy was further performed in the absence and presence

379

of the OH-PAHs. As shown in Fig. S4, the CD spectra of pure CAT exhibits two main

380

negative bands at approximately 210 and 220 nm, which characters the α-helix structure

381

of CAT (Wang et al., 2016). With the addition of 1-OHNap and 1-OHPyr, the intensity

382

of the two negative bands of CAT increased, while the intensity of the two bands

383

decreased in 9-OHPhe-CAT system, inferring considerable changes in the secondary

384

structure of CAT induced by OH-PAHs.

385

Furthermore, the CD data were quantitatively analyzed using the CDPro software

386

package, and the results of the secondary structural contents of CAT in each system

387

were listed in Table 2. As shown in Table 2, compared with pure CAT, the secondary

388

structural contents of CAT altered differently after the addition of OH-PAHs. With a

389

molar ratio of 1-OHNap or 1-OHPyr to CAT at 20 : 1, there is approximately a 1.7%

390

and 2.7% increase of α-helix, respectively, with a corresponding decrease of β-sheet. It

391

suggested that 1-OHNap and 1-OHPyr could stabilize the conformation of CAT to a

392

certain extent, mainly by increasing the helix content. While, the α-helix content

393

decreased approximately 2.4% with a molar ratio of 9-OHPhe to CAT at 20 : 1, with

394

an accompanying increase of β-sheet. It was probably because 9-OHPhe interacts with

395

certain amino acid residues in CAT, thus destroyed the hydrogen bonding networks of

396

the CAT, inducing the transformation of helical structure of the CAT to sheets (Xu et

397

al., 2018). Therefore, the three OH-PAHs induced varied structure changes of CAT,

398

which mainly due to their different molecular interactions with CAT.

399

3.6 Effects of the OH-PAHs on the activity of CAT 16

400

The biological activity of CAT can be used as a sensitive biomarker of

401

contaminant-induced oxidative stress in organisms (Glorieux et al., 2017). As the

402

structural changes of CAT induced by OH-PAHs may alter the activity of CAT, the

403

activity determination is needed for exploring the adverse effect of each OH-PAH on

404

CAT. The effects of different concentrations of OH-PAHs ranging from 0 to 1.0 × 10-

405

5

406

and the results are presented in Fig. 4 and Table S9. As can be seen from Fig. 4, the

407

activity of CAT had no obvious change at lower concentrations of 1-OHNap (from 0 to

408

2.0×10-5 mol L-1); while CAT activity trended to decrease with further increase of 1-

409

OHNap concentration, and decreased to 91.6% of the initial level at the highest

410

experimental concentration of 1-OHNap (5.0 × 10-5 mol L-1). The activity of CAT

411

increased gradually to 105.0% of its initial level at the concentration ratio of 9-OHPhe

412

to CAT below 6:1, and slightly decreased with the continuous increment of 9-OHPhe

413

(from 6.0 × 10-6 to 1.0 × 10-5 mol L-1). While, the relative activity of CAT remained

414

unchangeable when 1-OHPyr concentration was lower than 2.0 × 10-6 mol L-1, and

415

obviously decreased with further increase of 1-OHPyr concentration (to 86.8% of its

416

initial level at 1-OHPyr/CAT = 10:1). Combined with the CD spectral results, the

417

promotion effects of 9-OHPhe on CAT activity may be attributed to the decrease of the

418

helix of CAT and the loosening of CAT structure, which make the substrate channel of

419

CAT easier to get through for H2O2 and increase the chance for H2O2 to contact with

420

heme (Moradi, Divsalar, Saboury, Ghalandari, & Harifi, 2015; Rashtbari,

421

Dehghan, Yekta, Jouyban, & Iranshahi, 2017; Rui, Liu, & Zong, 2016). While, the

422

CAT activity slightly decreased at higher concentrations of 9-OHPhe, which was

423

probably because that the structure of the active center of CAT was destroyed by the

424

excess 9-OHPhe (Chen, Zhang, Zhu, & Zhang, 2018). In addition, 1-OHNap and 1-

mol L-1 or 5.0 × 10-5 mol L-1 on CAT activity (1.0 × 10-6 mol L-1) were tested in vitro

17

425

OHPyr induced the increase of the helix of CAT and thus make the structure of CAT

426

even tighter, which might hinder the contact of H2O2 with heme, and resulted in the

427

inhibition of CAT activity.

428

It can be seen from Table S9, compared the effects of the three OH-PAHs on CAT

429

activity at the identical concentration level (1.0 × 10-5 mol L-1), their influence degree

430

on CAT activity was in the order of 1-OHNap＜9-OHPhe＜1-OHPyr. This indicated

431

that the effects of the three OH-PAHs on the activity of CAT were associated with the

432

binding affinity of OH-PAHs to CAT, which increased with the increment of the

433

number of benzene rings of OH-PAHs. As the activity inhibition of CAT can cause the

434

enhanced generation of ROS (Lee et al., 2018; Najjar et al., 2016), 1-OHNap and 1-

435

OHPyr are more likely to induce oxidative stress than 9-OHPhe in vivo. Some research

436

had pointed out that PAHs could undergo oxidative metabolism in vivo and produced a

437

large amount of reactive oxygen species, hence the induced oxidative stress will change

438

the activity of CAT (Ranjit et al., 2016; Singh, Patel, Jyoti, Ram, Mathur, &

439

Siddiqui, 2008). Herein, the above results further suggested that the oxidative

440

metabolites of PAHs might also cause direct adverse effects on CAT in organism.

441 442

4. Conclusion

443

In this study, the interactions of three OH-PAHs with CAT and the corresponding

444

effects on the structure and function of CAT at the molecular level were investigated

445

using experimental studies and molecular docking. The results showed that 1) the

446

binding affinity of the three OH-PAHs with CAT increased with the increment of the

447

number of benzene rings at 17 and 27 ºC, as the hydrophobicity of OH-PAHs could

448

affect their binding ability to CAT. However, the order of the binding constants at 35

449

oC

was different from that at 17 and 27 oC, as the increase of temperature inhibited the 18

450

binding affinities of the three OH-PAHs with CAT to varied extent. Van der Waals’

451

force, hydrogen bonds and hydrophobic interactions played important roles in the

452

binding interactions of CAT with the three OH-PAHs. The binding distances of 1-

453

OHNap, 9-OHPhe, and 1-OHPyr with CAT were determined to be 3.28, 4.06 and 3.48

454

nm. 2) OH-PAHs with different numbers of aromatic rings can induce different

455

structural changes of CAT. Specifically, 1-OHNap and 9-OHPhe induced greater

456

perturbation of the microenvironment around Tyr residues, while 1-OHPyr showed a

457

greater effect on Trp residues. Both 1-OHNap and 1-OHPyr affected the secondary

458

structure of CAT by increasing the content of α-helix, while 9-OHPhe destroyed the

459

helix of CAT. 3) The activity of CAT can be inhibited by both 1-OHNap and 1-OHPyr

460

at experimental concentrations. While, 9-OHPhe has a continuous promotion effects on

461

CAT activity at lower concentrations (<6.0 × 10-6 mol L-1), but the promotion slightly

462

decreased at higher concentrations. At the concentration of 1.0 ×10-5 mol L-1, the effect

463

degree of 1-OHNap, 9-OHPhe and 1-OHPyr on CAT activity was in the order of 1-

464

OHNap ＜ 9-OHPhe ＜ 1-OHPyr. Overall, the results demonstrated that the varied

465

effects of the three OH-PAHs on CAT were closely related to the different molecular

466

interactions of the three OH-PAHs with CAT. The in-vitro adverse effects of OH-PAHs

467

on CAT indicated that OH-PAHs may cause the oxidative stress in vivo, and warned

468

the health risk of the PAHs through food intake.

469

Overview, this work provided useful data for clarifying the molecular interaction

470

mechanism of OH-PAHs with CAT. While, considering the limitations of this work,

471

further investigations need to be conducted on the issues which were not yet figured

472

out in the present study. For instance, the specific proportion of static and dynamic

473

quenching in combined quenching mechanism of 1-OHNap on CAT, the in-depth

474

reason for the inversions in fluorescence intensity of 1-OHNap and 9-OHPhe with and 19

475

without CAT, and the structural changes of CAT around its Trp or Tyr residues induced

476

by OH-PAHs and the corresponding effects on Trp or Tyr. Meanwhile, some other

477

techniques such as ITC, NMR and molecular dynamics simulations can be applied to

478

further study the molecular interactions between OH-PAHs and CAT. Moreover, more

479

research efforts are expected to investigate the molecular interactions of OH-PAHs with

480

other antioxidant enzymes as GPX, SOD and GST.

481 482 483

Conflict of interests The authors declare no conflict of interests.

484 485

Acknowledgments

486

We gratefully acknowledge the financial support of the National Major Scientific

487

Instruments Development Project of China [grant number 21627814], the National

488

Natural Science Foundation of China [grant number 21577110], the Natural Science

489

Foundation of Fujian Province [grant number 2018J05024] and the Education and

490

Scientific Research Projects of Young and Middle-Aged Teachers in Fujian Province

491

[grant number JAT170841].

492

References

493 494 495 496 497 498 499 500 501 502 503 504 505 506 507

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22

608

Figure captions

609

Fig. 1 Fluorescence quenching spectra of CAT in the presence of various

610

concentrations of 1-OHNap (A), 9-OHPhe (B) and 1-OHPyr (Chen et al., 2015)

611

(C) at 17 ºC (pH=7.40). Ex./Em. slit=5/10 nm (A and B); Ex./Em. slit=5/5 nm (C).

612

c(CAT)=1.0×10-6 mol L-1; c(CAT)=1.0×10-6 mol L-1; c(1-OHNap), a-g: (0, 0.5,1.0, 2.0,

613

3.0, 4.0, 5.0) ×10-5 mol L-1; c(9-OHPhe), a-g: (0, 1.0, 2.0, 4.0, 6.0, 8.0,10.0) ×10-6 mol

614

L-1; c(1-OHPyr), a-g: (0, 1.0, 2.0, 4.0, 6.0, 8.0,10.0) ×10-6 mol L-1

615 616

Fig. 2 The binding modes of three OH-PAHs to CAT. Binding site of 1-OHNap (A),

617

9-OHPhe (C), and 1-OHPyr (E) in CAT; Detailed illustration of the binding forces of

618

1-OHNap (B), 9-OHPhe (D), and 1-OHPyr (F) with the surrounding amino acid

619

residues

620 621

Fig. 3 Synchronous fluorescence spectra of the 1-OHNap-CAT (A), 9-OHPhe-

622

CAT (B) and 1-OHPyr-CAT (Chen et al., 2015) (C) systems at 17 ºC (pH=7.40).

623

c(CAT)=1.0×10-6 mol L-1; c(1-OHNap), a-g: (0, 0.5,1.0, 2.0, 3.0, 4.0, 5.0) ×10-5 mol

624

L-1; c(9-OHPhe), a-g: (0, 1.0, 2.0, 4.0, 6.0, 8.0,10.0) ×10-6 mol L-1; c(1-OHPyr), a-g:

625

(0, 1.0, 2.0, 4.0, 6.0, 8.0,10.0) ×10-6 mol L-1

626 627

Fig. 4 Effects of different concentrations of 1-OHNap, 9-OHPhe and 1-OHPyr on

628

the activity of CAT at 17 ºC (pH=7.40). c(CAT)=1.0×10-6 mol L-1; c(1-OHNap), 1-6:

629

(0, 1.0, 2.0, 3.0, 4.0, 5.0) × 10-5 mol L-1; c(9-OHPhe), 1-6: (0, 2.0, 4.0, 6.0, 8.0,10.0) ×

630

10-6 mol L-1; c(1-OHPyr), 1-6: (0, 2.0, 4.0, 6.0, 8.0,10.0) × 10-6 mol L-1. (n=6. *, P<

631

0.05 vs. control; **, P< 0.01 vs. control; ***, P< 0.001 vs. control)

632 23

633 634

Table captions

635

Table 1 Binding constants (Kb) and binding sites (n) of the 1-OHNap-CAT, 9-

636

OHPhe-CAT and 1-OHPyr-CAT interactions

637

Table 2 Secondary structure contents of CAT in presence of different molar ratios

638

of OH-PAHs

639 640 641 642 643 644 645 646 647

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

648 649 650 651 652 653

Highlights

654



1-OHNap, 9-OHPhe or 1-OHPyr can bind with catalase (CAT) to varying degrees

655



The specific interaction modes of the three OH-PAHs with CAT differed obviously

656



The three OH-PAHs induced varied conformational changes of CAT

657



The activity of CAT was promoted by 9-OHPhe, but inhibited by 1-OHNap or 1-

658

OHPyr

659 660 24

400

OH

1-OHNap

CAT

300 200 100 0 300

g a

g 350

400

450

500

(B)

400

500

a g

100 0 300

350

1-OHPyr g

OH

a

300

400

450

500

Emission wavelength / nm

a

200

g

a

200

400

100

g

300

550

(C)

300

9-OHPhe

HO

CAT

Emission wavelength / nm

0

662 663 664

500

a

Relative intensity / a.u.

661

(A)

Relative intensity / a.u.

Relative intensity / a.u.

500

CAT

400

350

450

Emission wavelength / nm

665

666

25

500

550

667

668

669 670 671 672

70

350

60 50

g

40 30 20 10 0 270

673

15 nm a

(A1)

Relative intensity / a.u.

Relative intensity / a.u.

80

280

290

300

Wavelength / nm

a

300 250

g

200 150 100 50 0 250

310

60 nm

(A2)

260

270

280

290

Wavelength / nm

26

300

310

15 nm

350

a

300

60

Relative intensity / a.u.

(B1)

70

50

g

40 30 20 10 0 270

280

674

Relative intensity / a.u.

80 70

290

Wavelength / nm

300

(C1)

15 nm

50

g

40 30 20

675 676

g

150 100 50 260

270

280

290

310

60 nm

(C2)

350

300

Wavelength / nm

a

300 250

g

200 150 100 50

10 0 270

60 nm a

200

0 250

a

60

(B2)

250

310

Relative intensity / a.u.

Relative intensity / a.u.

80

280

290

Wavelength / nm

300

0 250

310

260

270

280

290

Wavelength / nm

300

677

Relative activity / %

120 110 *

**

*

100

**

**

1-OHNap 9-OHPhe 1-OHPyr **

**

**

**

**

**

90

***

80 70 60

1

2

3

4

5

6

Serial number

678 679 680 681

Table 1 Binding constants (Kb) and binding sites (n) of the 1-OHNap-CAT, 9-OHPhe-CAT and 1-OHPyr-CAT interactions

682

1-OHNap-CAT

T / °C

Kb / L mol-1

n

R2

17

6.31×103

1.08

0.996

27

7.12×102

0.790

0.979

35

5.09×102

0.733

0.991

27

9-OHPhe-CAT

1-OHPyr-CAT

(Chen, et al., 2015)

17

1.03×104

0.948

0.997

27

1.63×103

0.782

0.978

35

3.49×102

0.654

0.985

17

2.96×105

1.11

0.995

27

1.62×105

1.07

0.994

35

5.54×104

0.982

0.995

683 684 685 686

Table 2 Secondary structure contents of CAT in presence of different molar

687

ratios of OH-PAHs OH-PAHs

1-OHNap

9-OHPhe

1-OHPyr

Secondary structural elements in CAT

c(OH-PAHs)/ c(CAT)

α-helix / %

β-sheet / %

β-turn / %

Random coil /%

0:1

20.1

27.3

21.1

31.5

1:1

20.9

27.0

20.9

31.3

20:1

21.8

26.4

20.6

31.2

1:1

19.8

27.8

20.7

31.7

20:1

17.7

29.1

20.9

32.3

1:1

21.2

25.6

21.8

31.4

20:1

22.8

24.2

22.7

30.3

688 689 690 691

28