Iron Porphyrin as a Cytochrome P450 Model for the Degradation of Dye

Organic dyes are widely used in the textile, biological, medical and other fields. However, a serious environmental problem has appeared because of the presence of organic dyes in industrial aqueous effluents. Thus, the efficient treatment of organic dyes in industrial wastewaters is currently in real demand. The current study investigated the oxidative degradation of the organic dye gentian violet by meso-tetra(carboxyphenyl) porphyriniron(III), [FeIII(TCPP)] as a cytochrome P450 model and iodosylbenzene (PhIO) as an oxidant at room temperature. The degradation reaction was monitored by UV–vis absorption spectroscopy via the observation of UV–vis spectral changes of the gentian violet. The results showed that the efficiency of catalyzed degradation reached more than 90% in 1 h, indicating the remarkable oxidative degradation capacity of the [FeIII(TCPP)]/PhIO system, which provided an efficient approach for the treatment of dyeing wastewater.


Introduction
As is well known, dyes are widely used in the papermaking, textile, food, cosmetics and other industries [1,2]. With the increase in material and cultural needs, the dye industry continues to develop and grow. About 300-400 million tons of industrial waste are discharged into rivers every year [3,4]. These persistent organic dyes found in industrial aqueous effluents can accumulate in the body after entering the human system and result in serious health problems in the cardiovascular, endocrine, nervous and immune systems, which may even be passed to the next generation. Among the various dyes, triphenylmethane dyes, for example gentian violet, are the largest and most versatile, and they play a predominant role in human and veterinary medicine and the textile processing industry [5]. However, gentian violet is difficult to degrade and can remain in the environment for a long period. It is toxic to aquatic and terrestrial life, and it is considered a mitotic poisoning agent and a potent clastogene [6]. Thus, the efficient treatment of gentian violet in industrial wastewaters is currently in real demand [7].
In the past few decades, people have invested a lot of energy into the treatment of dyecontaining effluents [8][9][10][11][12][13][14][15][16]. Various methods to degrade dyes, including chemical [17,18], physical [19][20][21][22] and biological [23,24] methods, have been developed and widely applied for the degradation of organic dyes. However, these traditional methods have certain limitations. For example, air-bubbling visible-light photocatalytic degradation can eliminate pollutants in wastewater under mild conditions [25][26][27]. However, due to the small active area of photocatalysts, insufficient light absorption and instantaneous electron hole separation, the photocatalytic degradation activity of this method is reduced. In addition, adsorption is the most common technology used to remove pollution from textile, printing

Preparation of Iodosylbenzene (PhIO)
Iodosylbenzene (PhIO) was prepared by a previously reported method [60]. A total of 8 g of (diacetoxyiodo)benzene (PhI(OAc)2) was weighed and placed in a 250 mL beaker and wrapped in tin foil for light protection. A total of 4.5 g of sodium hydroxide (NaOH) was weighed in a 50 mL beaker and dissolved with 30 mL H2O. The NaOH solution was slowly added to the beaker containing the solution of PhI(OAc)2 within 0.5 h, which was then stirred vigorously at room temperature for 1 h. Then, 20 mL H2O was added and continuously stirred for 1.5 h. The resulting solution was then filtered and washed with water until the pH value of filtrate reached 7, and then the precipitate was washed with dichloromethane and ethyl ether, respectively, dried and kept in a refrigerator for further use. Its purity was periodically controlled by iodometric titration [61].

Determination of Concentration of Gentian Violet
The concentration of gentian violet in the resulting solution was determined by a UV-vis spectrophotometer with a quartz cuvette (path length = 10 mm) in the wavelength range of 200-800 nm. The UV-vis spectrum of 0.05 mM gentian violet in MeOH exhibited a distinct absorption band at 578 nm (λmax) with an absorbance of 1.022 (Figure 2a), and its extinction coefficient (ε) was then calculated to be 20440 M −1 cm −1 using Beer's law, A = εc, where A is the absorbance at 578 nm and c is the concentration of gentian violet. Thus, the concentration of gentian violet in the reaction solution could be determined from the

Preparation of Iodosylbenzene (PhIO)
Iodosylbenzene (PhIO) was prepared by a previously reported method [60]. A total of 8 g of (diacetoxyiodo)benzene (PhI(OAc) 2 ) was weighed and placed in a 250 mL beaker and wrapped in tin foil for light protection. A total of 4.5 g of sodium hydroxide (NaOH) was weighed in a 50 mL beaker and dissolved with 30 mL H 2 O. The NaOH solution was slowly added to the beaker containing the solution of PhI(OAc) 2 within 0.5 h, which was then stirred vigorously at room temperature for 1 h. Then, 20 mL H 2 O was added and continuously stirred for 1.5 h. The resulting solution was then filtered and washed with water until the pH value of filtrate reached 7, and then the precipitate was washed with dichloromethane and ethyl ether, respectively, dried and kept in a refrigerator for further use. Its purity was periodically controlled by iodometric titration [61].

Determination of Concentration of Gentian Violet
The concentration of gentian violet in the resulting solution was determined by a UV-vis spectrophotometer with a quartz cuvette (path length = 10 mm) in the wavelength range of 200-800 nm. The UV-vis spectrum of 0.05 mM gentian violet in MeOH exhibited a distinct absorption band at 578 nm (λ max ) with an absorbance of 1.022 (Figure 2a), and its extinction coefficient (ε) was then calculated to be 20,440 M −1 cm −1 using Beer's law, A = εc, where A is the absorbance at 578 nm and c is the concentration of gentian violet. Thus, the concentration of gentian violet in the reaction solution could be determined from

Degradation of Gentian Violet
The stock solution of gentian violet with a concentration of 10 mM and [Fe III (TCPP)] with a concentration of 0.315 mM in MeOH were prepared, which were kept for further use. It should be noted that the stock solution of PhIO (10 mM) was reconfigured with each use.

General Procedure for the Effect of PhIO Concentration on Degradation of Gentian Violet
In a typical experiment, 160 μL of the stock solution of [Fe III (TCPP)] and 50 μL of the stock solution of gentian violet were put in 10 mL of MeOH to give a final concentration of 0.005 mM [Fe III (TCPP)] and 0.05 mM gentian violet. Then, 1 equiv, 2 equiv, 3 equiv, 4 equiv and 5 equiv of PhIO (relative to gentian violet, same as below) were added into the solution, respectively. The reaction mixture was stirred in a water bath at 303 K for 2.5 h. After that, the concentration of gentian violet in the resulting solution was determined by an ultraviolet-visible (UV-vis) spectrophotometer. Control experiments using [Fe III (TCPP)] or PhIO alone in gentian violet solution were performed in parallel. Reactions were performed at least in triplicate, and the data reported represent the average of these reactions.

General Procedure for the Effect of Reaction Time on Degradation of Gentian Violet
In a typical experiment, 240 μL of the stock solution of [Fe III (TCPP)] and 75 μL of the stock solution of gentian violet were put in 15 mL of MeOH, and then 1-5 equiv of PhIO were added into this solution, respectively. Then, the reaction mixture was stirred in a water bath at 303 K. A total of 2 mL of each solution was taken at 0.5 h, 1 h, 1.5 h, 2 h and 2.5 h, respectively, to measure the UV-vis spectra. Reactions were performed at least in triplicate, and the data reported represent the average of these reactions.

Results and Discussion
The UV-vis spectra of the gentian violet and [Fe III (TCPP)] in MeOH exhibited a distinct absorption band at 578 nm and 414 nm, respectively, which indicated that their absorption peaks had no effect on each other. Thus, using UV-vis spectroscopy to monitor the concentration changes in the gentian violet was a suitable method. The degradation of the gentian violet was examined at fixed concentrations of catalyst [Fe III (TCPP)] and gentian violet dye in MeOH at 303 K using m-CPBA and PhIO as the oxidant, respectively, at preliminary experiments. No spectral change was observed when using 5 equiv of m-

Degradation of Gentian Violet
The stock solution of gentian violet with a concentration of 10 mM and [Fe III (TCPP)] with a concentration of 0.315 mM in MeOH were prepared, which were kept for further use. It should be noted that the stock solution of PhIO (10 mM) was reconfigured with each use.

General Procedure for the Effect of PhIO Concentration on Degradation of Gentian Violet
In a typical experiment, 160 µL of the stock solution of [Fe III (TCPP)] and 50 µL of the stock solution of gentian violet were put in 10 mL of MeOH to give a final concentration of 0.005 mM [Fe III (TCPP)] and 0.05 mM gentian violet. Then, 1 equiv, 2 equiv, 3 equiv, 4 equiv and 5 equiv of PhIO (relative to gentian violet, same as below) were added into the solution, respectively. The reaction mixture was stirred in a water bath at 303 K for 2.5 h. After that, the concentration of gentian violet in the resulting solution was determined by an ultraviolet-visible (UV-vis) spectrophotometer. Control experiments using [Fe III (TCPP)] or PhIO alone in gentian violet solution were performed in parallel. Reactions were performed at least in triplicate, and the data reported represent the average of these reactions.

General Procedure for the Effect of Reaction Time on Degradation of Gentian Violet
In a typical experiment, 240 µL of the stock solution of [Fe III (TCPP)] and 75 µL of the stock solution of gentian violet were put in 15 mL of MeOH, and then 1-5 equiv of PhIO were added into this solution, respectively. Then, the reaction mixture was stirred in a water bath at 303 K. A total of 2 mL of each solution was taken at 0.5 h, 1 h, 1.5 h, 2 h and 2.5 h, respectively, to measure the UV-vis spectra. Reactions were performed at least in triplicate, and the data reported represent the average of these reactions.

Results and Discussion
The UV-vis spectra of the gentian violet and [Fe III (TCPP)] in MeOH exhibited a distinct absorption band at 578 nm and 414 nm, respectively, which indicated that their absorption peaks had no effect on each other. Thus, using UV-vis spectroscopy to monitor the concentration changes in the gentian violet was a suitable method. The degradation of the gentian violet was examined at fixed concentrations of catalyst [Fe III (TCPP)] and gentian violet dye in MeOH at 303 K using m-CPBA and PhIO as the oxidant, respectively, at preliminary experiments. No spectral change was observed when using 5 equiv of m-CPBA and reacting for 2.5 h. However, the color of the reaction solution changed from purple to colorless when using 5 equiv of PhIO and reacting for 2.5 h (Figure 3). Therefore, herein, PhIO and [Fe III (TCPP)] were employed as the oxidant and catalyst, respectively, for the degradation of the gentian violet.
CPBA and reacting for 2.5 h. However, the color of the reaction solution changed from purple to colorless when using 5 equiv of PhIO and reacting for 2.5 h (Figure 3). Therefore, herein, PhIO and [Fe III (TCPP)] were employed as the oxidant and catalyst, respectively, for the degradation of the gentian violet.  Figure 4. The absorbance band at 578 nm, which was due to the gentian violet, decreased when increasing the concentration of PhIO, and it provided a 98% degradation efficiency when adding 5 equiv of PhIO (Table 1). The absorbance at 414 nm, which was due to the [Fe III (TCPP)], exhibited almost no change (Supplementary Materials Figure S1), which indicated that the catalyst [Fe III (TCPP)] was stable during the degradation of the gentian violet. No spectral changes were observed in the absence of [Fe III (TCPP)] or PhIO (Supplementary Materials, Figure S2). The spectral measurements confirmed that the presence of both [Fe III (TCPP)] and PhIO was essential for the degradation of the gentian violet and a high concentration of PhIO was beneficial in improving the degradation efficiency.  Figure 4. The absorbance band at 578 nm, which was due to the gentian violet, decreased when increasing the concentration of PhIO, and it provided a 98% degradation efficiency when adding 5 equiv of PhIO (Table 1). The absorbance at 414 nm, which was due to the [Fe III (TCPP)], exhibited almost no change (Supplementary Materials Figure S1), which indicated that the catalyst [Fe III (TCPP)] was stable during the degradation of the gentian violet. No spectral changes were observed in the absence of [Fe III (TCPP)] or PhIO (Supplementary Materials, Figure S2). The spectral measurements confirmed that the presence of both [Fe III (TCPP)] and PhIO was essential for the degradation of the gentian violet and a high concentration of PhIO was beneficial in improving the degradation efficiency.     The effect of the reaction time on the degradation of the gentian violet was further examined at fixed concentrations of [Fe III (TCPP)] and gentian violet in the [Fe III (TCPP)]/PhIO system, and the results are presented in Figure 5, Table 2 and Figures S3-S6 in the Supplementary Materials. As shown in Figure 5 and Table 2, when using 1 equiv of PhIO, the degradation rate was the slowest and the degradation efficiency of gentian violet was the lowest compared with that of 2-5 equiv of PhIO. The degradation efficiency was basically unchanged after 2 h due to the insufficient amount of oxidant. When using 2 equiv and 3 equiv oxidants, although the corresponding degradation efficiency of gentian violet increased, the degradation rates were slow, and the degradation efficiency was only 65% and 79% after reacting for 2.5 h, respectively. When 4 equiv oxidant was used, with the increase in the reaction time, the degradation efficiency of gentian violet increased to 90%, but it remained almost unchanged after 2 h. When using 5 equiv oxidant, the degradation efficiency of gentian violet reached 75% within 0.5 h and 94% within 1 h, showing a remarkable degradation of gentian violet. The degradation products of the gentian violet produced by the [Fe III (TCPP)]/PhIO system after color removal were detected by GC-MS (Supplementary Materials, Figure S7). However, only iodobenzene (PhI) with a molecular weight of 204 was shown in the GC-MS results, which came from the reduction of PhIO. No product of the gentian violet was observed, which may have been due to the formation of high-boiling-point products, which could not be detected by GC-MS.

The effect of PhIO concentration on the degradation of the gentian violet was first studied at fixed concentrations of [Fe III (TCPP)] and gentian violet in the [Fe III (TCPP)]/PhIO system. A typical set of PhIO concentration-dependent spectral absorptions recorded for the [Fe III (TCPP)]-catalyzed degradation of gentian violet in MeOH at 303 K is shown in
The above experimental results are summarized in Figure 6, which indicated that with the increase in PhIO concentration and reaction time, the degradation efficiency of gentian violet increased, and it was up to more than 90% within 1 h when using 5 equiv of PhIO, showing a high catalytic degradation efficiency of the [Fe III (TCPP)]/PhIO system. Generally, the iron(IV)-oxo porphyrin π-cation radical species [Fe IV (O)(porph +• )] was proposed as the active species, which plays a major role in the catalytic oxidation process of iron porphyrin. Kong et al. proposed that an iron(IV)-oxo porphyrin π-cation radical, generated from the heterolytic cleavage of the O-O bond in H 2 O 2 catalyzed by hemin, may play a major role in dye degradation [62]. Meyer et al. investigated the oxidative degradation of azo dyes by [Fe III (tmpyp)] and m-CPBA in aqueous solution at room temperature and suggested that [Fe IV (O)(tmpyp +• )] was capable of oxidatively breaking down the naphthalene core of azo dyes [52]. Thus, based on the previously suggested mechanisms [52,62]   The above experimental results are summarized in Figure 6, which indicated that with the increase in PhIO concentration and reaction time, the degradation efficiency of  and suggested that [Fe IV (O)(tmpyp +• )] was capable of oxidatively breaking down the naphthalene core of azo dyes [52]. Thus, based on the previously suggested mechanisms [52,62]

Conclusions
Various methods have been developed and widely applied for the degradation of gentian violet. However, these traditional methods have certain limitations. The traditional physical method is inefficient for eliminating dyes, especially from dilute solutions. The photo-oxidation method is relatively expensive and not appropriate for the treatment of large flows. Microbial treatment is considered to be an economical and feasible method without secondary pollution, yet biodegradation has the disadvantages of being unstable and time-consuming. Metalloporphyrins, as a representative model of cytochrome P450, have unparalleled catalytic specificity and efficiency, and they can be used as a catalyst to overcome the disadvantages of the above methods used to degrade dyes. In this work, a ferric porphyrin complex, [Fe III (TCPP)], as a cytochrome P450 model, was investigated for its potential to degrade dyes, where [Fe III (TCPP)] in combination with an oxidant, PhIO,

Conclusions
Various methods have been developed and widely applied for the degradation of gentian violet. However, these traditional methods have certain limitations. The traditional physical method is inefficient for eliminating dyes, especially from dilute solutions. The photo-oxidation method is relatively expensive and not appropriate for the treatment of large flows. Microbial treatment is considered to be an economical and feasible method without secondary pollution, yet biodegradation has the disadvantages of being unstable and time-consuming. Metalloporphyrins, as a representative model of cytochrome P450, have unparalleled catalytic specificity and efficiency, and they can be used as a catalyst to overcome the disadvantages of the above methods used to degrade dyes. In this work, a ferric porphyrin complex, [Fe III (TCPP)], as a cytochrome P450 model, was investigated for its potential to degrade dyes, where [Fe III (TCPP)] in combination with an oxidant, PhIO, was used for the degradation of the dye gentian violet. UV-vis absorption spectroscopy was used to monitor the concentration changes in the gentian violet via its UV-vis spectral changes. The [Fe III (TCPP)]/PhIO system provided a high catalytic degradation efficiency, where the degradation efficiency reached more than 90% in 1 h when using 5 equiv of PhIO at 303 K. Thus, as a cytochrome P450 model, iron porphyrins can play a great role in the degradation of dyes, which provides an efficient approach for the treatment of dye-containing wastewater.