Peroxidase-Catalyzed Oxidative Coupling of Phenols in the Presence

Oxidative coupling processes in subsurface systems comprise a form of natural contaminant attenuation in which hydroxylated aromatic compounds (HACs) ...
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Environ. Sci. Technol. 2002, 36, 596-602

Peroxidase-Catalyzed Oxidative Coupling of Phenols in the Presence of Geosorbents: Rates of Non-extractable Product Formation QINGGUO HUANG, HILDEGARDE SELIG, AND WALTER J. WEBER, JR.* Environmental and Water Resources Engineering, Department of Chemical Engineering, and Department of Civil and Environmental Engineering, The University of Michigan, Ann Arbor, Michigan 48109-2125

Oxidative coupling processes in subsurface systems comprise a form of natural contaminant attenuation in which hydroxylated aromatic compounds (HACs) are incorporated into soil/sediment organic matter matrices. Here we describe the oxidative coupling of phenol catalyzed by horseradish peroxidase (HRP) in systems containing two geosorbents having organic matter of different composition; specifically Chelsea soil, a near-surface geologically young soil having a predominantly humictype soil/sediment organic matter (SOM) matrix, and Lachine shale, a diagenetically older natural material having a predominantly kerogen-type SOM matrix. It was found that each of these two different types of natural geosorbents increased the formation of non-extractable coupling products (NEPs) over that which occurred in solids-free systems. The extent of coupling was higher in the systems containing humic-type Chelsea SOM than in those containing kerogentype Lachine SOM. It was observed that HRP inactivation by free radical attack was significantly reduced in the presence of each geosorbent. A rate model was developed to facilitate quantitative evaluation and mechanistic interpretation of such coupling processes. Experimental rate measurements revealed that the greater extent of reaction observed in the presence of Chelsea soil than in the presence of Lachine shale can be attributed to two factors: (i) more effective protection of HRP from inactivation by the Chelsea SOM and (ii) the greater reactivity of Chelsea SOM with respect to cross-coupling. Interrelationships among enzyme protection, cross-coupling reactivity, and SOM chemistry are discussed.

Introduction Hydroxylated aromatic compounds (HACs) are common constituents of contaminated surface and subsurface aquatic systems. This generally soluble and environmentally mobile class of compounds can be introduced through a variety of agricultural and industrial activities, such as pesticide applications, industrial discharges, and as a result of the incomplete biodegradation of certain other widespread contaminants, e.g., polyaromatic hydrocarbons. A particularly intriguing aspect of the environmental behavior of this class of organic contaminants is that they are able to undergo * Corresponding author phone: (734)763-2274; fax: (734)936-4391; e-mail: [email protected]. 596

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extensive oxidative coupling reactions both among themselves and with other organic substances, reactions mediated by such naturally occurring catalysts as peroxidases, laccases, tyrosinases, and oxides of iron and manganese (1-9). Enhancement of the enzymatic coupling reactions of HACs has in fact been examined as a potentially promising means for both subsurface immobilization of these compounds (1, 2) and their transformation in wastewater treatment processes (10-16). Two principal reaction stages in the peroxidase-catalyzed oxidative coupling of HACs in aqueous solutions can be identified: (i) an enzymatic reaction stage in which phenolic substrates are oxidized to form phenoxyl free radicals and (ii) a post-enzymatic reaction stage in which these phenoxyl radicals may couple with each other (3, 10, 17-22). In the presence of hydrogen peroxide, peroxidase catalyzes the generation of phenoxyl radicals by a catalytic cycle involving several intermediate forms of the enzyme (19, 20). Two phenoxyl radicals are formed, and one H2O2 is consumed upon one catalytic cycle. Soluble coupling products, formed in the post-enzymatic stage, may still serve as phenolic substrates and undergo further oxidative coupling until larger polymers that precipitate from solution are formed. The stoichiometric ratio between phenol and peroxide thus shifts from 2:1 and approaches 1:1 as the polymeric coupling products grow in size (19, 20). In aquatic systems that include such geosorbents as soils and sediments, phenoxyl radicals generated in the enzymatic reaction stage may also cross-couple with soil/sediment organic matter (SOM). Oxidative coupling reactions of this type are indeed known to occur naturally as part of the formation or humification processes of SOM (1). Both selfand cross-coupling reactions are expected to decrease the environmental mobility and toxicity of the compounds by polymer precipitation or by SOM binding (1, 17, 23). Crosscoupling of phenolic contaminants with model dissolved humic SOM precursors has been studied in homogeneous aqueous phases (17, 24, 25), but research relating to such reactions in the presence of soils, sediments, and other geosorbents is limited. The potential influences that natural geosorbents may have on the catalytic behavior of peroxidase and on reaction rates are not clear. It was, therefore, with the objective of clarifying these effects that the present study of phenol coupling catalyzed by horseradish peroxidase (HRP) in the presence of selected natural geosorbents was initiated. The generation of phenoxyl radicals in the enzymatic reaction stage of HRP catalysis has been described by the “Ping-Pong” rate equation (20), which has the general form of a Michaelis-Menten equation for multiple substrates that can be reduced to single-substrate form when all but one is present in excess. HRP-catalyzed phenol coupling processes in aqueous phase are reasonably well described by rate models based on the Ping-Pong equation (19, 20). The postenzymatic reaction rate is generally neglected in these cases, and phenol disappearance is calculated by assuming a unit stoichiometric ratio between H2O2 and phenol. However, this unit ratio cannot be expected to hold for systems in which phenol cross-coupling with soil/sediment SOM may be more prevalent than simple homo-polymer growth. Furthermore, neglect of the post-enzymatic reactions precludes evaluation of the potential effects of SOM reactivity. An additional factor of importance in rates of phenol coupling is enzyme inactivation during reaction. HRP inactivation is believed to occur predominantly through binding with phenoxyl free radicals (10, 19, 26). In the absence of a reductive substrate, inactive enzyme states may also be 10.1021/es010512t CCC: $22.00

 2002 American Chemical Society Published on Web 12/29/2001

TABLE 1. Relevant Properties of Natural Geosorbents Studied sorbent Lachine shale Chelsea soil

surface % organic area, m2/g matter 12.6 3.98

8.27 5.45

particle size

type of organic matter

No soil, suggesting a possible relationship between reaction extent and type of SOM. Because blank controls with denatured enzyme showed that phenol was completely extractable in all systems in the absence of H2O2, the enhanced NEP formation in the presence of the geosorbents can be VOL. 36, NO. 4, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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tion of the enzyme; (ii) self-coupling of radicals; and/or (iii) cross-coupling of radicals with SOMs. In Figure 3, the generation of phenoxyl radicals in the enzymatic reaction step under conditions of excess hydrogen peroxide can be represented by the single substrate Michaelis-Menten equation

rE )

FIGURE 2. Time profiles for HRP inactivation in systems with and without geosorbents. Initial phenol concentration, 50 µM; [E]0 ) 0.1 unit/mL; [H2O2] ) 1 mM; sorbent/water ratio, 7 g/L. Data points are means of three measurements with 1 SD error bars.

kE[E][AH2] KM + [AH2]

where kE represents the enzymatic reaction rate constant, KM is the Michaelis constant, and [E] denotes the total active enzyme concentration. Equation 1 can be further simplified to a first-order rate equation with respect to the enzyme concentration at the conditions of enzyme saturation (i.e. [AH2].KM). The condition of enzyme saturation can be evaluated by the NEP formations at varying phenol substrate concentrations. Our preliminary experiments showed that NEP formations increase with increasing phenol concentration until it levels off indicating the saturation of the enzyme. Enzyme saturation at the experimental conditions employed in this study was confirmed in our preliminary experiments (not described here), so that eq 1 can be converted into eq 2.

rE ) kE[E]

FIGURE 3. Schematic illustration of processes involved in peroxidase-catalyzed phenol coupling. attributed in large part to the increased cross-coupling of phenol with the associated SOMs. The reduction of enzyme inactivation observed in the presence of the geosorbents, an important finding of this study, also appears however to contribute importantly to enhancement of NEP formation. Figure 2 clearly illustrates that HRP inactivation was significantly reduced by the Chelsea soil and Lachine shale relative to that which occurred in the soil-free system. Note in Figure 2 that the order of the residual enzyme activities in the three experimental systems was the same as that for NEP formation; i.e., Chelsea soil > Lachine shale > No soil. This suggests strongly that the greater extent of NEP formation in the presence of geosorbents is at least partially attributable to reduced enzyme inactivation. It is apparent that further elucidation of the roles of geosorbents and other natural materials in reducing enzyme inactivation and/or inducing cross-coupling reactions is essential to development of a detailed understanding of peroxidase-catalyzed phenol coupling in natural and engineering systems. A rate analysis of experimental data and observations based on an appropriate conceptual model can often shed light on such complex processes, and an initial attempt at such an analysis follows. Rate Model Development. A conceptual schematic model of relationships involved in the HRP-catalyzed formation of non-extractable coupling products of HACs in the presence of geosorbents is presented in Figure 3. This model presumes that substrate radicals (AH‚) generated in a reversible enzymatic reaction can participate in several post-enzymatic reactions (30), including: (i) attack and consequent inactiva598

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(1)

(2)

The process of reverse electron transfer, depicted in Figure 3, represents the reverse of the radical generation process. It is known that free radicals, generated in radical-pairs, can easily recombine to form the original molecule (31, 32). This radical-pair recombination represents the reverse of the radical generation process, which releases large amounts of free energy and is geometrically advantaged. Peroxidase catalyzes the generation of radicals involving single-electron transfer from the substrates to the enzymes, by which radicalpair is also formed between the substrate radical and the activated enzyme intermediate (33). The reversibility of the catalytic reaction mediated by peroxidase has, recently, been experimentally proven, and the reversal process is believed to be due to the reverse electron transfer within the radicalpair (33). The process of reverse electron transfer can be described by a first-order rate expression because the partners of the radical-pair are not randomly distributed but right next to each other (31, 34).

rr ) - kr[AH‚]

(3)

It can be further assumed that the reverse electron-transfer process predominates the disappearance of radicals over the radical scavenging in post-enzymatic reactions because of the energy- and geometrical advantages of the radical-pair recombination and also the fact that our experiments were conducted at very low substrate concentrations. Combination of eqs 2 and 3 at steady-state condition gives

[AH‚] )

kE [E] kr

(4)

Equation 4 states that the instantaneous steady-state concentration of phenoxyl radicals generated by HRP catalysis is linearly proportional to the active enzyme concentration under the condition of excess H2O2 and enzyme saturation. As indicated in Figure 3, the phenoxyl radicals can attack and inactivate the HRP enzyme (10), for which an appropriate rate expression can be written as

-

d[E] ) kin[E][AH‚] dt

(5)

As indicated earlier, HRP inactivation can occur via three different mechanisms: (1) radical attack, (2) reaction with H2O2, and (3) sorption by polymeric products. Radical attack would appear to be the principal enzyme inactivation mechanism under the experimental conditions of this study. In one of our preliminary experiments, the enzyme inactivation, at three different H2O2 concentrations (0.5, 1.0, and 1.5 mM) with other conditions identical ([E]0 ) 0.1 unit/mL, [AH2]0 ) 50 µM), follows the same trend. The relative insignificance of H2O2-dependent enzyme inactivation may be attributable to (i) faster enzyme inactivation by phenoxylradical attack than by the H2O2-dependent inactivation (19, 26) and/or (ii) suppression of the H2O2-dependent inactivation in the presence of phenolic substrates due to their competition for the enzyme active sites (19, 21, 22, 35). The third mechanism is not relevant because the amounts of polymeric precipitates formed at the micromolar substrate concentrations used in this study were very small. A second-order rate equation that describes HRP inactivation can then be obtained by substituting eq 4 into 5 to give

d[E] ) k ′in[E]2 dt

(6)

where k in ′ ) kinkE/kr is a combined rate constant related to enzyme inactivation. Enzyme Inactivation Rates. Time profiles for enzyme inactivation at varying initial enzyme activities in systems with and without Lachine shale are shown in Figure 4. Model simulations based on the integral form of eq 6 may be observed to reasonably describe the loss of enzyme activity over time for both soil-free (Figure 4A) and Lachine shalecontaining systems (Figure 4B) under the experimental conditions employed. As shown in Figure 4 (parts A and B), the overall enzyme inactivation rate constant, k ′in, obtained for the Lachine shale-containing systems (0.3 mL/unit-min) is largely reduced in comparison to that for systems without the shale (1.6 mL/unit-min). Reduction of k in ′ was also observed in experiments with the Chelsea soil. According to eq 6, k in ′ is comprised by three intrinsic rate constants (k ′in ) kinkE/kr). The recombination rate constant, kr, should not be affected by the presence of geosorbents because reverse electron transfer occurs predominantly at the radical-pair stage. The enzymatic rate constant, kE, can also be assumed to remain stable upon addition of geosorbents considering that the activity of most soil enzymes tend not to be affected in matrices of geological materials. Relatively stable enzymatic reaction rate constants for peroxidase in systems containing various geosorbents have been reported (36). Reduction of the apparent enzyme inactivation rate constant (i.e, lower k in ′ ) in the presence of geosorbents can therefore be attributed largely to decreases in the intrinsic inhibition rate constant, kin, which suggests that the susceptibility of HRP to phenoxyl-radical attack can be reduced by some protection mechanisms associated with the geosorbents. Similar protection of HRP from inactivation has been found in previous studies with the organic additives poly(ethylene glycol) (PEG) and gelatin (12-14, 18), which have been shown to promote peroxidase efficiency in wastewater treatment applications. Figure 5 shows the variation of k ′in as a function of soil/ water ratios for both Lachine shale and Chelsea soil. It can be seen that k in ′ decreases rapidly upon addition of geosorbents (i.e., enzyme inactivation is reduced), asymptotically approaching minimum values as soil/water ratios increase. This dependency of k ′in on soil/water ratios may suggest that certain forms of association such as ionic or hydrogen bonding between HRP and solution-phase or soil-phase SOM are effective in protecting the enzyme. We assume that the phenoxyl radical attacks a turn-off site, different from the

FIGURE 4. HRP inactivation in the absence (A) and presence (B) of Lachine shale (7 g/L). Initial phenol concentration, 50 µM; [H2O2] ) 1 mM; varying [E]0. Solid lines show model simulations (integral form of eq 6) and symbols are experimental data points. active site, on the enzyme leading to its inactivation. SOM may associate with HRP in a way that the phenoxyl radical, which would otherwise attack the turn-off site, can be blocked or intercepted, so that the susceptibility of enzyme to radical attack is reduced. The dependency of k ′in on soil/water ratios shown in Figure 5 is similar in trend to that observed for the protection effects of organic additives (18), suggesting that enzymes may be protected by organic additives and geosorbent SOM in a similar fashion. This in fact seems logical in that HRP, a peptide-like molecule, can form complexes by hydrogen bonding or electrostatic forces with hydroxyl or carboxylic groups contained in either the organic additives or the SOMs associated with the geosorbent materials. Enzyme Protection Mechanisms. The role of natural materials in protecting HRP from inactivation can be further illustrated by examining the observed effects of variable soil/ water ratios. Variations in the overall enzyme inactivation rate constant, k in ′ , may be related to the formation of certain HRP-SOM associations

E + SOM T E - SOM VOL. 36, NO. 4, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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mL/unit-min for Lachine shale and Chelsea soil at soil/water ratio ) 30 g/L were employed. NEP Formation Rates and SOM Effects. As illustrated in Figure 3, phenoxyl radicals can form non-extractable products (NEPs) by either self-coupling or cross-coupling processes. However, the NEP formation by self-coupling may be largely suppressed in the presence of geosorbents due to the fact that the cross-coupling reaction, in effect, terminates the growth of polymer. This suppression may be especially significant at relatively low enzyme concentration with low radical productivity because the self-coupling reaction is second-order dependent on the phenoxyl radical concentration. If we assume that the cross-coupling reaction dominates the formation of NEP, the rate of NEP formation can be described in terms of a first-order rate equation

FIGURE 5. Variation of k ′in as a function of sorbent/water ratios for Lachine shale and Chelsea soil. Initial phenol concentration, 50 µM; [E]0 ) 0.1 unit/mL; [H2O2] ) 1 mM. Solid lines show model simulations by the k ′in expression given in eq 13 and symbols are experimental data points. such that the total enzyme, ET , is distributed into two forms (i.e., uncomplexed or free form, Ef , and complexed form, Es)

[ET] ) [Ef] + [Es]

(8)

which, at equilibrium, are interrelated by

[Es] ) β[Ef][SOM]

(9)

where β is the formation constant. If radical attack is the main enzyme inactivation mechanism, the inactivation rates of both Ef and Es can still be represented by eq 5, but the rate constants are expected to be different for each form. After substituting the expression for the steady-state radical concentration given in eq 4, the inactivation rates for Ef and Es can be written as

-

d[Ef] ) k ′in,aq [Ef][ET] dt

(10)

-

d[Es] ) k ′in,so[Es][ET] dt

(11)

where k in,aq ′ and k ′in,so are inactivation rate constants for the free and complexed enzymes, respectively. By adding eqs 10 and 11 and incorporating the distributions given in eqs 8 and 9, we obtain an expression relating the overall enzyme inactivation rate constant to the soil organic matter concentration; i.e.,

d[ET] ) k ′in [ET]2 dt

-

(12)

and

k in ′ )

(

)

k ′in,aq + k ′in,so β[SOM] 1 + β[SOM]

(13)

As illustrated in Figure 5, k ′in decreases as soil/water ratios increase, suggesting that more enzymes become protected by complexation. When the soil/water ratio reaches a certain level (Figure 5), the complexed form of the enzyme becomes dominant and k in ′ approaches k ′in,so. Model simulations using the expression for k in ′ given in eq 13 describe this trend very well (solid line in Figure 5). For this simulation, the value of k in,aq ′ ) 1.6 mL/unit-min determined for soilfree systems, and the respective k ′in,so values of 0.25 and 0.15 600

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d[NEP] ) kc[AH‚] dt

(14)

where [NEP] stands for the concentration of non-extractable product expressed as a phenol equivalent concentration in solution and kc is the cross-coupling rate constant. It should be mentioned that when phenoxyl radical production is higher (e.g., at higher enzyme activities), or when SOM concentrations are relatively low, the self-coupling process may become important. In such cases the reaction order may be greater than one, and eq 14 may not be valid. After substituting the free radical expression given in eq 4 into eq 14 and combining it with eq 12, an expression for NEP formation as a function of time of the integral form

[NEP] )

k ′c [ln(1 + [ET]0k ′int)] k ′in

(15)

is obtained, where k c′ ) kckE/kr is a combined rate constant related to the coupling rates and [ET]0 is the initial enzyme activity. Figure 6 presents time profiles for NEP formation in the presence of Lachine shale (Figure 6A) and Chelsea soil (Figure 6B) at different initial enzyme activities. It is shown in this figure that the simulations given by eq 15 reasonably well describe the NEP formation rates observed in experiments at two initial enzyme activities ([E]0 ) 0.05 and 0.1 unit/mL) chosen for purposes of illustration. The difference in the k ′c values obtained for the experiments with Lachine shale and Chelsea soil (i.e., 2.9 ( 0.5, and 4.5 ( 0.01 µM-mL/unit-min, respectively) suggests that the characteristics of the SOM may have an effect on coupling. Differences in k ′c can be translated to different intrinsic reactivities (kc) for crosscoupling between phenoxyl radicals and different natural materials, because, as elaborated earlier, kE and kr were relatively constant for our experimental systems. The rate analysis conducted in this study reveals that differences in NEP formation and in enzyme protection effects exist between the two geosorbents studied. Chelsea soil has a stronger HRP-protection effect than does Lachine shale, as indicated by a lower inactivation rate constant for complexed enzyme, k in,so ′ (Table 2). In addition, Chelsea soil has a higher reactivity with phenoxyl radicals, as suggested by a larger cross-coupling rate constant (i.e., higher k ′c ) than for Lachine shale. These observations are consistent with fundamental differences in the SOM chemistry and reactivity of the two geosorbents. Lachine shale, a diagenetically mature material, contains relatively reduced and chemically inert SOM. Chelsea soil SOM, on the other hand, is comprised of younger humic-type organic matter containing a variety of functional groups (e.g., carboxylic, phenolic, and amino). These functionalities may activate the organic matter by electronic or resonance effects so that the humic-type SOM should be more susceptible to phenoxyl-radical attack and

as successful, however, in describing all of the data, and they led to several irresolvable conflicts in logic. Given the complexity of the system studied, and the unknowns and unresolved issues that yet remain, it would be premature to conclude that inclusion of the “reverse electron transfer” concept is a completely valid approach to analysis of such reactions under all conditions. There is need for further verification of certain relevant issues, and we are pursuing this in ongoing work in our laboratories. These and similar efforts in other laboratories will hopefully lead to a more conclusive assessment of the modeling approach advanced here.

Acknowledgments We thank the three anonymous reviewers for their thoughtful comments and critiques; one reviewer in particular provided a thorough critique of the rate analysis and a number of constructive suggestions. Three EWRE graduate students at Michigan also are acknowledged for their contributions: T. Michael Keinath for valuable discussions and measurements of the surface area of the materials, and Deborah Ann Ross and Carl W. Lenker for experimental assistance. This research was supported in part under Grant No. DE-FG07-96ER14719 Environmental Management Science Program, Office of Science and Technology, Office of Environmental Management, United States Department of Energy (DOE). The opinions, findings, conclusions, or recommendations expressed herein are those of the authors, however, and do not necessarily reflect the views of DOE.

Literature Cited

FIGURE 6. Time profiles for phenol coupling at different initial enzyme activities ([E]0 ) 0.05 and 0.10 unit/mL) in the presence of Lachine shale (A) and Chelsea soil (B). Initial phenol concentration, 50 µM; [H2O2] ) 1 mM; sorbent/water ratio, 7 g/L. Solid lines show model simulations (eq 15), and data points are means of three replicates with 1 SD error bars.

TABLE 2. Enzyme Inactivation and NEP Formation Rate Constants k ′in,so (mL/unit-min) k ′c (µM-mL/unit-min)

Lachine shale

Chelsea soil

0.25 2.9

0.15 4.5

subsequent attachment, resulting in a greater degree of crosscoupling reactivity. The stronger HRP-protection effect of humic-type SOM may also be attributed to the functionalities. The hydrophilic functional groups of the humic-type SOMs may have a greater tendency to complex with HRP, so that the enzyme can be protected more efficiently. Moreover, the humic-type SOM in complex with HRP is expected to be more effective, due to its higher cross-coupling reactivity, in intercepting phenoxyl radicals that would otherwise bind to the enzyme, resulting in lower susceptibility of the enzyme to radical attack. A major difference between the rate analysis presented here and more traditional analyses of peroxidase-catalyzed reactions is that the “reverse electron transfer” concept, an important recent development (33), is incorporated. This concept has proven instrumental in interpreting our data. Attempts to model the data without invoking the “reverse electron transfer” concept were successful in capturing certain aspects of the observed rate behavior. They were not

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Received for review January 8, 2001. Revised manuscript received October 5, 2001. Accepted October 30, 2001. ES010512T