Dye-Decolorizing Peroxidase of Streptomyces coelicolor (ScDyPB) Exists as a Dynamic Mixture of Kinetically Different Oligomers

Dye-decolorizing peroxidases (DyPs) are heme-dependent enzymes that catalyze the oxidation of various substrates including environmental pollutants such as azo dyes and also lignin. DyPs often display complex non-Michaelis–Menten kinetics with substrate inhibition or positive cooperativity. Here, we performed in-depth kinetic characterization of the DyP of the bacterium Streptomyces coelicolor (ScDyPB). The activity of ScDyPB was found to be dependent on its concentration in the working stock used to initiate the reactions as well as on the pH of the working stock. Furthermore, the above-listed conditions had different effects on the oxidation of 2,2′-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) and methylhydroquinone, suggesting that different mechanisms are used in the oxidation of these substrates. The kinetics of the oxidation of ABTS were best described by the model whereby ScDyPB exists as a mixture of two kinetically different enzyme forms. Both forms obey the ping-pong kinetic mechanism, but one form is substrate-inhibited by the ABTS, whereas the other is not. Gel filtration chromatography and dynamic light scattering analyses revealed that ScDyPB exists as a complex mixture of molecules with different sizes. We propose that ScDyPB populations with low and high degrees of oligomerization have different kinetic properties. Such enzyme oligomerization-dependent modulation of the kinetic properties adds further dimension to the complexity of the kinetics of DyPs but also suggests novel possibilities for the regulation of their catalytic activity.


■ INTRODUCTION
Dye-decolorizing peroxidases (EC 1.11.1.19,DyPs) are enzymes that catalyze the oxidation of various substrates using H 2 O 2 as an electron acceptor.Although the physiological substrate has not been identified yet, these peroxidases are known to oxidize anthraquinone and azo dyes, 1 β-carotene, 2 and different phenolic compounds 3 including lignin.There has been a growing interest in DyPs due to their biotechnological potential in bioremediation of industrial dyes and valorization of lignin. 4o date, over 50 different DyPs have been characterized. 5,6−10 All DyPs adopt a similar dimeric ferredoxin-like fold consisting of β-sheets and peripheral α-helices, distinct from well-known peroxidases, such as horseradish peroxidase. 11−14 Heme is held in the heme cavity, positioned in the C-terminal domain of the monomer, where it is ligated by a proximal conserved histidine residue. 9,15eing able to degrade bulky textile dyes and even polymeric lignin, DyPs differ from classic peroxidases. 4,8,9,16However, like other heme peroxidases, DyPs obey the ping-pong kinetic mechanism, by cycling between the ferric resting state and its high-valent intermediates compound I (Cpd I) and compound II (Cpd II). 8 Smaller reducing substrates as well as the H 2 O 2 cosubstrate can access the heme through channels that connect heme with the protein exterior. 17,18To catalyze the oxidation of substrates with a bulkier size, DyPs are known to apply longrange electron transfer pathways.In this case, the electrons are first transferred from the reducing substrate to the solventaccessible amino acid residues at the surface (usually Trp or Tyr residues) from where they are further transferred to the high-valent intermediate of heme. 19,20ike for other heme peroxidases, the general ping-pong kinetic mechanism of DyPs is well studied and generally accepted.At the same time, the molecular mechanisms behind the non-Michaelis−Menten kinetics that are often observed with DyPs and characterized by the presence of substrate inhibition 18,21−26 and cooperative effects 15,22,27,28 remain largely unknown.The oligomeric state of DyPs varies from monomers to hexamers.Most of the fungal DyPs are monomeric; 1,22,29−33 however, the existence of dimers has also been described. 2,34It has been suggested that loop insertions and the increasing complexity of fungal DyPs favor the monomeric form by hindering the oligomerization. 11,35In contrast to their fungal counterparts, the bacterial DyPs exist mostly as oligomers, including dimers, 11,28,35−41 tetramers, 26,42,43 pentamers, 44 and hexamers. 11,45,46−53 The DyP from Thermomonospora curvata was shown to exist as a mixture of dimer, tetramer, and octamer. 27Furthermore, many bacterial DyPs are often loaded as cargo proteins into the encapsulin nanoparticles, 45,46,53−55 where they exist at an even higher degree of oligomerization. 54Although it has been shown that the DyP−encapsulin complex has higher lignin degrading activity compared to nonencapsulated DyP 45 and that monomeric forms may be deficient in the binding of heme, 50,52,53 little is known about the relations between the degree of oligomerization of DyPs and their catalytic activity.
Here, we performed an in-depth kinetic characterization of the subfamily B DyP of the bacterium Streptomyces coelicolor (ScDyPB) along with analyses of its size distribution.Our results suggest that ScDyPB exists as a mixture of enzyme forms with different degrees of oligomerization, which differed in their catalytic properties.

■ MATERIALS AND METHODS
ABTS (lot no.SLBT0759) and methylhydroquinone MHQ (lot no.BCBH9920 V) were purchased from Sigma-Aldrich.BSA (lot no.K00113-2235, Fraction V) was obtained from GE Healthcare.The concentration of the H 2 O 2 stock solution (Honeywell, lot # SZBG2070) was determined spectrophotometrically at 240 nm using an extinction coefficient of 39.4 M −1 cm −1 . 56Dilutions of a H 2 O 2 stock solution were prepared in water before use.Milli-Q (mQ) ultrapure (type 1) water was used in all experiments.
Recombinant ScDyPB.Dye-decolorizing peroxidase from S. coelicolor A3(2) (ScDyPB, UniProtKB Q9FBY9) was overexpressed in Escherichia coli BL21 (DE3), purified, and reconstituted with hemin as described in Pupart et al. 23 The concentration of ScDyPB was determined by the absorbance of the heme at 406 nm using an extinction coefficient of 100,000 M −1 cm −1 or by the absorbance at 280 nm using an extinction coefficient of 18,450 M −1 cm −1 .The concentration of the enzyme storage stock was 18.1 ± 0.6 μM (based on the absorbance at 406 nm) and 39.0 ± 1.6 μM (based on the absorbance at 280 nm).In all experiments, the enzyme was dosed based on its concentration measured by the absorbance of heme.ScDyPB was stored at −80 °C as frozen aliquots in 20 mM Tris-HCl (pH 7.5) containing 0.1 M NaCl.ScDyPB working stocks were made by the dilution of the storage stock to the appropriate buffer containing 0.1 g L −1 BSA and 0.1 M NaCl.
Measuring the Activity of ScDyPB.ABTS and MHQ were used for the kinetic characterization.If not stated otherwise, the activity measurements were performed in 50 mM sodium acetate (pH 4.0) in a spectrophotometer (Shimadzu UV-1900i UV−vis) cuvette at 25 °C in a total volume of 1.0 mL.The oxidation of ABTS was measured by the increase in the absorbance at 420 nm (ε 420 = 36,000 M −1 cm −1 ) and that of MHQ by the increase in the absorbance at 251 nm (ε 251 = 21,450 M −1 cm −1 ). 57Reactions were started by the addition of the enzyme from its working stock to a cuvette containing a mixture of the reducing substrate and H 2 O 2 .The nonenzymatic oxidation of substrates was measured in the experiments without the enzyme, and all activity measurements were corrected for this background.If not stated otherwise, the oxidation of ABTS was measured using 1 mM ABTS and 100 μM H 2 O 2 , and the oxidation of MHQ was measured using 1 mM MHQ and 1 mM H 2 O 2 .The oxidation of textile dyes reactive blue 4 (RB4) and 19 (RB19) was tested using 0.15 μM ScDyPB, 0.1 mM, dye, and 1.0 mM H 2 O 2 in 50 mM NaAc (pH 4.0) at 25 °C.Oxidation of the RB4 was measured by the decrease of the absorbance at 610 nm using an ε 610 of 4200 M −1 cm −1 and by that of RB19 at 595 nm using an ε 595 of 10,000 M −1 cm −1 .The data were analyzed by using STATISTICA 8.0 and GraphPad Prism 5 software.
Inactivation of ScDyPB by H 2 O 2 .1.5 μM ScDyPB in 50 mM NaAc (pH 4.0) or 20 mM Tris-HCl (pH 7.5) (both supplemented with 0.1 g L −1 BSA) was preincubated with H 2 O 2 (0−5 mM) at 25 °C.At selected times, a 10 μL aliquot was withdrawn and added to the cuvette containing 990 μL of the mixture of 1 mM ABTS and 0.1 mM H 2 O 2 and the activity was measured by the increase in the absorbance at 420 nm.
Gel Filtration Chromatography.A Superdex 75 Increase 10/300 GL column (GE Healthcare) was equilibrated with 20 mM Tris-HCl and 0.1 M NaCl, pH 7.5.100 μL of 18.1 μM ScDyPB was injected, and the column was eluted with equilibration buffer at a flow rate of 0.5 mL min −1 .Elution of the proteins was monitored by the absorbance at 280 nm.The high-molecular weight gel filtration calibration kit (Cytiva) contained ovalbumin (43 kDa), conalbumin (75 kDa), aldolase (158 kDa), and ferritin (440 kDa).The standard proteins were dissolved in Tris-HCl buffer (20 mM, pH 7.5, supplemented with 0.1 M NaCl) and used for calibration.
Dynamic Light Scattering.Dynamic light scattering (DLS) analyses were performed using a Zetasizer Nano S particle size analyzer (Malvern Panalytical) with a constant 173 C scattering angle at 25 °C and the laser wavelength of 633 nm.Before DLS analysis, the buffers (20 mM Tris-HCl, pH 7.5, 0.1 M NaCl, or 50 mM NaAc mM, pH 4.0) were filtered through a sterile syringe filter with 0.22 μm pore size.The sample volume used for analysis was 80 μL, and the concentration of ScDyPB was 1.5 μM.The scattering intensity data were processed using instrumental software to obtain the hydrodynamic diameter (D h ) and the size distribution of scatters in each sample.
Spectra of ScDyPB.The UV−vis absorption spectra of ScDyPB were recorded at 250−700 nm using a Shimadzu UV-1900i UV−vis spectrophotometer at 25 °C.After recording the spectrum of 3.6 μM ScDyPB in 4 mM Tris-HCl (pH 7.5, supplemented with 20 mM NaCl) in 0.5 mL total volume, the pH was brought to 4.0 by the addition of 26 μL of 1.0 M NaAc (pH 4.0) and spectra were recorded again.Finally, 150 μL of 1.0 M Tris-HCl (pH 7.5) was added, and spectra were recorded.

■ RESULTS
Measuring the Activity of ScDyPB.Kinetic characterization of ScDyPB was performed using two reducing substrates: a conventional peroxidase substrate, 2,2′-azinodi(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), and a phenolic substrate, methylhydroquinone (MHQ).ABTS is a one electron-donating substrate, and its oxidation can be followed by the absorbance of the ABTS cation radical product (ABTS +• ) at 420 nm.MHQ is a two electron-donating substrate, and its oxidation can be followed by the absorbance of the methylquinone (MQ) product at 251 nm (Scheme 1).
All peroxidase reactions were carried out in a spectrophotometer cuvette at 25 °C.Reactions were started by the addition of the enzyme from its working stock to the cuvette containing the mixture of the reducing substrate and H 2 O 2 , and all rates correspond to the initial rates (measured between 30 and 60 s, Figure 1A,B).The pH optima of the oxidation of both substrates were around 4 (Figure 1C), and all further activity measurements were made in 50 mM sodium acetate (NaAc), pH 4.0.
Dependency of the Activity on the Concentration of ScDyPB.As expected for the enzyme-catalyzed reactions, the rate of the oxidation of both ABTS and MHQ scaled linearly with the concentration of the ScDyPB in the cuvette (Figure 2A,B).However, we found that the activity of ScDyPB was dependent on its concentration in the working stock used for the initiation of the enzyme reactions in the cuvette as well as on the pH of the working stock.Furthermore, the presence and the direction of these effects were dependent on the substrate used for the activity measurements.With ABTS as the substrate, the activity of the enzyme from the working stock made in NaAc pH 4.0 was about 1.5−2.5-fold(depending on the concentration of ScDyPB in the working stock) higher than that from the working stock made in Tris pH 7.5 (Figure 2A).The opposite was true for the activity measured with MHQ where the reactions that started from the working stock made in NaAc pH 4.0 had about sixfold lower activity compared to those made in Tris pH 7.5 (Figure 2B).
With ABTS as the substrate, we made a series of activity measurements using different ScDyPB concentrations in the working stock.The activity seemed to reach a plateau value with an increasing concentration of ScDyPB in the working stock (Figure 2C).An apparent half-saturating concentration of ScDyPB in the working stock made in 20 mM Tris pH 7.5 was about 0.38 μM, whereas the corresponding figure for the working stock made in 50 mM NaAc pH 4.0 was below 0.03 μM (Figure 2C).We note that in all cases, the concentration of ScDyPB in the cuvette was 15 nM.The simplest explanation for this phenomenon would be a nonspecific binding of ScDyPB to the laboratory plastics, like microcentrifuge tubes used for preparing enzyme working stocks.The results of the preliminary experiments, indeed, suggested that such nonspecific binding exists but not in the presence of BSA that was present (at 0.1 g L −1 ) in the working stocks of ScDyPB used in all experiments shown in this work.Furthermore, one may expect that the nonspecific binding to laboratory plastics would have similar effects on the activities measured using different  substrates such as ABTS and MHQ, which was clearly not the case in this study (Figure 2A,B).
The higher activity measured in the reactions that started from the working stocks with higher enzyme concentrations can be explained by the enzyme being active as an oligomer.In the case of reversible binding, the relative concentration of the oligomeric form is expected to increase with an increasing total concentration of the enzyme.However, the relaxation to a possible binding equilibrium between different oligomeric states must be slow enough not to be achieved during the activity measurements in the cuvette.The results of several control experiments made using different preincubation times of ScDyPB working stocks suggested that the establishment of the possible new equilibrium upon the dilution of ScDyPB working stocks was relatively slow (compared to the time frame of the activity measurement).Furthermore, higher activity was observed when ABTS was present in preincubation of the ScDyPB working stock (Figure S1).These results suggest that equilibrium between different possible oligomeric forms of ScDyPB that has been established in its working stock is at least partly retained during the activity measurement in the cuvette.Since higher total enzyme concentration is expected to favor the association, the higher activity observed in the case of higher concentration of ScDyPB in its working stock (Figure 2C) suggests that higher oligomeric forms have higher ABTS oxidizing activity.If that is the case, the lower pH of the working stock seems to favor the higher oligomeric forms of ScDyPB (Figure 2A,C).Although there was little dependency between the MHQ oxidizing activity and the concentration of ScDyPB in its working stock, the lower activity observed with working stocks made at pH 4.0 (compared to those made in pH 7.5) (Figure 2B) suggests that contrary to the ABTS, the higher oligomeric forms have lower MHQ oxidizing activity.We also tested the oxidation of two textile dyes, RB4 and RB19, by ScDyPB (using 0.1 mM dye and 1.0 mM H 2 O 2 ), but the activity with these substrates was low with apparent rates of 0.66 ± 0.04 and 0.58 ± 0.02 s −1 for RB4 and RB 19, respectively.
Effects of Additives on the Activity of ScDyPB.As shown above, the pH of the ScDyPB working stock had differential effects depending on which substrate, ABTS or MHQ, was used for the activity measurement (Figure 2A,B).
Here, we tested the effects of different additives (ammonium sulfate, DMSO, methanol, Tween-20, and ethylene glycol) in the ScDyPB working stock (1.5 μM ScDyPB in 20 mM Tris-HCl pH 7.5 supplemented with 0.1 g L −1 BSA and 0.1 M NaCl) to the ABTS and MHQ oxidizing activity.Among the compounds tested, only the presence of ammonium sulfate in the working stock of ScDyPB had a significant effect on the activity (Figure 3).The presence of 1.0 M ammonium sulfate in the working stock resulted in about a twofold increase in  ABTS oxidizing activity, whereas there was about a fourfold decrease in the MHQ oxidizing activity (Figure 3).Control experiments made with 0.01 M ammonium sulfate in the cuvette showed no effect on the activity (data not shown), confirming that the observed effects were caused by the presence of ammonium sulfate in the working stock of ScDyPB.The opposite effects of ammonium sulfate on the activity with ABTS and MHQ corroborate with the effects of the pH of the working stock (Figure 2A,B) and suggest that different oligomeric forms of ScDyPB may be responsible for the oxidation of ABTS and MHQ.
Kinetics of the Oxidation of ABTS.All experiments described above were performed using ABTS and H 2 O 2 concentrations of 1.0 and 0.1 mM, respectively.Here, we performed experiments with varied concentrations of ABTS (0.01−3.0 mM) and H 2 O 2 (0.01−1.0 mM).The concentration of ScDyPB in the cuvette was 15 nM, and the reactions were started by the addition of ScDyPB from the working stock with 1.5 μM ScDyPB in 20 mM Tris pH 7.5 (supplemented with 0.1 g L −1 BSA and 0.1 M NaCl).The dependency of the rates on the concentration of ABTS shows substrate inhibition by ABTS with the effect being more prominent at low concentration of H 2 O 2 (Figure 4A, see Figure S2A for the zoom-in of the data at the region of low H 2 O 2 concentrations).Substrate inhibition by one substrate at a low concentration of the other substrate is a kinetic signature of the enzymes obeying a ping-pong kinetic mechanism such as heme peroxidases.There was no substrate inhibition by H 2 O 2 at any concentration of ABTS (Figure 4B).However, when incubated with H 2 O 2 in the absence of ABTS, the ScDyPB was irreversibly inactivated (Figure S3).The rate of inactivation increased with increasing concentration of H 2 O 2 , and the second order rate constants of 4.9 ± 0.3 and 6.2 ± 0.9 M −1 s −1 were found for H 2 O 2 -driven inactivation of ScDyPB at pH 4.0 and 7.5, respectively (Figure S3).
When the concentration of H 2 O 2 was treated as a variable, the rate of ABTS oxidation was well in line with the simple Michaelis−Menten equation (Figure S2B and eq S1).
However, the dependency of apparent parameters for H 2  ) on the concentration of ABTS (Figure 4C−E) was more complex than expected for ping-pong peroxidase kinetics with substrate inhibition by ABTS.The dependency of k cat app (Figure 4C) on [ABTS] does not follow simple saturation with ABTS according to the hyperbola (eq S2) but shows a drop in the parameter value between 0.1 mM and 0.2 mM ABTS.Furthermore, an apparent k cat /K M(Hd 2 Od 2 ) does not approach zero with increasing [ABTS] (eq S3) but levels to a constant value of 65.7 ± 2.7 mM −1 s −1 after the initial drop with increasing [ABTS] (Figure 4E).When the concentration of ABTS was treated as a variable, both the Michaelis−Menten equation and the equation accounting for the substrate inhibition failed to describe the kinetics.The characteristic feature of the kinetics of ABTS oxidation was a slight drop or retardation in the rates observed around ABTS concentrations of 0.1−0.2mM, which was followed by the increase in rates with a further increase in [ABTS].This kinetic phenomenon was best revealed in the series made at higher H 2 O 2 concentrations (Figure 4A).
The simplest kinetic mechanism that can account for the above-described phenomenon assumes the enzyme to be active as two independent, kinetically different forms.One form of the enzyme (form II, E II ) is a subject of substrate inhibition by ABTS, whereas the other form (form I, E I ) follows the Michaelis−Menten saturation kinetics.Assuming that the two forms are independent, the rate equation can be written as a sum of the two steady-state rate equations (eq 1) In eq 1, the enzyme forms I and II and corresponding kinetic parameters are designated with superscripts I and II, respectively.The enzyme form I follows ping-pong kinetics, whereas form II follows ping-pong kinetics with substrate inhibition by ABTS.This equation was able to account for experimentally observed kinetic peculiarities, like a "kink in the curve" observed after 0.1 mM ABTS (Figure 4A).Global nonlinear regression analysis of the data in Figure 4A  I values of 59 ± 3 s −1 , 1.0 ± 0.12 mM, and 0.89 ± 0.06 mM, respectively (for the fit, see Figure S4A).However, because of the interdependency between the parameters, the values of the kinetic parameters for enzyme form II came with a large standard deviation.Precise determination of the parameter values for two different enzyme forms apparently assumes measurements under the experimental conditions where one of the forms is predominant and the contribution by the other is insignificant.Since the activity of ScDyPB was higher (with 1.0 mM ABTS and 0.1 mM H 2 O 2 ) when the reactions were started from the working stock made in 50 mM NaAc pH 4.0 (Figure 2C) instead of 20 mM Tris pH 7.5, we also tested the 1.5 μM ScDyPB working stock made in pH 4.0 in making the series with varying [ABTS] and [H 2 O 2 ].Although the general activity was higher, apparent biphasic kinetics persisted also in these experiments, suggesting that ScDyPB existed in two kinetically different forms also in 50 mM NaAc pH 4.0 (data not shown).
In order to evaluate the relative abundancy of enzyme forms, we further assume that the two enzyme forms have the same values of kinetic parameters and they differ only by the presence of substrate inhibition in the case of form E II .In this case, eq 1 simplifies to Global nonlinear regression analysis of the data in Figure 4A according to eq 2 predicted the relative abundancy of forms I ([E I ]/[E] 0 ) and II (1 − [E I ]/[E] 0 ) of 0.18 ± 0.01 and 0.82 ± 0.01, respectively.The estimates of common parameter values for both forms were 327 ± 28 s −1 , 0.95 ± 0.09 mM, and 0.89 ± 0.06 mM, for k cat , K M(ABTS) , and K M(Hd 2 Od 2 ) , respectively.The estimate of the K i(ABTS) was 4.12 ± 0.36 μM.Despite having two parameters less, the fitting according to eq 2 was not significantly worse than that according to eq 1 with R 2 values of 0.9957 and 0.9962, respectively (for the comparison of fits, see Figure S4A,B).The high k cat value obtained from the analysis according to eq 2 is a result of the low abundancy of the nonsubstrate inhibited form.Despite strong substrate inhibition, the enzyme form E II has significant contribution to the overall activity at [ABTS] below 0.1 mM (Figure S4C,D).ScDyPB Exists in Oligomeric Forms with Different Sizes.The results of the kinetic studies described above suggested that ScDyPB may exist as a mixture of different oligomeric forms.Here, we analyzed the size distribution of ScDyPB using gel filtration chromatography and dynamic light scattering (DLS).Analysis using a Superdex-75 column showed that at pH 7.5, the ScDyPB eluted as two peaks.A dominant peak with an apparent molecular weight of 91 kDa and a smaller peak eluted close to the void volume of the column (MW of about 450 kDa) (Figure 5A).Considering the molecular weight of ScDyPB of 34 kDa, the dominant peak corresponds to an apparent trimer.The shoulder in the region of the expected elution of the ScDyPB monomer suggests that the dominant peak may correspond to the mixture of monoand trimeric ScDyPB.Unfortunately, the gel permeation chromatographic analysis at pH 4.0 but also at pH 7.5 but in the presence of 1.0 M ammonium sulfate was not possible.At pH 4.0, ScDyPB precipitated at high concentrations necessary for this analysis (14.5 μM) and at pH 7.5, but in the presence of 1.0 M ammonium sulfate, the elution of ScDyPB was retarded because of the interaction with the column matrix.
DLS analyses were also applicable at pH 4.0 and 7.5 in the presence of 1.0 M ammonium sulfate.In the case of all conditions tested, the ScDyPB existed as a complex mixture of particles with different sizes with the diameter ranging from 9 nm to more than 4 μm.However, although intensity-based size distribution revealed the presence of large aggregates (Figure 5B), the relative contribution of ScDyPB engaged in these aggregates was less than 1% as judged by the volume-based size distribution analysis (Figure 5C).The majority of ScDyPB appeared in the population of oligomers with an average size of around 10 nm (Figure 5C and Table 1).The lowest average size of ScDyPB oligomers was observed at pH 7.5 followed by pH 4.0 and pH 7.5 but in the presence of 1.0 M ammonium sulfate (Figure 5B,C and Table 1).Although the average size of oligomers depends on which distribution, intensity, or volume was used for the size calculation, the trends were the same.Addition of ammonium sulfate led to the increase in the average size of ScDyPB oligomers, and higher oligomers were observed at pH 4.0 compared to pH 7.5 (Table 1).
We also recorded the UV−vis spectra of ScDyPB at pH 7.5 and pH 4.0 (Figure S5).At both pH values, there was a clear absorbance of the Soret band around 400 nm, characteristic for heme proteins.Changing the pH from 7.5 to 4.0 resulted in an increase in the absorbance at all wavelengths, but the effect was more prominent at shorter wavelengths, suggesting the contribution of the light scattering.This observation is corroborated by the higher abundancy of large particles observed in DLS spectra at pH 4.0 compared to pH 7.5 (Figure 5B,C).Of note, the pH-dependent changes in the UV−vis spectrum were reversible as the adjustment of pH from 4.0 back to 7.5 restored the initial absorbance spectrum measured at pH 7.5 (Figure S5).

■ DISCUSSION
DyP peroxidases often display complex, non-Michaelis− Menten kinetics with substrate inhibition by H 2 O 2 , 21,24,25 by the reducing substrate, 18,22,23 or by both. 22An apparent positive cooperativity with the reducing substrate has also been observed with many DyP peroxidases. 15,22,27,28Kinetics of heme peroxidases are well studied, and they obey a ping-pong kinetic mechanism.Catalysis is initiated by the binding of H 2 O 2 to the heme in its resting state (Fe 3+ ) followed by twoelectron oxidation of the heme and formation of the reactive intermediate known as compound I (Cpd I). 58Cpd I is reduced back to the resting state via two consecutive oneelectron transfer steps from the reducing substrate.−61 The latter strategy is used in the oxidation of bulky substrates that cannot pass through the heme access channel(s).
Substrate inhibition is a phenomenon that is often observed with enzymes obeying a ping-pong kinetic mechanism, and it happens when substrates bind to the "wrong form" of the enzyme.In the case of DyP peroxidases, it assumes the binding of the reducing substrate to the enzyme resting state in a way that competes with the binding of H 2 O 2 .The substrate inhibition by H 2 O 2 occurs when H 2 O 2 binds to Cpd I and restricts its reduction by the reducing substrate.The ScDyPB studied here was substrate-inhibited by ABTS but not by H 2 O 2 (Figure 4A,B).Analysis of the structure of ScDyPB (PDB: 4GU7) reveals the presence of one heme access channel, a propionate pocket with a bottleneck radius of 2.45 Å that can possibly accommodate ABTS (Figure 6A).The heme access channel of ScDyPB is similar to the well-studied DtpB of the bacterium Streptomyces lividans. 62Thus, it seems plausible that substrate inhibition of ScDyPB involves the entrance of the ABTS through the propionate pocket and binding to the heme resting state.Inactivation of ScDyPB upon incubation with H 2 O 2 (Figure S3) in the absence of ABTS suggests that H 2 O 2 can interact with Cpd I.Such binding would compete with the direct binding of ABTS to the heme but not with the possible binding to the surface site.In latter case, the oxidation of ABTS would be kinetically favored by the factor of more than 10 4 (k cat app /K M(Hd 2 Od 2 ) app for the oxidation of ABTS of 65.7 ± 2.7 mM −1 s −1 Figure 4E, versus rate constant for inactivation by H 2 O 2 of 0.0049 ± 0.0003 mM −1 s −1 , Figure S3) and provides possible explanation for the absence of substrate inhibition by H 2 O 2 .
Characteristic to the enzyme catalyzed reactions, the rate of the oxidation of ABTS and MHQ was proportional to the concentration of the ScDyPB in the reaction (Figure 2A,B).However, we found that the rates of ABTS oxidation were dependent on the concentration of ScDyPB in its working stock used to initiate the reactions.Higher rates were observed at higher concentrations of ScDyPB in the working stock (Figure 2C).The in-depth kinetic characterization of the oxidation of ABTS (Figures 4 and S4) suggests that ScDyPB exists as a mixture of at least two kinetically different enzyme forms, with one being inhibited by ABTS but not the other one.Size exclusion chromatography and DLS analyses revealed that ScDyPB, indeed, exists as a complex mixture of oligomers and aggregates with different sizes (Figure 5).Furthermore, the lower pH (pH 4.0 versus 7.5) and supplementation with ammonium sulfate seemed to favor a higher degree of oligomerization (Figure 5B,C and Table 1).It is also important to note that while ScDyPB working stocks made at pH 4.0 resulted in higher ABTS oxidizing activity when compared to the working stocks made at pH 7.5 (Figure 2A), the opposite was true for the oxidation of MHQ (Figure 2B).Similarly, the presence of 1.0 M ammonium sulfate in the working stock of ScDyPB increased the ABTS oxidizing activity but decreased Calculated based on the size distribution of intensity (Figure 5B) and volume (Figure 5C).Mean sizes are calculated from at least four DLS scans, and error bars show SD.
the MHQ oxidizing activity (Figure 3).Collectively, these results suggest that different mechanisms are used in the oxidation of ABTS and MHQ and that these mechanisms are differently affected by the degree of oligomerization of ScDyPB.
The simplest mechanism that would explain the results of this study is depicted in Figure 6B, and it relies on the four following assumptions.(i) ScDyPB exists as a mixture of two enzyme forms (we note that assuming just two enzyme forms may be an oversimplification, but including more forms leads to the overparametrization of the rate equations that do not permit quantitative analyses) with different degrees of oligomerization.One form has a high degree of oligomerization (HDO form), whereas the other form has a low degree of oligomerization (LDO form).(ii) ABTS can be oxidized at the surface binding site through the long-range electron transfer, but there is no such possibility with smaller substrate MHQ.Oxidation of MHQ takes place in a direct contact with Cpd I (note that there is no need to exclude this possibility for the oxidation of ABTS).(iii) Substrate inhibition by ABTS involves the binding of ABTS to the heme resting state competing with the binding of H 2 O 2 and Cpd I formation.(iv) Direct access of ABTS and MHQ to the heme and Cpd I is possible only with the LDO form of ScDyPB, but H 2 O 2 has access to the heme of ScDyPB in its both LDO and HDO forms.
Relative contribution of the HDO form increases with an increasing concentration of ScDyPB in its working stocks.Since the direct access of reducing substrates to heme in the HDO form is blocked, the substrate inhibition by ABTS is relieved, while the oxidation through long-range electron transfer is unaffected.The net result is increased ABTS The heme is shown as a stick model.Nitrogen, oxygen, and iron atoms are colored blue, red, and brown, respectively.The inset shows the heme access channel of ScDyPB, the propionate pocket (left).The ScDyPB hexamer (trimer of dimers), with dimers shown in different colors and the approximate dimensions of the hexamer (right).(B) Simplest mechanism of the catalysis by ScDyPB that explains the experimental observations of this study.ScDyPB exists as an equilibrium (slow compared with the time frame used for the activity measurements) of the enzyme forms with low (LDO form) and high degrees of oligomerization (HDO form).For the simplicity of visualization of the concept, the LDO-and HDO forms are represented by the monomer and dimer, respectively (in a real system, the degree of oligomerization of both forms is much higher).In their resting state (RS), both forms of the enzyme can be oxidized by H 2 O 2 to form an active intermediate, compound I (Cpd I).In the case of ABTS as the reducing substrate, the RS is restored by the long-range electron transfer to Cdp I.For MHQ, there is no surface binding site, and Cpd I is reduced via direct electron transfer to the heme iron.The heme access channel is assumed to be inaccessible for the reducing substrate in the enzyme in its HDO form.Therefore, the oxidation of MHQ occurs only with the enzyme in its LDO form.Substrate inhibition by ABTS results from the nonproductive binding of ABTS to the heme (NP) and is possible only with the enzyme in its LDO form.Thus, the conditions that favor the HDO form of ScDyPB will decrease the MHQ oxidizing activity through blockage of the access to the heme and increase ABTS oxidizing through relieving substrate inhibition.Note that possible substrate inhibition by MHQ and oxidation of ABTS through direct electron transfer do not change the general outcome of the model and are omitted for simplicity.
oxidizing activity with increasing concentration of ScDyPB in its working stocks (Figure 2).Without long-range electron transfer, as proposed for the MHQ substrate, the blockage of the heme access channel in the HDO form will abolish the MHQ oxidizing activity, and it decreases with increasing concentration of ScDyPB in its working stocks (Figure 2).As indicated by the increased average size of ScDyPB oligomers, the lower pH of the ScDyPB working stock as well as the presence of ammonium sulfate in it also seems to increase the relative concentration of the HDO form (Table 1).The result is increased ABTS but decreased MHQ oxidizing activity in the experiments with the ScDyPB working stock made at pH 4.0 (Figure 2) but also at pH 7.5 in the presence of 1.0 M ammonium sulfate (Figure 3).
Long-range electron transfer involves accessible Trp or Tyr residues at the surface of the enzyme.There are two Trp and five Tyr residues in ScDyPB, and all of them, except Tyr 141, are in the solvent-accessible surface.Although the use of surface binding site(s) by ScDyPB remains to be experimentally validated, its ability to oxidize bulky dyes RB4 and RB19 and polymeric lignin 23 suggests the presence of this possibility.The core dimensions of a ScDyPB (PDB: 4GU7) dimer in the crystal structure are 6 × 8 × 4 nm 3 .Assessment of the structure of ScDyPB using PISA analysis revealed that the most probable multimeric state of ScDyPB would be a hexamer (trimer of dimers) with a buried surface area of 23,290 Å 2 (27.5% of the total surface area of six monomers, Figure 6A).The hexamer had a diameter of 8 nm × 8 nm × 9 nm (Figure 6A).The maximum particle dimensions D max and the radius of gyration R g are 86 and 27 Å for the dimer and 99 and 36 Å for the hexamer, respectively, as calculated with GNOM based on the theoretical solution small-angle X-ray scattering curves generated with FoXS. 63,64Thus, the ScDyPB population with the diameter of around 10 nm observed in DLS scans (Figure 5B,C and Table 1) may well correspond to a hexamer.However, considering the heterogeneity of enzyme populations revealed in consecutive DLS scans and relatively big differences between intensity-and volume distribution-based sizes (Table 1), identification of this population as a dimer cannot be excluded.Since more than 99% of ScDyPB appears in this peak (Table 1), it is evident that both LDO and HDO forms are merged to this single peak.Unfortunately, our data do not enable us to derive the sizes of LDO and HDO forms separately as we see only an average size of the mixture of different oligomers (Table 1).All in all, the structural as well as biophysical analyses (Figure 5) suggest that ScDyPB exists as a mixture of enzyme populations with different degrees of oligomerization.Our results suggest that lower pH (pH 4.0 compared to pH 7.5) supports the formation of the HDO form.The most probable candidate for changing its protonation state upon shifting from pH 7.5 to 4.0 would be a histidine with a pK a value of 6.0−7.0.Although there is one His residue (His 137) in the contact area of adjacent ScDyPB monomers, this residue is not involved in salt bridge formation.Increased contribution of the HDO form in the presence of 1.0 M ammonium sulfate suggests that hydrophobic interactions may be involved in the formation of oligomers.An important assumption of our model was that the heme access channel is not accessible for the reducing substrates, ABTS and MHQ, in the HDO form of ScDyPB (Figure 6B).Similar to the observations made with the oligomerization of other DyPs, 11,35 the heme access channel in the ScDyPB hexamer is not located in the subunit interface and seems to be accessible.However, a possible blockage of heme access channels upon formation of oligomers with a higher degree of oligomerization that were observed in both gel filtration and DLS analyses (Figure 5) cannot be excluded.Further studies are needed to judge the plausibility of the assumptions underlying the mechanism in Figure 6B.
The biological role of DyP peroxidases and the nature of their native reducing substrates are not known.Many DyP peroxidases are secreted as cargo proteins in the interior of the shell made from encapsulins. 45,46,53,54As an example, the DyP of Mycobacterium smegmatis is loaded into the encapsulin shell as a dodecamer made from two hexamers. 54However, there are no encapsulin-coding genes in the genome of S. coelicolor, suggesting that oligomerization of ScDyPB is not related to the packing into encapsulin nanoparticles.Although the exact mechanism and biological role remain to be revealed, the enzyme oligomerization-dependent modulation of the kinetic properties observed here expands the complexity of the kinetics of DyP peroxidases but also suggests novel possibilities for the regulation of their catalytic activity.
Effects of preincubation time of ScDyPB working stocks before the measurement of the activity with ABTS, kinetics of the oxidation of ABTS, inactivation of ScDyPB by H 2 O 2 , comparison of the fitting of the data of ABTS oxidation according to eqs 1 and 2, contribution of the enzyme forms EI and EII, and absorbance spectra of ScDyPB (PDF)

Figure 1 .
Figure 1.Dependency of initial rates of reducing substrate oxidation on pH by ScDyPB.The time curves of the oxidation of (A) ABTS and (B) MHQ.The pH values are given in the plot.(C) Dependency of the initial rates (measured between 30 and 60 s) of the oxidation of ABTS and MHQ on pH.All reactions were performed at 25 °C.Buffers were 50 mM sodium citrate (for pH 3.0 and 3.5), 50 mM sodium acetate (for pH 4.0−5.0),and 50 mM Bis−Tris−HCl (for pH 5.5 and 6.0).Reactions were initiated by the addition of the ScDyPB from its working stock (1.5 μM ScDyPB in 20 mM Tris-HCl pH 7.5 supplemented with 0.1 g L −1 BSA and 0.1 M NaCl) to the cuvette containing the mixture of the substrate and H 2 O 2. The final concentration of the ScDyPB in the cuvette was 15 nM.The oxidation of ABTS was measured using 1 mM ABTS and 100 μM H 2 O 2 .The oxidation of MHQ was measured using 1 mM MHQ (note that MHQ preparation had a high background absorbance at pH 6.0) and 1 mM H 2 O 2 .Data are presented as average values (n = 3, independent experiments), and error bars show SD.For clarity, the error bars are not shown for the traces in panels (A) and (B).

Figure 2 .
Figure 2. Dependency of the activity on the concentration of ScDyPB in the cuvette and in its working stock.All reactions were performed in NaAc buffer (50 mM, pH 4.0) at 25 °C.The oxidation of ABTS was measured using 1 mM ABTS and 100 μM H 2 O 2 and the oxidation of MHQ with 1 mM MHQ and 1 mM H 2 O 2 .The rates of oxidation of (A) ABTS and (B) MHQ at different concentrations of ScDyPB in the cuvette.The concentration of ScDyPB in its working stock and the pH of the working stock are shown in the figure.Solid lines show the linear regression of the data.(C) Dependency of the ABTS oxidizing activity of ScDyPB on its concentration in the working stock.The pH of the ScDyPB working stock is shown in the figure.The concentration of the ScDyPB in the cuvette was 15 nM.The working stocks of ScDyPB, with concentrations of 30 nM−5 μM, were prepared in 20 mM Tris pH 7.5, 0.1 M NaCl, or 50 mM NaAc pH 4.0 buffers (both supplemented with 0.1 g L −1 BSA).Data are presented as average values (n = 3, independent experiments), and error bars show SD.

Figure 3 .
Figure 3. Effects of different additives in the working stock of ScDyPB on the ABTS and MHQ oxidizing activity.Prior to the activity measurements, the ScDyPB working stock (1.5 μM ScDyPB in 20 mM Tris-HCl pH 7.5 supplemented with 0.1 g L −1 BSA and 0.1 M NaCl) was incubated for 30 min at 25 °C in the presence of different compounds as indicated in the figure.The activity was measured in 50 mM NaAc at pH 4.0 using 15 nM ScDyPB (100-fold dilution of the working stock to the cuvette).The activity was measured using 1 mM ABTS and 100 μM H 2 O 2 or 1 mM MHQ and 1 mM H 2 O 2 .Data are presented as average values (n = 3, independent experiments), and error bars show SD.

Figure 4 .
Figure 4. Kinetics of the oxidation of ABTS by ScDyPB.Dependency of initial rates of the oxidation of ABTS on the concentration of (A) ABTS and (B) H 2 O 2 and the dependency of apparent parameters (C) k cat app , (D) K M(Hd 2 Od 2 ) app , and (E) k cat app /K M(Hd 2 Od 2 ) app on the concentration of ABTS.Reactions were made in 50 mM NaAc (pH 4.0) at 25 °C.The concentration of ScDyPB was 15 nM, and the reactions were initiated by the addition of ScDyPB from the working stock with 1.5 μM ScDyPB in 20 mM Tris pH 7.5 (supplemented with 0.1 g L −1 BSA and 0.1 M NaCl).Solid lines in (A) and (B) show the global nonlinear regression of the data according to eq 2. The concentration of the substrate that has been kept constant within the series is indicated in the plot.The values of apparent kinetic parameters for H 2 O 2 shown in panels (C−E) were derived by nonlinear regression analysis of the data in panel (B) according to the Michaelis−Menten equation (eq S1, for the fit, see Figure S2B).Data are presented as average values (n = 3, independent experiments), and error bars show SD.
according to eq 1 predicted the [E I ]k cat I /[E] 0 , K M(ABTS) I , and K M(Hd 2 Od 2 )

Figure 5 .
Figure 5. Analysis of the size distribution of ScDyPB.(A) Gel filtration chromatogram of ScDyPB in 20 mM Tris-HCl (pH, 7.5) (supplemented with 100 mM NaCl).Black squares show the elution volume of standard proteins: ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (44 kDa).The red line shows linear regression analysis of the mobility of the standard proteins used for calibration.The red square shows the expected elution volume of the ScDyPB monomer.(B, C) Dynamic light scattering (DLS) analysis of 1.5 μM ScDyPB in 50 mM NaAc pH 4.0, in 20 mM Tris pH 7.5, and in 20 mM Tris pH 7.5 supplemented with 1.0 M ammonium sulfate (as indicated in the plot).Size distribution based on the intensity (B) or volume (C).Traces show the average of at least four consecutive scans.

Figure 6 .
Figure 6.Structure of ScDyPB and simplest possible mechanism of the formation of kinetically different forms of the enzyme.(A) Structure of ScDyPB (PDB: 4GU7).The structure of the ScDyPB monomer, where αhelices, β-sheets, and loops are colored blue, pink, and tan, respectively.The heme is shown as a stick model.Nitrogen, oxygen, and iron atoms are colored blue, red, and brown, respectively.The inset shows the heme access channel of ScDyPB, the propionate pocket (left).The ScDyPB hexamer (trimer of dimers), with dimers shown in different colors and the approximate dimensions of the hexamer (right).(B) Simplest mechanism of the catalysis by ScDyPB that explains the experimental observations of this study.ScDyPB exists as an equilibrium (slow compared with the time frame used for the activity measurements) of the enzyme forms with low (LDO form) and high degrees of oligomerization (HDO form).For the simplicity of visualization of the concept, the LDO-and HDO forms are represented by the monomer and dimer, respectively (in a real system, the degree of oligomerization of both forms is much higher).In their resting state (RS), both forms of the enzyme can be oxidized by H 2 O 2 to form an active intermediate, compound I (Cpd I).In the case of ABTS as the reducing substrate, the RS is restored by the long-range electron transfer to Cdp I.For MHQ, there is no surface binding site, and Cpd I is reduced via direct electron transfer to the heme iron.The heme access channel is assumed to be inaccessible for the reducing substrate in the enzyme in its HDO form.Therefore, the oxidation of MHQ occurs only with the enzyme in its LDO form.Substrate inhibition by ABTS results from the nonproductive binding of ABTS to the heme (NP) and is possible only with the enzyme in its LDO form.Thus, the conditions that favor the HDO form of ScDyPB will decrease the MHQ oxidizing activity through blockage of the access to the heme and increase ABTS oxidizing through relieving substrate inhibition.Note that possible substrate inhibition by MHQ and oxidation of ABTS through direct electron transfer do not change the general outcome of the model and are omitted for simplicity.

Table 1 .
DLS Analysis of the Mean Size and Relative Contribution of ScDyPB Oligomers a