Characterization of the glutathione‐dependent reduction of the peroxiredoxin 5 homolog PfAOP from Plasmodium falciparum

Abstract

Peroxiredoxins use a variety of thiols to rapidly reduce hydroperoxides and peroxynitrite. While the oxidation kinetics of peroxiredoxins have been studied in great detail, enzyme‐specific differences regarding peroxiredoxin reduction and the overall rate‐limiting step under physiological conditions often remain to be deciphered. The 1‐Cys peroxiredoxin 5 homolog PfAOP from the malaria parasite Plasmodium falciparum is an established model enzyme for glutathione/glutaredoxin‐dependent peroxiredoxins. Here, we reconstituted the catalytic cycle of PfAOP in vitro and analyzed the reaction between oxidized PfAOP and reduced glutathione (GSH) using molecular docking and stopped‐flow measurements. Molecular docking revealed that oxidized PfAOP has to adopt a locally unfolded conformation to react with GSH. Furthermore, we determined a second‐order rate constant of 6 × 10⁵ M⁻¹ s⁻¹ at 25°C and thermodynamic activation parameters ΔH ^‡, ΔS ^‡, and ΔG ^‡ of 39.8 kJ/mol, −0.8 J/mol, and 40.0 kJ/mol, respectively. The gain‐of‐function mutant PfAOP^L109M had almost identical reaction parameters. Taking into account physiological hydroperoxide and GSH concentrations, we suggest (a) that the reaction between oxidized PfAOP and GSH might be even faster than the formation of the sulfenic acid in vivo, and (b) that conformational changes are likely rate limiting for PfAOP catalysis. In summary, we characterized and quantified the reaction between GSH and the model enzyme PfAOP, thus providing detailed insights regarding the reactivity of its sulfenic acid and the versatile chemistry of peroxiredoxins.

1. INTRODUCTION

Peroxiredoxins are highly versatile thiol‐dependent hydroperoxidases that can also act as redox sensors and chaperones. ¹ , ² , ³ , ⁴ In addition to their subcellular localization, members of the peroxiredoxin family are grouped into three different mechanistic and six major structural subfamilies. ¹ , ⁵ , ⁶ , ⁷ The human malaria parasite Plasmodium falciparum has four peroxiredoxin isoforms, ⁸ one of which, termed antioxidant protein (PfAOP), is a member of the peroxiredoxin 5 subfamily. ⁹ PfAOP is dually localized in the cytosol and the apicoplast of P. falciparum. ¹⁰ Although PfAOP is supposed to protect P. falciparum from hydroperoxides, deletion of the encoding gene was shown to have no effect on the fitness of the asexual blood stages and their susceptibility toward diamide, tert‐butyl hydroperoxide (tBuOOH) or the antimalarial endoperoxide artemisinin. ¹¹ Recombinant PfAOP forms predominantly dimers, has a so‐called 1‐Cys mechanism because it requires only one cysteine residue for catalysis, and has become a model enzyme for glutathione/glutaredoxin‐dependent peroxiredoxins. ¹² , ¹³ , ¹⁴ , ¹⁵ Reduced PfAOP reacts with a hydroperoxide or peroxynitrite, yielding a sulfenic acid enzyme species. ¹² , ¹⁴ Reduced glutathione (GSH) and the glutaredoxin PfGrx are probably the physiological reductants of cytosolic PfAOP in accordance with high peroxidase activities in reconstituted hydroperoxidase assays in vitro. ¹² , ¹³ While GSH is the first reducing agent for the sulfenic acid species of PfAOP, PfGrx deglutathionylates PfAOP and therefore acts as the second reducing agent. ¹² , ¹³ Even though GSH is also present in the apicoplast, the exact reduction mechanism in this organelle remains unknown. Using yeast as a cellular test tube, H₂O₂‐dependent oxidation of heterologous PfAOP in the mitochondrial matrix was shown to directly or indirectly result in the formation of glutathione disulfide (GSSG). ¹⁵ Previous analyses revealed that the active site residue C117 of PfAOP can be glutathionylated in vitro and in Escherichia coli, yielding a mixed PfAOP‐SSG disulfide. ¹² Furthermore, kinetic measurements with recombinant wild‐type and mutant PfAOP, including the gain‐of‐function mutant PfAOP^L109M, suggested that conformational changes and the first reduction of oxidized PfAOP by GSH are rate limiting for catalysis. ¹² , ¹³ However, while the oxidation of PfAOP has been characterized for different substrates and mutants in vitro and in yeast, ¹⁴ the isolated reduction of PfAOP has not been studied to date.

Here, we qualitatively reconstituted and monitored the catalytic cycle of PfAOP in vitro. Furthermore, using stopped‐flow measurements, we quantified the second‐order rate constant and estimated the activation energy and thermodynamic activation parameters ΔH ^‡, ΔS ^‡, and ΔG ^‡ for the rapid GSH‐dependent reduction of oxidized PfAOP, thus providing insights into the reactivity of its sulfenic acid and the transition state of the first reduction step of the catalytic cycle of peroxiredoxins.

2. RESULTS

2.1. Hydroperoxide‐ and glutaredoxin‐dependent reversible glutathionylation of PfAOP

To qualitatively study the proposed hydroperoxidase mechanism for PfAOP (Figure  1a), we reconstituted the reaction with recombinant proteins and monitored it by western blot analyses and gel mobility shift assays (Figure 1b–d). Treatment of reduced wild‐type enzyme (PfAOP^WT) with 1 equiv of tBuOOH and subsequent addition of 1 equiv of GSH yielded PfAOP‐SSG (Figure 1b). PfAOP has two cysteine residues, the peroxidatic residue C117 and the nonessential residue C143. ¹² , ¹³ Comparative studies with PfAOP^C143S and PfAOP^C117S revealed a specific glutathionylation of active site residue C117 (Figure 1c). The glutathionlyation of residue C117 was reversed by the addition of reduced PfGrx^C32S/C88S. This glutaredoxin mutant was used because it is catalytically fully active and has only one cysteine residue so that alternative parallel or subsequent thiol‐disulfide exchange reactions cannot occur. ¹² , ¹⁶ , ¹⁷ The addition of equimolar amounts of PfGrx^C32S/C88S resulted in an almost complete loss of the glutathione signal for PfAOP^C143S‐SSG, while only traces of the mixed disulfide between PfAOP^C143S and PfGrx^C32S/C88S were formed, suggesting a preferential nucleophilic attack of PfGrx^C32S/C88S at the glutathione sulfur atom of PfAOP^C143S‐SSG. Increasing the ratio between PfGrx^C32S/C88S and PfAOP^C143S to 2:1 also resulted in the loss of the glutathione signal but yielded more mixed disulfide between both proteins (Figure 1c). Since the anti‐glutathione antibody did not detect glutathionylated PfGrx^C32S/C88S, we confirmed the transfer of the glutathione‐moiety from PfAOP to the single cysteine residue of PfGrx^C32S/C88S by labeling available thiol groups with a 1.24 kDa methyl ether poly(ethylene glycol)₂₄ maleimide (mmPEG₂₄) (Figure 1d). The thiol group of pure reduced PfGrx^C32S/C88S was modified by mmPEG24 and resulted in an altered SDS‐PAGE mobility as expected. Incubation of PfGrx^C32S/C88S with GSSG or PfAOP‐SSG prevented the modification of PfGrx^C32S/C88S by mmPEG₂₄ in accordance with the formation of PfGrx^C32S/C88S‐SSG. In summary, the reconstituted reaction in vitro supports a stepwise oxidation, glutathionylation, and PfGrx‐dependent deglutathionylation of residue C117 of PfAOP.

FIGURE 1.

In vitro reconstitution of the catalytic cycle of PfAOP. (a) Proposed catalytic cycle for cytosolic PfAOP (E) with GSH and PfGrx (Grx). Helix α2 of PfAOP adopts two different conformations, a folded conformation (fo) with residue C117 facing the active‐site pocket (shown as a dotted circle), and a locally unfolded (lu) conformation with residue C117 facing the protein surface. Oxidized and reduced reactants are shown in red and blue, respectively. The oxidation state of the C117 sulfur atom is indicated for each enzyme species. Enzyme species with an intermediate sulfur oxidation state (−1) are shown in green. (b) GSH glutathionylates recombinant tBuOOH‐oxidized PfAOP^WT as revealed by nonreducing SDS‐PAGE, ponceau‐staining (shown on the left), and western‐blot analysis (shown on the right). A faint glutathionylation signal was also detected following treatment of reduced PfAOP^WT with an excess of GSSG. In contrast to correctly folded PfAOP, denatured tBuOOH‐oxidized PfAOP^WT can form the intramolecular disulfide species PfAOP(S₂). ¹² , ¹³ (c) Reversal of the glutathionylation of tBuOOH‐oxidized PfAOP depends on active‐site residue C117 and the concentration of PfGrx^C32S/C88S. (d) SDS‐PAGE redox mobility shift assays reveal a glutathione transfer from PfAOP to PfGrx^C32/88S. Treatment of reduced PfGrx^C32/88S with GSSG (left side) or with glutathionylated PfAOP^WT (right side) prevented an alkylation by mmPEG₂₄. The calculated molecular masses of PfAOP and PfGrx^C32S/C88S are 22.6 kDa and 13.7 kDa, respectively. Reductions with 2‐mercaptoethanol (2‐ME) served as controls. Band numbering and identified protein species refer to the literature ¹²

2.2. Molecular docking of GSH to fully folded PfAOP

Next, we performed molecular docking experiments to address whether GSH can only reduce the sulfenic acid of PfAOP in the locally unfolded protein conformation (Figure 1a) or whether GSH might also attack the C117 sulfur atom of the fully folded protein. A first round of docking experiments for GSH and the fully folded conformation of hyperoxidized PfAOP^{WT 9} revealed GSH‐C117 sulfur–sulfur distances ≥3.8 Å. Since these distances were much longer than the sulfur–sulfur distances of disulfide bonds around 2.0 Å, we optimized the position of the side chain of C117 by rotating it toward the opening of the active site and performed 10 more simulation runs that resulted in 84 different binding modes. Although PfAOP accommodated GSH in a groove on top of the active site pocket, shortest sulfur–sulfur distances were between 3.1 and 3.3 Å (Figure 2a). Binding energies were between −2 and −5 kJ/mol and did not reveal a favorable GSH orientation. Lower binding energies around −20 kJ/mol were actually obtained for the most unfavorable sulfur–sulfur orientations (Figure 2b). In summary, GSH probably cannot get close enough to attack the sulfur atom of C117 in the fully folded enzyme conformation.

FIGURE 2.

Molecular docking of GSH to fully folded PfAOP. (a) Selection of three different GSH binding modes with the shortest sulfur–sulfur distances (orientation 1–3 from top to bottom). The two subunits of PfAOP are shown as space‐filling models in orange and lilac on the left. GSH and the sulfonic acid of residue C117 are highlighted as ball‐and‐stick models. The electrostatic surface potential is depicted in the middle (red −4.0; white 0; blue +4.0). The sulfur–sulfur distances are shown on the right. (b) Selection of three different GSH binding modes with the lowest binding energies (orientation 4–6 from left to right)

2.3. Direct monitoring of the reaction between GSH and oxidized PfAOP

We previously showed that the oxidation of reduced PfAOP by hydroperoxides can be monitored using stopped‐flow fluorescence measurements. ¹⁴ Here, we applied the same technique to determine the reaction kinetics of the GSH‐dependent first reduction step for recombinant PfAOP (Figure 3). Stopped‐flow kinetics with tBuOOH‐oxidized PfAOP^WT in the first syringe and variable concentrations of GSH in the second syringe revealed an altered tryptophan fluorescence after mixing. Higher GSH concentrations did not affect the total change of fluorescence but accelerated the reaction (Figure 3a). Similar kinetics were observed for the tBuOOH‐oxidized gain‐of‐function mutant PfAOP^L109M, although the total change of fluorescence was smaller than for PfAOP^WT (Figure 3b) (akin to the smaller change of fluorescence during the oxidation of PfAOP^L109M in comparison with PfAOP^{WT 14} ). The altered tryptophan fluorescence of PfAOP was coupled to the reduction of the sulfur atom of C117. This was demonstrated for the inactive mutant PfAOP^C117S, which lacks the active‐site thiol group and, therefore, served as a negative control to exclude unspecific GSH interactions or redox‐independent changes of fluorescence (Figure 3c). The stability of tBuOOH‐oxidized PfAOP^WT on ice was analyzed for up to 3 hr. Assays with 5‐thio‐2‐nitrobenzoate (TNB) as a reducing agent (which reacts with neither the sulfinic nor the sulfonic acid species) revealed that 94 ± 4% of PfAOP^WT was reversibly oxidized by tBuOOH and could be reduced again by TNB. This value did not decrease during the 3‐hr incubation. Furthermore, stopped‐flow measurements with tBuOOH‐oxidized PfAOP and GSH also showed no activity loss when the oxidized protein was stored on ice. Thus, tBuOOH‐oxidized PfAOP was stable enough for subsequent in‐depth test series. Although we could not specify whether reversibly tBuOOH‐oxidized PfAOP comprised just its sulfenic acid species or also a putative cyclic sulfenamide species, we interpret the change of tryptophan fluorescence in accordance with a reaction between GSH and the locally unfolded conformation of the sulfenic acid species. In summary, a redox‐dependent reaction between GSH and the oxidized active site of PfAOP can be directly monitored by stopped‐flow fluorescence measurements.

FIGURE 3.

Stopped‐flow kinetics of the reaction between oxidized PfAOP and GSH. Recombinant reduced PfAOP (2 μM) was oxidized with 1 equiv tBuOOH and subsequently mixed 1:1 with variable concentrations of GSH at pH 7.4 and 25°C. Representative kinetic traces are shown for 1 μM PfAOP^WT with (a) 1 μM GSH or 10 μM GSH. (b) Reaction kinetics between GSH and the gain‐of‐function mutant PfAOP^L109M. (c) Reaction kinetics between GSH and the redox‐inactive negative control PfAOP^C117S

2.4. Kinetic and thermodynamic reaction parameters

We applied the stopped‐flow fluorescence measurements to determine the rate constant for the reaction between GSH and tBuOOH‐oxidized PfAOP^WT (assuming that it is predominantly present in its unfolded conformation as a sulfenic acid species) at five different temperatures between 5 and 25°C. The change of fluorescence within the first second after mixing followed first‐order kinetics and was fitted accordingly, yielding a k _obs value for each GSH concentration. Plotting the k _obs values versus the GSH concentration revealed a linear correlation, which allowed us to calculate temperature‐dependent second‐order rate constants k ₂ from the slopes of the linear fits (Figure 4a and Table 1). These values of up to 6.1 × 10⁵ M⁻¹ s⁻¹ at 25°C were used to calculate the activation energy and pre‐exponential factor A according to Arrhenius theory (Figure 4b) and to estimate the thermodynamic activation parameters ΔH ^‡ and ΔS ^‡ according to Eyring–Polanyi theory (Figure 4c and Table 2). Note that these estimates have to be interpreted with care because they are based on macroscopic measurements in aqueous buffers with a postulated transmission coefficient κ and activity coefficients of 1. The activation energy E _a and the enthalpy of activation ΔH ^‡ were both around 40 kJ/mol. The entropy of activation ΔS ^‡ was close to zero and, therefore, the Gibbs free energy of activation ΔG ^‡ was temperature independent (Table 2). In summary, tBuOOH‐oxidized PfAOP^WT rapidly and directly reacts with GSH with a second‐order rate constant of 6.1 × 10⁵ M⁻¹ s⁻¹ at 25°C, E _a and ΔH ^‡ values around 40 kJ/mol, and a ΔS ^‡ value close to zero, resulting in a temperature‐independent ΔG ^‡ value of 40 kJ/mol.

FIGURE 4.

Arrhenius and Eyring–Polanyi plots for the reaction between oxidized PfAOP and GSH. (a) Temperature‐dependency of the k _obs values for the reaction between variable concentrations of GSH and 1 μM PfAOP^WT that was pre‐oxidized with 1 equiv tBuOOH. The data sets at the indicated temperatures were fitted by linear regression and the slopes yielded the second‐order rate constants k ₂ that are listed in Table 1. (b) The temperature‐dependent rate constants from Table 1 were plotted according to the Arrhenius equation. Following linear regression analysis, the pre‐exponential factor A and the activation energy E _a were calculated from the y‐axis intercept and the slope, respectively. (c) The rate constants from Table 1 were also plotted according to Eyring–Polanyi theory to estimate the thermodynamic activation parameters ΔH ^‡ and ΔS ^‡. The value for ΔS ^‡ was calculated with a transmission coefficient κ = 1. ΔS ^‡ increases to +5.0 J/mol for κ = 0.5. (d–f) Analogous analyses for the reaction between 1 μM oxidized PfAOP^L109M and GSH. Data in panels (a) and (d) were obtained from three or four independent biological replicates

TABLE 1.

Temperature‐dependent second‐order rate constants from Figure 4 for the reaction between oxidized PfAOP and GSH

	PfAOPWT	PfAOPL109M
T (°C)	k 2 (mM−1 s−1)	k 2 (mM−1 s−1)
5	182 ± 3	197 ± 3
10	234 ± 3	276 ± 5
15	337 ± 4	336 ± 7
20	449 ± 5	475 ± 8
25	607 ± 5	652 ± 11
37 a	1,161	1,189

^a Calculated from data in Figure 4.

TABLE 2.

Activation energy and thermodynamic activation parameters from Figure 4 for the reaction between oxidized PfAOP and GSH

	E a	A	ΔH ‡	ΔS ‡	ΔG ‡ @25°C/37°C
T (°C)	(kJ/mol)	(M/s)	(kJ/mol)	(kJ/mol)	(kJ/mol)
PfAOPWT	42.2	1.4 × 1013	39.8	−0.8 × 10−3	40.0/40.0
PfAOPL109M	40.5	7.8 × 1012	38.1	−6.2 × 10−3	39.9/40.0

2.5. Comparison with the gain‐of‐function mutant PfAOP^L109M

Point mutations at the bottom of the active site were shown to affect the enzyme kinetic parameters of PfAOP. While PfAOP^WT and PfAOP^L109M had very similar k _cat ^app/K _m ^app values and second‐order rate constants with hydroperoxide substrates, ¹³ , ¹⁴ k _cat ^app/K _m ^app values of PfAOP^WT with GSH from steady‐state measurements were more than one order of magnitude smaller than for the gain‐of‐function mutant PfAOP^L109M. ¹³ To address a potential effect of the L109M mutation on the direct reaction between tBuOOH‐oxidized PfAOP^L109M and GSH, we determined the k _obs values and compared the second‐order rate constants and thermodynamic activation parameters of PfAOP^L109M with the values and parameters of PfAOP^WT (Figure 4d–f and Tables 1 and 2). The second‐order rate constants and values for E _a, A, ΔH ^‡, ΔS ^‡, and ΔG ^‡ were extremely similar between both data sets. Minor changes of ΔS ^‡ still had no effect on the temperature‐independent ΔG ^‡ value. In summary, the kinetics and thermodynamic activation parameters for the GSH‐dependent reduction of tBuOOH‐oxidized PfAOP^L109M and PfAOP^WT do not indicate significant differences between both enzymes and are in accordance with identical mechanisms. The most likely explanation is that both enzymes already adopted the locally unfolded conformation when they were mixed with GSH.

3. DISCUSSION

The substrate specificity for selected reducing agents remains one of the unresolved topics in peroxiredoxin catalysis. In contrast to 2‐Cys peroxiredoxins, the sulfenic acid of 1‐Cys peroxiredoxins cannot react with a resolving cysteine and could, therefore, be exposed to a variety of physiological thiols. If and how 1‐Cys peroxiredoxins prevent the formation of nonspecific disulfide bonds probably depends on unknown structural and kinetic features. Here, we reconstituted the catalytic cycle for the model enzyme PfAOP and confirmed a stepwise oxidation, glutathionylation, and PfGrx‐dependent deglutathionylation of residue C117. Furthermore, stopped‐flow measurements provided detailed insights into the kinetics and transition state of the GSH‐dependent first reduction of the oxidized enzyme (Figure 5a). While previous steady‐state kinetic measurements showed that the GSH‐dependent first reduction of PfAOP is rate limiting in vitro, they could not discriminate between (a) conformational changes, including local unfolding of helix α2, and (b) the reduction of the sulfenic acid. ¹³ An infinite “true” k _cat value for GSH suggested that the enzyme‐substrate complex cannot be saturated, and previous kinetic as well as structural analysis of the gain‐of‐function mutant PfAOP^L109M with its much lower K _m ^app value for GSH already pointed toward local unfolding of PfAOP as the rate‐limiting step during the catalytic cycle. ¹² , ¹³ Here, we provide four more arguments to support this model. The following interpretations are based on the assumption that tBuOOH‐oxidized enzyme was present in its sulfenic acid state in accordance with the generally accepted catalytic cycle of peroxiredoxins ² , ⁵ , ⁷ and the fact that protein sulfenic acids, for example, in bovine Prx6, can be stable for several minutes or even hours in the absence of thiols or peroxides. ¹⁸ , ¹⁹ , ²⁰ (a) Docking analyses argue against a direct reaction between GSH and residue C117 of the fully folded conformation of PfAOP. Thus, assuming a similar structure of fully folded PfAOP in its sulfenic and sulfonic acid state, ⁹ helix α2 has to partially unfold for the reaction to occur. (b) While bulky TNB was shown to react with bovine Prx6 only in the presence of the detergent sodium dodecyl sulfate (SDS), which is indicative for a buried sulfenic acid, oxidized PfAOP readily reacted with TNB in the absence of SDS in accordance with an accessible sulfenic acid that is exposed in the locally unfolded conformation. (c) The GSH‐dependent k _cat ^app/K _m ^app values of PfAOP^L109M from steady‐state kinetic measurements were roughly 10 times higher than the values for PfAOP^WT, ¹³ whereas the stopped‐flow second‐order rate constants for the reaction between GSH and tBuOOH‐oxidized PfAOP^WT or PfAOP^L109M were almost identical (Table 1). In accordance with the kinetics for PfAOP^WT and PfAOP^L109M, one would expect that residue 109 at the bottom of the active site can affect the stability of the fully folded conformation, and, therefore, influence the K _m ^app value, but the residue should not affect the reactivity of the sulfenic acid in the locally unfolded protein conformation. (d) The stopped‐flow second‐order rate constants for the GSH‐dependent reduction of tBuOOH‐oxidized PfAOP^WT and PfAOP^L109M at pH 7.4 and 25°C were one to three orders of magnitude higher than the k _cat ^app/K _m ^app values from the biphasic GSH‐dependent steady‐state kinetics at pH 8.0 and 25°C (Figure 5a). ¹² , ¹³ Thus, the reaction between the sulfenic acid and GSH is too fast to be rate limiting for this part of the catalytic cycle. In summary, PfAOP first has to undergo a slow conformational change so that the sulfenic acid can rapidly react with GSH (Figure 5a). On the one hand, this explains why glutaredoxin, plasmoredoxin, and thioredoxin could also transfer electrons to oxidized PfAOP in vitro (Figure 1c), ¹² , ²¹ or why oxidized PfAOP was efficiently reduced by fused roGFP2 in yeast. ¹⁴ On the other hand, this raises once again the question if and how 1‐Cys peroxiredoxins prevent the formation of disulfide bonds with other physiological thiols.

FIGURE 5.

Updated model and quantitative assessment of the catalytic cycle of PfAOP. (a) Assignment of measured rate constants. Values for k ₂ and a combination of k ₃ and k ₋₃ are from stopped‐flow measurements at pH 7.4 and 25°C from the literature. ¹⁴ Values for k ₇ are from k _cat ^app/K _m ^app values from steady‐state measurements at pH 8.0 and 25°C from refs. 12, 13. (b) Estimated reaction velocities for the hydroperoxide‐dependent oxidation (v _ox) and GSH‐dependent first reduction (v _red) of PfAOP based on the values from panel (a) and substrate concentrations in vivo and in vitro

Differences in the Gibbs free energy of activation ΔG ^‡ could explain the versatile thiol chemistry and substrate specificity. However, thermodynamic activation parameters for peroxiredoxin catalysis are scarce. Studies on the alkyl hydroperoxide reductase E from Mycobacterium tuberculosis revealed a ΔH ^‡ value for the reduction of H₂O₂ of 20 kJ/mol and an unfavorable ΔS ^‡ value of −80 J/mol, resulting in a ΔG ^‡ value of 44 kJ/mol at 25°C in accordance with a rather moderate second‐order rate constant of 8 × 10⁴ M⁻¹ s⁻¹. ²² The ΔG ^‡ value of 40 kJ/mol for the GSH‐dependent first reduction of PfAOP is temperature independent and smaller in accordance with higher second‐order rate constants (Tables 1 and 2). First‐order rate constants for the disulfide bond formation in different 2‐Cys peroxiredoxins were shown to differ by two orders of magnitude. ²³ , ²⁴ Furthermore, reported second‐order rate constants for the reaction between protein or low‐molecular‐weight sulfenic acids and low‐molecular‐weight thiols such as cysteine or GSH were shown to differ by at least five orders of magnitude. ¹⁸ , ¹⁹ , ²⁵ Our second‐order rate constant of 6 × 10⁵ M⁻¹ s⁻¹ for the reaction between oxidized PfAOP and GSH is on the upper limit of the reported values. This is not a general feature of peroxiredoxins. For example, the reduction of peroxiredoxin 6‐type enzymes usually occurs very slowly in the presence of GSH or other common reducing agents. ⁷ , ²¹ , ²⁶ , ²⁷ , ²⁸ , ²⁹ , ³⁰ Deciphering the underlying cause for the different reactivity will require much more structural and kinetic information. We therefore suggest to compare not only the structures but also the rate constants and thermodynamic activation parameters of alternative combinations of 1‐Cys peroxiredoxins and thiol substrates to better understand this part of the reaction cycle.

Is the hydroperoxide‐dependent oxidation, the GSH‐dependent first reduction, or the Grx‐dependent second reduction of PfAOP faster at physiological substrate concentrations? Rate constants k ₂ for the reaction between reduced PfAOP and hydroperoxides at pH 7.4 and 25°C were between 5 × 10⁶ M⁻¹ s⁻¹ and 3 × 10⁷ M⁻¹ s⁻¹, ¹⁴ which is one or two orders of magnitude higher than the value of k ₅ for GSH (Figure 5a). If we (a) take into account that the enzyme has ping‐pong kinetics with separated half‐reactions, ¹² , ¹³ (b) use estimated intracellular hydroperoxide concentrations between 1 and 10³ nM as well as GSH concentrations between 1 and 10 mM, ³¹ and (c) assume similar steady‐state concentrations of oxidized and reduced PfAOP (which are unknown variables that remain to be determined), the rate constants translate into an estimated reaction velocity ratio ν _red:ν _ox between 20 and 10⁶ (Figure 5b). Thus, at physiological substrate concentrations, the GSH‐dependent first reduction of the locally unfolded oxidized enzyme could be faster than the hydroperoxide‐dependent oxidation of the fully folded reduced enzyme. A more balanced estimated ν _red:ν _ox ratio between 0.2 and 12 is obtained using the same numbers except for a hydroperoxide concentration of 100 μM based on in vitro experiments. ¹² , ¹³ The second‐order rate constant for the PfGrx‐dependent second reduction of cytosolic PfAOP‐SSG is probably in the range between 10⁵ M⁻¹ s⁻¹ and 10⁶ M⁻¹ s⁻¹ in accordance with measured k _cat ^app/K _m ^app values for PfAOP ¹² , ¹³ and PfGrx reaction kinetics with glutathionylated model substrates. ¹⁶ Whether the deglutathionylation can become rate limiting under physiological conditions therefore depends on the PfGrx concentration and the steady‐state concentrations of the involved enzyme species. What also remains to be analyzed for PfAOP catalysis is (a) the reduction of PfAOP‐SSG in the apicoplast, including the identification and characterization of the physiological second reducing agent, and (b) to which degree the macroscopic k _cat ^app/K _m ^app values for hydroperoxides and GSH (which are all much smaller than the second‐order rate constants from the stopped‐flow measurements in Table 1, ¹² , ¹³ , ¹⁴ ) translate into microscopic rate constants that include the rate‐limiting conformational changes of PfAOP. Such insights, for example, from stopped‐flow circular dichroism measurements, might also explain the biphasic kinetic patterns with GSH from steady‐state kinetic measurements ¹² , ¹³ that were absent in the stopped‐flow kinetic measurements (Figure 4).

In conclusion, we generated a reference data set of the thermodynamic activation parameters for the reaction between GSH and a 1‐Cys peroxiredoxin 5‐type model enzyme with implications for the reactivity and substrate specificity of peroxiredoxin sulfenic acids.

4. MATERIALS AND METHODS

4.1. Materials

Recombinant N‐terminally MRGSH₆GS‐tagged PfAOP^WT, PfAOP^C117S, PfAOP^C143S, PfAOP^L109M, and PfGrx^C32S/C88S were produced in Escherichia coli strain XL1‐Blue and purified by Ni‐NTA affinity chromatography as described previously. ¹² , ¹³ , ³² Diethylenetriaminepentaacetic acid (DTPA), tBuOOH, and 1,4‐dithiothreitol (DTT) were from Sigma Aldrich, the anti‐glutathione antibody was from Thermo Fisher Scientific, the goat anti‐mouse antibody‐HRP conjugate was from Bio‐Rad, and mmPEG₂₄ (CAS# 88504‐24‐9) was from Iris Biotech. A 500 mM stock solution of mmPEG₂₄ was prepared in DMSO under inert conditions (N₂). All enzymes and reactants were diluted/dissolved in ice‐cold assay buffer, containing 100 mM Na_xH_yPO₄, 0.1 mM DTPA, pH 7.4.

4.2. Sample preparation and western blot analysis

Freshly purified PfAOP^WT, PfAOP^C117S, PfAOP^C143S, and PfGrx^C32S/C88S were reduced with 5 mM DTT on ice for 30 min. Excess DTT and imidazole were removed on a PD‐10 desalting column (Merck), and the reduced proteins were eluted with 3.5 ml ice‐cold assay buffer. PfAOP concentrations of the eluates, which were usually around 0.2 mM, were determined spectrophotometrically using a molar extinction coefficient ε_280 nm of 21.43 mM/cm (as calculated at http://web.expasy.org/protparam). The reduced proteins were diluted with assay buffer to a final concentration of 20 μM and incubated for 30 min on ice with either buffer, 50 equiv of GSSG, 1 equiv of tBuOOH, or 1 equiv of GSH. Aliquots of tBuOOH‐oxidized PfAOP were subsequently incubated with 1 equiv of GSH for 30 min on ice, and, if indicated, for another 30 min with 0–2 equiv of PfGrx^C32S/C88S. Subsequent redox reactions were blocked by the addition of 20 mM iodoacetamide in Laemmli buffer. The protein samples were heated for 5 min at 95°C, separated by nonreducing SDS‐PAGE, blotted onto PVDF membranes, and stained with ponceau. Destained membranes were subsequently probed with a commercial monoclonal mouse anti‐glutathione antibody (diluted 1:1000) and a commercial goat anti‐mouse antibody‐HRP conjugate.

4.3. Electrophoretic mobility shift assays

Reduced 20 μM PfGrx^C32S/C88S was incubated with either buffer or 1 or 100 equiv of GSSG for 30 min on ice. Reduced 20 μM PfAOP^WT was obtained as outlined above. The protein was either incubated with buffer or was oxidized, glutathionylated, and deglutathionylated by the stepwise addition of 1 equiv of tBuOOH, 1 equiv of GSH, and 1 equiv of PfGrx^C32S/C88S as described above. Subsequent redox reactions were blocked by the addition of the indicated concentrations of mmPEG₂₄ in Laemmli buffer. The protein samples were heated for 5 min at 95°C and separated by nonreducing SDS‐PAGE.

4.4. Stopped‐flow kinetic measurements

PfAOP^WT or PfAOP^L109M was freshly purified, reduced, and desalted in assay buffer as described above. PfAOP^WT or PfAOP^L109M were subsequently diluted to 2.0 μM with assay buffer containing 1 equiv of tBuOOH and incubated on ice for 30 min. Stopped‐flow measurements were performed either at 25°C or at the indicated temperature in a thermostatted SX‐20 spectrofluorometer (Applied Photophysics) with 2.0 μM oxidized enzyme in the first syringe and 2.0 μM to 2.0 mM GSH in assay buffer in the second syringe. The change of fluorescence was measured for 10 s after mixing (total emission at an excitation wavelength of 295 nm with a slit width of 2 mm). The values of three consecutive measurements were averaged and fitted by single exponential regression using the Pro‐Data SX software (Applied Photophysics). Rate constants k _obs were plotted against the substrate concentration in SigmaPlot 13.0 to obtain second‐order rate constants from the slopes of the linear fits. The second‐order rate constants were subsequently plotted and fitted according to Arrhenius and Eyring–Polanyi theory.

4.5. TNB oxidation assay

To assess the stability of tBuOOH‐oxidized PfAOP^WT, the concentration of reversibly oxidized enzyme was quantified with TNB as described previously. ²⁰ , ³³ A solution with 50 μM TNB was freshly generated by adding 25 μM DTT to 25.6 μM 5,5′‐dithiobis(2‐nitrobenzoic acid) in assay buffer. Samples with 20 μM freshly tBuOOH‐oxidized PfAOP^WT were incubated for 0, 30, 60, 120, or 180 min on ice and were subsequently mixed with an equal volume of the 50 μM TNB solution. The reduction of oxidized PfAOP^WT by TNB was monitored at 412 nm (ε = 13.6 mM/cm) using a thermostated Jasco V‐550 UV/vis spectrophotometer at 25°C.

4.6. Molecular docking

A structure data file (SDF) for GSH was downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/compound/124886) and converted in PyMol v2.5.2 into a PDB file. The PDB file for GSH was opened in AutoDockTools v1.5.6 (https://ccsb.scripps.edu/mgltools/downloads) to define flexible bonds and to alter the protonation state in accordance with a physiological pH. The PDB file for PfAOP^WT (entry 1XIY ⁹ ) was also opened in AutoDockTools v1.5.6 to remove all water molecules, add hydrogen atoms, and calculate Kollmann‐charges. The files were stored in PDBQT format and docking was performed with AutoDock Vina v1.1.2. ³⁴ , ³⁵ The grid was centered around the sulfur atom of C117 of PfAOP, and the grid size was set to 15 in each dimension. The exhaustiveness was set to 24. Binding modes with a negative binding affinity were visualized in PyMol (https://pymol.org/2) and Swiss PDB viewer v4.1.0 ³⁶ to determine sulfur–sulfur distances. The electrostatic surface potential of PfAOP was computed with Swiss PDB viewer using atomic partial charges with a dielectric constant of 78.54 for the solvent (Poisson–Boltzmann computation with a dielectric constant of 4 for the protein and a solvent ionic strength of 0.1 M). For a second round of experiments, the side chain of C117 was rotated toward the opening of the active site using the Swiss PDB viewer to further optimize and shorten the sulfur–sulfur distance. The file was converted again to PDBQT format and used for docking as described above.

AUTHOR CONTRIBUTIONS

Robin Schumann: Data curation (equal); formal analysis (equal); investigation (equal); methodology (lead). Lukas Lang: Data curation (equal); formal analysis (equal); investigation (equal); methodology (lead); writing – review and editing (supporting). Marcel Deponte: Conceptualization (lead); data curation (supporting); formal analysis (supporting); funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead).

Schumann R, Lang L, Deponte M. Characterization of the glutathione‐dependent reduction of the peroxiredoxin 5 homolog PfAOP from Plasmodium falciparum . Protein Science. 2022;31(5):e4290. 10.1002/pro.4290