Molecular Mechanism of Oxidative Stress Perception by the Orp1 Protein*

In this study we investigated the molecular mechanism by which the Orp1 (Gpx3) protein in Saccharomyces cerevisiae senses and reacts with hydrogen peroxide. Upon exposure to H2O2 Orp1Cys36 forms a disulfide-bonded complex with the C-terminal domain of the Yap1 protein (Yap1-cCRD). We used 4-nitrobenzo-2-oxa-1,3-diazole to identify a cysteine sulfenic acid (Cys-SOH) modification that forms on Cys36 of Orp1Cys36 upon exposure to H2O2. Under similar conditions, neither Cys82 of Orp1Cys82 nor Cys598 of Yap1 forms Cys-SOH. A homology-based molecular model of Orp1 suggests that the structure of the active site of Orp1 is similar to that found in mammalian selenocysteine glutathione peroxidases. Proposed active site residues Gln70 and Trp125 form a catalytic triad with Cys36 in the Orp1 molecular model. The remainder of the active site pocket is formed by Phe38, Asn126, and Phe127, which are evolutionarily conserved residues. We made Q70A and W125A mutants and tested the ability of these mutants to form Cys-SOH in response to H2O2. Both mutants were unable to form Cys-SOH and did not form a H2O2-inducible disulfide-bonded complex with Yap1-cCRD. The pKa of Cys36 was determined to be 5.1, which is 3.2 pH units lower than that of a free cysteine (8.3). In contrast, Orp1 Cys82 (the resolving cysteine) has a pKa value of 8.3. The pKa of Cys36 in the Q70A and W125A mutants is also 8.3, demonstrating the importance of these residues in modulating the nucleophilic character of Cys36. Finally, we show that S. cerevisiae strains with ORP1 Q70A and W125A mutations are less tolerant to H2O2 than those containing wild-type ORP1. The results of our study suggest that attempts to identify novel redox-regulated proteins and signal transduction pathways should focus on characterization of low pKa cysteines.

(HO ⅐ ), and alkyl hydroperoxides (ROOH). For example, exposure to the prevalent environmental toxicant arsenic induces oxidative stress in the form of increased levels of ROS (6,7). If allowed to accumulate unchecked, these molecules exceed the normal antioxidant buffering capacity of the cell, leading to indiscriminate damage of cellular components, including DNA, proteins, and lipids (8 -11).
Recent studies have demonstrated that ROS, such as hydrogen peroxides and alkyl hydroperoxides, can regulate signal transduction pathways, transcription factors, and gene expression in a variety of prokaryotic and eukaryotic organisms (12)(13)(14)(15)(16)(17)(18)(19). Of particular relevance to this work are the common cysteine thiol switches that can regulate the biological function of proteins (20,21). For example, the Escherichia coli transcription factor OxyR contains two conserved cysteine residues that are essential for oxidant perception and regulation (22). Upon exposure to H 2 O 2 , Cys 199 of OxyR reacts with H 2 O 2 to form a transient Cys-SOH intermediate (23). The Cys 199 Cys-SOH immediately reacts with Cys 208 and forms a disulfide bond, which results in increased DNA binding affinity and OxyR target gene expression.
The budding yeast Saccharomyces cerevisiae is a valuable eukaryotic model for understanding the molecular and biochemical mechanisms involved in H 2 O 2 -regulated signal transduction pathways (24). The major H 2 O 2 response pathway in S. cerevisiae involves the transcription factor Yap1 and the oxidant receptor protein Orp1 (12,13,(25)(26)(27). The Yap1 oxidative stress response pathway is regulated by a novel disulfide bondrelay cascade and controls the expression of ϳ70 genes in response to H 2 O 2 (28,29). Orp1, which is homologous to glutathione peroxidase enzymes (hGpx), is responsible for H 2 O 2 perception and subsequent catalysis of disulfide bond formation in Yap1 (12). The proposed peroxidase mechanism for Orp1 involves the initial H 2 O 2 reaction with Cys 36 (12). Based on the homology of Orp1 with selenocysteine containing hGpx enzymes, it has been proposed that Orp1 forms a sulfenic acid intermediate upon reaction with peroxides. Ultimately the Cys-SOH intermediate is resolved by Cys 82 to form an intramolecular disulfide bond. This disulfide bond is then reduced via the thioredoxin pathway (12). For this reason, this class of non-selenocysteine hGpx-like enzymes has been classified as thioredoxin-dependent peroxidases or thiol peroxidases, with a similar function and mechanism to the peroxiredoxin family of proteins (30). The role of Orp1 in regulation of Yap1 involves the reaction of Cys 598 in Yap1 with Cys 36 resulting in a mixed disulfide bond complex. This complex has been captured in vivo through site-directed mutagenesis of the resolving Cys 303 residue in Yap1 (12). The mixed disulfide is then resolved by Cys 303 of Yap1 to form an intramolecular disulfide bond in Yap1. The Yap1 disulfide bond results in the masking of a nuclear export sequence in Yap1, nuclear accumulation of Yap1, and increased gene expression of Yap1 target genes (18,28).
The molecular mechanism by which the Orp1 class of peroxiredoxins perceives hydrogen peroxide and alkyl hydroperoxide has not been characterized. In this study we demonstrate that upon reaction with H 2 O 2 , Orp1 Cys36 forms a transient Cys-SOH on Cys 36 . Our data suggest that the reactivity of Cys 36 is because of its extremely low pK a of 5.1. We also present a homology model of the Orp1 structure, which indicates that it has an active site structure similar to mammalian glutathione peroxidase. Mutation of the putative Gln 70 -or Trp 125 -active site residues to alanine abolishes H 2 O 2 -induced Cys 36 Cys-SOH formation and reverts the pK a of Cys 36 to 8.3.
Protein Cloning, Expression, and Purification-The ORP1 gene was cloned from S. cerevisiae genomic DNA, subcloned into the pRSET vector, and purified as described previously (13). Orp1 single and double point mutants were made using standard PCR-based mutagenesis. The peroxidase activity was measured following established methodology (13). The Yap1-cCRD protein was designed and purified as described previously. 3 Briefly, Cys 598 of Yap1 can then react with the Cys 36 -SOH intermediate to form a mixed disulfide-bonded complex between Orp1 and Yap1 and ultimately results in the inhibition of a nuclear export sequence in Yap1 (18,28). This complex has been identified with mass spectrometry in vivo (12). Additionally, a C310A/C315A/C620A/C629T mutant form of Yap1 has been shown to form disulfide bonds in vivo in a manner similar to wild-type Yap1 (12). These data indicated to us that a truncated Yap1 protein with only Cys 598 could serve as a useful tool in examining the interaction of Yap1 with H 2 O 2 -activated Orp1. After purification, all proteins were dialyzed into 25 mM Tris (pH 7.5), 100 mM NaCl, and 1 mM EDTA and stored at Ϫ80°C.
Orp1 and Yap1-cCRD Interaction Assay-Prior to use, Orp1 and Yap1-cCRD proteins were reduced with 50 mM DTT for 2 h at 25°C. After reduction, each protein was exchanged into 25 mM Tris buffer (pH 7.5), 100 mM NaCl, and 1 mM ETDA using a Hi-Trap desalting column (GE Healthcare) operating on an AKTA purifier fast protein liquid chromatograph. The protein concentrations of Yap1-cCRD and Orp1 proteins were determined using the BCA assay (Pierce). Yap1-cCRD and Orp1 were mixed together in equal molar ratios to a final concentration of 4 M in a total reaction volume of 100 l. As controls, reactions containing Yap1-cCRD or Orp1 alone were prepared. All reactions were initiated with the addition of 8 M H 2 O 2 and allowed to react for 1 min. Reactions were stopped with the addition of trichloroacetic acid and precipitated on ice for 20 min. Precipitated proteins were pelleted with centrifugation and resuspended in 100 mM Tris (pH 8.0), 1% SDS, and 20 mM iodoacetamide. Resuspended samples were incubated in the dark for 30 min at 25°C, and 4ϫ SDS-PAGE buffer was added. Reactions were incubated at 95°C for 5 min, separated by nonreducing or reducing SDS-PAGE, and proteins visualized with Coomassie Brilliant Blue.
Detection of Cysteine Sulfenic Acid Modifications with NBD-Cl-NBD-Cl was used to detect the formation of Cys-SOH on purified proteins in response to H 2 O 2 exposure (31). 100 mM stocks of NBD-Cl were prepared in Me 2 SO and stored in the dark at Ϫ20°C. All experiments were performed aerobically. Each protein was reduced with 50 mM DTT for 2 h at 25°C. After reduction, proteins were exchanged into 25 mM potassium phosphate (pH 6.0) and 1 mM ETDA using a Hi-Trap desalting column (GE Healthcare) operating on an AKTA purifier fast protein liquid chromatograph. Proteins were concentrated to a final concentration of 40 M with an Amicon Ultra-4 (10-kDa cutoff). 200-l oxidation reactions were initiated with the addition of H 2 O 2 followed immediately with NBD-Cl, to a final concentration of 80 and 250 M, respectively. Reactions were incubated for 1 h at 25°C, and unreacted NBD-Cl was separated from protein with a 5-ml Hi-trap desalting column. NBD-Cl-labeled proteins were concentrated to 40 M and analyzed with UV-visible spectroscopy on a Varian Cary 50 spectrophotometer.
pK a Determination of Orp1 Sulfhydryls with PDT-Bimane-The reaction of PDT-Bimane with cysteine forms pyridine-2thione, which has a maximum absorption wavelength of 343 nm (32). This reaction has been used previously to determine the pK a value of cysteine residues in proteins (33). The reduced Orp1 proteins were diluted to 10 M in sodium citrate or phosphate buffer ranging from pH 3.5 to 11.5. Reactions were started by addition of PDT-Bimane to a final concentration of 40 M and rapidly mixed, and the absorbance at 343 nm was recorded over 120 min with a Varian Cary 50 Bio UV-visible spectrophotometer. The resulting 343 nm curves were fit to a single exponential (Y ϭ Y 0 ϩ Ae Ϫ x/t1 ) function. The values for the inverse of the first-order rate constant (t 1 ) were plotted against pH. The resulting curves were fit to the Henderson-Hasselbach equation, and the pK a value was determined.
Homology Model of Orp1-The three-dimensional model of Orp1 was calculated based on the x-ray crystal structures of human hGpx2 and hGpx5. The structures of each protein have yet to be published but are deposited in the Protein Data Bank codes 2HE3 and 2I3Y, respectively. The model was constructed using MODELLER 9 version 1 following the detailed steps described in the MODELLER advanced tutorial (34,35). Briefly, the primary sequences of the Gpx2 and Gpx5 templates were initially aligned using the align multiple structure/sequence alignment module within MODELLER. The Orp1 sequence was then aligned with the aligned sequences of hGpx2 and hGpx5 using the multiple alignment module. Five models of Orp1 were calculated using the model module and were analyzed with the discrete optimized protein energy function. We selected the lowest energy structure for modeling purposes and used MacPyMOL for displaying the structure.
Analysis of Orp1 Mutations in Vivo-Yeast strains YPH499 (MATa ura3-52 lys2-801_amber ade2-101_ochre trp1-⌬63 his3-⌬200 leu2-⌬1) and YMJW22 described previously (YPH499:⌬orp1::Kan R ) were used and have been described previously (18). For Orp1 expression in S. cerevisiae, we used the ppp81 vector, which allows for stable integration of the plasmid DNA into the ADH1 promoter region (36). The pADH1-ORP1 overexpression constructs were constructed by amplifying the ORP1 coding region by PCR using oligonucleotides primers containing NotI (5Ј) and BglII (3Ј) sites. The PCR products were digested with NotI and BglII and ligated to ppp81 digested with the same restriction enzymes. The resulting plasmids were verified by DNA sequencing. The wild-type and mutant ORP1 expression strains were created by transforming PacI linearized pADH1-ORP1 into the YMJW22 strain. As a control we also constructed a vector-only strain by transforming PacI linearized ppp81 into the YMJW22 strain. The integration of the ORP1 gene into the ADH1 locus was confirmed by colony PCR analysis. For H 2 O 2 sensitivity analysis, each strain was grown to mid-log phase (A 600 ϭ 0.7) in either YPD media or SC-URA minimal media. YPD plates were freshly prepared with 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM H 2 O 2 and were used immediately. Each strain was serially diluted, and 3 l of each dilution was plated onto YPD plates. The plates were incubated at 30°C for 2 days.

The Formation of Orp1 Cys36 and Yap1-cCRD Disulfide Bond
Complex-The goal of this study was to elucidate the molecular mechanism by which Orp1 senses peroxides and ultimately reacts with Yap1. Therefore, we developed a gel shift assay for monitoring the Orp1-Yap1 protein interaction. A Yap1-cCRD expression construct was prepared, which included Yap1 amino acids Asn 565 to Asn 650 and where Cys 620 and Cys 629 were mutated to alanine and threonine, respectively. For this assay we used the Orp1 Cys36 protein, which only contains the active site Cys 36 and has Cys 64 and Cys 82 mutated to serine. Orp1 Cys36 is incapable of forming a Cys 36 -Cys 82 intramolecular disulfide bond but can still catalyze disulfide bond formation in Yap1 in response to H 2 O 2 in vivo (12). Both Orp1 Cys36 and Yap1-cCRD were purified to homogeneity, reduced with DTT, and exchanged into buffer containing EDTA. It was observed that the presence of EDTA minimized protein oxidation, thus allowing all experiments to be performed aerobically. These assays were also conducted anaerobically and showed similar results (data not shown). Yap1-cCRD and Orp1 Cys36 were mixed in equal molar ratios, and H 2 O 2 was added to the reactions. Reactions were stopped with trichloroacetic acid after 1 min, separated with reducing or nonreducing SDS-PAGE, and stained with Coomassie Brilliant Blue (Fig. 1). The gel shows that Orp1 Cys36 alone treated with H 2 O 2 forms a small amount of disulfide-bonded dimer. This has also been observed with Orp1 homologs from poplar trees (37). Yap1-cCRD appears unchanged by H 2 O 2 treatment and migrates at the same appar-ent molecular weight. However, mixtures of Orp1 Cys36 and Yap1-cCRD form a higher molecular weight complex upon H 2 O 2 treatment. This complex migrates at the appropriate molecular weight of an Orp1 Cys36 and Yap1-cCRD disulfide bond-linked complex. The band corresponding to the Orp1 Cys36 -Yap1-cCRD complex is not observed in the individual Orp1 Cys36 or Yap1-cCRD reactions. When these reactions are run under reducing conditions the Orp1 Cys36 -Yap1-cCRD complex is not observed, further demonstrating that Orp1 Cys36 and Yap1-cCRD are covalently linked via a disulfide bond. These experiments provide evidence that Yap1-cCRD can specifically react with Orp1 in an H 2 O 2 -inducible manner, and we hypothesized that an intermediate of the reaction involved Cys-SOH formation on Orp1.
Identification of a Cys-SOH Intermediate on Cys 36 of Orp1-To elucidate the molecular mechanism by which Orp1 senses H 2 O 2 , we wanted to determine whether the redox-active cysteines in Orp1 and Yap1-cCRD formed Cys-SOH in response to H 2 O 2 . We used the chemical NBD-Cl to probe Cys-SOH modification of specific cysteines on Orp1 and Yap1-cCRD. NBD-Cl has been used previously to monitor Cys-SOH modification on proteins (31). One limitation of this technique is that to obtain unambiguous results, it requires the use of proteins that contain only one cysteine residue. Therefore, we constructed and purified two forms of Orp1 that contained either Cys 36 or Cys 82 and Yap1-cCRD that contained only Cys 598 . We first reacted Orp1 Cys36 with H 2 O 2 and NBD-Cl. As controls we prepared samples of Orp1 Cys36 in parallel that were untreated and treated with NBD-Cl alone. Unreacted NBD-Cl was removed from the reactions by gel filtration chromatography, and the recovered protein samples were concentrated prior to UV-visible spectrophotometry ( Fig. 2A). For untreated Orp1 Cys36 , we did not observe any absorbance peaks between 300 and 500 nm. Orp1 Cys36 samples that were treated with NBD-Cl showed a peak with maximal absorbance at 420 nm. This is the expected peak for the reaction of NBD-Cl with a free sulfhydryl group (R-S-NBD). Upon treatment with both H 2 O 2 and NBD-Cl, Orp1 Cys36 showed a new peak with maximal absorbance at 347 nm. This peak is indicative of formation of Cys-SOH on Each lane represents a reaction that was precipitated with trichloroacetic acid and separated with either reducing or nonreducing SDS-PAGE. Only when Orp1 Cys36 and Yap1-cCRD are exposed to H 2 O 2 is appreciable disulfide bond formation initiated. Under reducing conditions, the Orp1 Cys36 and Yap1-cCRD disulfide-linked complex is not observed. OCTOBER 26, 2007 • VOLUME 272 • NUMBER 43

Molecular Mechanism of Oxidative Stress Perception by Orp1
Cys 36 (R-SO-NBD). Both the 347 and 420 nm peaks disappeared upon reduction with DTT, indicating that the NBD-Cl molecule was connected via Cys 36 (data not shown). We repeated these sets of experiments with both Orp1 Cys82 and Yap1-cCRD (Fig. 2, B and C). In both cases the spectra of NBD-Cl-treated samples looked the same with absorbance peak maxima at 420 nm, regardless of whether H 2 O 2 was added or not. These data suggest that Orp1 reacts with H 2 O 2 at Cys 36 (12,38). This differs from their human counterparts, which are glutathionedependent, contain a seleno-cysteine in their active sites, and form tetramers in solution (39). We used the x-ray crystallographic structure of hGpx2 and hGpx5 as templates for constructing the Orp1 model. The overall fold of the lowest energy model of Orp1 is shown in Fig. 3A. The side chains of the Cys 36active site and Cys 82 are displayed. Also shown are the side chains of the conserved Gln 70 and Trp 125 residues, which are located around Cys 36 in the active site. These residues are also located in the active site of human seleno-cysteine glutathione peroxidases. In the Orp1 model, Cys 82 is located on the surface of the protein and is partially solvent-exposed. It is ϳ10 Å away   36 , we prepared Q70A and W125A mutants of Orp1 that only contained the Cys 36 . We monitored the hydrogen peroxidase enzymatic activity of Orp1, Orp1 Q70A , and Orp1 W125A using a previously established NADPH-linked assay (13). This assay showed that Orp1 Q70A is no longer active as a peroxidase and that the Orp1 W125A mutant retained ϳ10% of wild-type peroxidase activity (Fig. 4A). Next we examined the ability of Orp1 Cys36/Q70A and Orp1 Cys36/W125A to form Cys-SOH in response to H 2 O 2 . NBD-Cl modification experiments were conducted as described previously with similar controls. Wild-type Orp1 formed Cys-SOH as judged by the absorbance peak at 347 nm (Fig. 4B) Analysis of the pK a of Orp1 Sulfhydryls-We hypothesized that the sulfhydryl pK a of Cys 36 is lowered by its molecular environment. To quantify the pK a of Orp1 Cys36 and Orp1 Cys82 , we used a thiol reactivity assay described previously by Brennan and co-workers (33) for measuring the pK a value of the active site cysteine in the OhrR transcription factor. Briefly, the chemical PDT-Bimane is reacted with a protein that contains a single cysteine residue over a range of pH values (32). The reaction is monitored at 343 nm as a function of time and fit to a first-order exponential function. We performed this analysis on the Orp1 Cys36 protein over a range of pH values from 3.5 to 8.5 (Fig.  5A). These data show that at pH values of 3.5 and 4.0, the reaction of Orp1 Cys36 with PDT-Bimane is slow and largely unchanged. This is what would be expected for a cysteine sulfhydryl that is protonated. Between the pH values of 4.5 and 6.0, the t 1 for the reactions decreased ϳ90-fold. At pH values 7.0 and above, the reaction is extremely fast and complete within 2 min. The t 1 values were normalized and plotted as function of pH (Fig. 5B). These points were fit to the Henderson-Hassel-  OCTOBER 26, 2007 • VOLUME 272 • NUMBER 43 bach equation, and a pK a value of 5.1 was determined. This value is 3.2 pH units lower than the pK a of free cysteine and indicates that Cys 36 would be in the thiolate form at physiological pH. We performed the similar PDT-Bimane reaction pK a analysis for Orp1 Cys82 , Orp1 Cys36/Q70A , and Orp1 Cys36/W125A (data not shown). The t 1 values for the resulting first-order reaction curves were normalized and plotted with those for Orp1 Cys36 (Fig. 5B). These data show that the pK a for Cys 82 in Orp1 is 8.3, which is very close to the pK a of an isolated cysteine molecule. Remarkably, mutation of either Gln 70 or Trp 125 to alanine results in the reversion of the pK a of Cys 36 to 8.3. These data strongly suggests that Gln 70 and Trp 125 stabilize Cys 36 in its thiolate form.

Molecular Mechanism of Oxidative Stress Perception by Orp1
Analysis of Orp1 Mutations in Vivo-We sought to characterize the phenotypes of the Orp1 Q70A and Orp1 W125A S. cerevisiae mutants. To this end, we transformed an S. cerevisiae strain in which the ORP1 gene had already been deleted, with integrating plasmids containing the wild-type ORP1, ORP1 Q70A , ORP1 W125A , or ppp81 as a vector control. Correct integration of the ORP1 genes into the ADH1 promoter was verified with colony PCR analysis (data not shown). The result-ing strains were assayed for their resistance to increasing concentrations of H 2 O 2 peroxide. The parental wild-type YPH499 strain was used as a positive control. This strain showed colony forming ability up to 3.0 mM H 2 O 2 (Fig. 6). The ⌬orp1 strain showed increased sensitivity to H 2 O 2 . This strain was able to form colonies up to 2.0 mM H 2 O 2 . As expected, the ppp81transformed strain showed similar H 2 O 2 resistance levels as the ⌬orp1 strain. Expression of Orp1 resulted in complementation of the ⌬orp1 strain, and this strain had H 2 O 2 resistance levels that were similar to that of the parental YPH499 strain. Expression of either Orp1 Q70A or Orp1 W125A resulted in strains that displayed H 2 O 2 resistance phenotypes similar to the ⌬orp1 strain.

DISCUSSION
The discovery that disulfide bond formation in the Yap1 transcription factor is regulated by a peroxidase-like enzyme has led to the realization that peroxidases and peroxiredoxin enzymes are not only involved in ROS scavenging but also function as peroxide sensors in vivo. The Orp1 protein is a member of a thiol peroxidase family of enzymes that is homologous to human glutathione peroxidases, yet it is functionally similar to the peroxiredoxin protein family (12). These classes of proteins have evolved to specifically react with and reduce peroxides, such as H 2 O 2 and the less polar alkyl hydroperoxides. Additionally, as part of their catalytic mechanism, these enzymes form oxidized disulfide bonds either between cysteine pairs within the protein or with glutathione. It is thus not surprising that these classes of enzymes have evolved to use similar biochemical mechanisms to form mixed disulfide bonds with other proteins, thereby activating disulfide bond relay cascades and modulating the function of transcription factors such as Yap1 and Pap1 (12,13,17,42).
The goal of this study was to investigate the molecular mechanism by which the Orp1 class of peroxiredoxin-like enzymes senses H 2 O 2 . We initially established a disulfide bond formation assay between Orp1 and a portion of the Yap1 protein that   has been shown to react with Orp1 in vivo. A disulfide-bonded complex was observed between Orp1 and Yap1-cCRD immediately following treatment with H 2 O 2 . We showed that a mutant form of Orp1, in which only the active site Cys 36 is present, forms a Cys-SOH intermediate upon H 2 O 2 exposure using an NBD-Cl chemical modification assay. Cys-SOH intermediates have been shown to have extremely short half-lives and have proven difficult to identify in proteins. For this reason, the remaining nucleophilic cysteines need to be removed from the protein being studied. In the case of Orp1, Cys 36 -SOH can be attacked by Cys 82 of Orp1 to form an intramolecular disulfide bond or by Cys 598 of Yap1 to form a mixed disulfidebonded complex. We also examined the ability of Cys 82 of Orp1 or Cys 598 of Yap1 to form Cys-SOH in response to H 2 O 2 . Our data showed that neither cysteine could form Cys-SOH as judged by NDB-Cl reactivity. These data provided support for the idea that Cys-SOH could be used as a reliable biochemical marker for Orp1 activity. It also suggests that H 2 O 2 -induced Cys-SOH formation may be a general characteristic of redoxactive cysteine residues.
In this study we provide a comprehensive analysis of the correlation between cysteine sulfhydryl pK a , peroxidase activity, H 2 O 2 -induced Cys-SOH formation, and Orp1-active site structure. It had been shown previously that the selenocysteine of the human glutathione peroxidase enzymes reacts with H 2 O 2 to form a selenol intermediate prior to reaction with glutathione (40). In addition, the seleno-cysteine-active site is formed by a catalytic triad involving Gln 83 and Trp 157 . Mutation of either of these amino acids was shown to decrease the enzymatic activity of hGpx and reduced the ability of iodoacetic acid to inhibit hGpx activity (41). Our homology model of Orp1 showed that the homologous Gln 70 and Trp 125 were part of an active site pocket formed around Cys 36 (Fig. 3, A and B). One side of the pocket was entirely hydrophobic, lined with the conserved phenylalanine residues Phe 38 and Phe 127 . These residues have van der Waals interactions with Cys 36 and appear to effectively shield Cys 36 from the bulk solvent. On the other side of the pocket are the side chains of Gln 70 , Trp 125 , and Asn 126 , which are all conserved residues containing side chains that can make hydrogen bonds with the sulfhydryl-active site. Our results strongly suggest that the role of Gln 70 and Trp 125 in Orp1 reactivity is to stabilize the thiolate form of Cys 36 . In the absence of H 2 O 2 , mutation of either residue abolishes the low pK a of Cys 36 . Although the Cys 36 -active site pocket is accessible to solvent, this pocket does not appear to be able to accommodate Cys 36 -SOH. Therefore, upon oxidation the Cys-SOH would protrude from the active site pocket into the solvent, making it more vulnerable to nucleophilic attack by another cysteine.
The global identification of redox-active cysteine residues in proteins has been the focus of many recent investigations (43)(44)(45). Gladyshev and co-workers (44) used a novel bioinformatics approach to identify redox-active cysteines in the proteomes of multiple organisms. Their approach identified many previously characterized oxidoreductase proteins, such as the glutathione peroxidase family, which contain a nucleophilic active site cysteine. Proteins that contain cysteines with abnormally low pK a values have historically been characterized by their increased reactivity with haloacetic acids. Use of haloacetic acid modification in conjunction with monitoring enzymatic activity is a well established approach for measuring cysteine pK a (46,47). One example is Cys 25 in the sulfhydryl protease papain that has a pK a of ϳ4 (47). Our results and prior studies on nucleophilic active site cysteine suggest that one common biochemical feature of these active site cysteines is a lowered sulfhydryl pK a . In the case of Orp1, we determined the pK a of Cys 36 to be ϳ3.2 pH units lower than that of free cysteine. It is this feature alone that contributes to the reactivity of Orp1 toward peroxides and enables it to form Cys-SOH and, eventually, disulfide bonds.
There appear to be multiple ways that enzymes and transcription factors have evolved lower pK a cysteines, thereby increasing their reactivity toward peroxides. In the case of the OxyR transcription factor, the reactivity of Cys 199 appears to be modulated via an ion pair with Arg 266 . The x-ray crystal structure of OxyR shows that the side chain of Arg 266 is buried in the hydrophobic core behind the Cys 199 -active site (48). Although the pK a of Cys 199 has not been determined, it has been shown that Cys 199 forms Cys-SOH in response to H 2 O 2 (23). The active site composition of OxyR suggests that the positively charged and buried Arg 266 side chain stabilizes Cys 199 in the thiolate form. Two additional examples of redox-active cysteines that use ion pairing to lower their pK a can be seen in ArsC, an arsenate reductase found in E. coli, and in AhpC, a peroxiredoxin found in E. coli (49). In the case of ArsC, the pK a of Cys 12 in its active site is 6.4 and is stabilized by the positive charge on His 8 . Mutation of His 8 to glycine resulted in reversion of the Cys 12 pK a back to that of free cysteine. Finally, an example of a low pK a cysteine that is neither part of a catalytic triad nor an ion pair can be found in the OhrR transcription factor from Bacillus subtilis (50). When reduced, OhrR can cooperatively bind its promoter and repress transcription (33). Upon reaction with organic hydroperoxides, OhrR is oxidized at Cys 15 and forms a Cys-SOH. Using similar pK a determination techniques as those used in the present study, Brennan and co-workers (33) showed that OhrR has a pK a value of 5.2. The x-ray crystal structure of reduced OhrR shows that Cys 15 sits at the end of an ␣-helix and is adjacent to two tyrosine residues. Brennan and co-workers (33) suggest that the positive macrodipole of the ␣-helix could explain the lower pK a value of Cys 15 , but biochemical studies to confirm this hypothesis have not been performed. What remains a major challenge in the field of protein redox regulation is the identification of proteins with low pK a cysteines. Identification of these cysteines will undoubtedly lead to the discovery of novel proteins that are regulated by thiol oxidation and reduction as well as other enzymes that utilize nucleophilic sulfhydryls as part of their catalytic mechanism.