The Oxidation of Yeast Alcohol Dehydrogenase-1 by Hydrogen

we exposed YADH-1 to hydrogen peroxide in vitro and assessed the oxidized protein by LC-MS/MS ... of the zinc-containing ADH family.1 The YADH family ...
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The Oxidation of Yeast Alcohol Dehydrogenase-1 by Hydrogen Peroxide in Vitro Lijie Men and Yinsheng Wang* Department of Chemistry-027, University of California, Riverside, California 92521-0403 Received July 28, 2006

Yeast alcohol dehydrogenase (YADH) plays an important role in the conversion of alcohols to aldehydes or ketones. YADH-1 is a zinc-containing protein, and it accounts for the major part of ADH activity in growing baker’s yeast. To gain insight into how oxidative modification of the enzyme affects its function, we exposed YADH-1 to hydrogen peroxide in vitro and assessed the oxidized protein by LC-MS/MS analysis of proteolytic cleavage products of the protein and by measurements of enzymatic activity, zinc release, and thiol/thiolate loss. The results illustrated that Cys43 and Cys153, which reside at the active site of the protein, could be selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and cysteine sulfonic acid (Cys-SO3H). In addition, H2O2 induced the formation of three disulfide bonds: Cys43Cys153 in the catalytic domain, Cys103-Cys111 in the noncatalytic zinc center, and Cys276-Cys277. Therefore, our results support the notion that the oxidation of cysteine residues in the zinc-binding domain of proteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO2H and Cys-SO3H is also possible. Furthermore, most methionines could be oxidized to methionine sulfoxides. Quantitative measurement results revealed that, among all the cysteine residues, Cys43 was the most susceptible to H2O2 oxidation, and the major oxidation products of this cysteine were Cys-SO2H and Cys-SO3H. The oxidation of Cys43 might be responsible for the inactivation of the enzyme upon H2O2 treatment. Keywords: cysteine oxidation • zinc binding protein • alcohol dehydrogenese

Introduction Yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) is a member of the zinc-containing ADH family.1 The YADH family from baker’s yeast Saccharomyces cerevisiae contains three isoenzymes: YADH-1, YADH-2, and YADH-3. YADH-1 is found in the cytoplasm, and it constitutes the major alcohol dehydrogenase activity in growing baker’s yeast.2 YADH-1 is a tetrameric protein composed of four identical subunits with a subunit molecular mass of 36 kDa.3,4 Each subunit contains two zinc ions. The zinc ion coordinates with two cysteines (i.e., cysteines 43 and 153) and one histidine to form the catalytic center (Zn1Cys2His1), which is essential for the conversion of ethanol and other primary alcohols to the corresponding aldehydes. The fourth coordination position of the zinc ion is occupied by the hydroxyl group of ethanol, which is replaced with water in the absence of substrate.5 The second zinc ion is bound with four cysteine residues (i.e., cysteines 97, 100, 103, and 111), but this zinc ion assumes only a conformational role.6,7 Those six cysteines mentioned above are highly conserved in YADH and in nearly all ADH families.1 It is well-known that intracellular oxidative stress and reactive oxygen species (ROS) are produced by growth factors as well as by exogenous agents.8,9 Accumulation of ROS has been associated with a diverse range of diseases in humans.9-11 * To whom correspondence should be addressed. E-mail: [email protected]. Tel: (951)827-2700. Fax: (951)827-4713.

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Sulfur-containing amino acids, cysteine and methionine, are the major targets in proteins for modification by a variety of ROS.12-14 Oxidation of protein sulfhydryls can lead to structural, conformational, and catalytic consequences. Zinc finger proteins constitute one of the largest classes of DNA-binding proteins and are very susceptible to oxidative stress since they utilize thiolate functional group of cysteines and sometimes, together with histidines, to coordinate the zinc ion.15,16 For instance, the oxidation of zinc finger-bearing replication protein A, a protein involved in DNA replication, repair, and recombination, could compromise its DNA-binding function.17 In addition, the RING- and PHD-finger domains, which are rich in cysteine residues and bind to zinc ions, play important roles in mediating protein-protein interactions.18-20 Studying the behavior of zinc-binding proteins under oxidative stress is essential for us to understand the mechanism of cellular events with which this group of proteins are involved. YADH-1 will be a perfect model for such study because the zinc-binding domains of YADH-1 share many structural similarities with typical zinc finger proteins, and it is commercially available. Previous studies showed that this enzyme could be deactivated upon treatment with H2O2 or peroxynitrite, and the peroxynitrite-induced enzyme activity loss was correlated with thiol/thiolate oxidation and zinc release.21 However, the identities of the cysteine oxidation products resulting from the oxidation reactions remain elusive. Here, we examined the oxidation of YADH-1 upon exposure to H2O2 in vitro by rigorous 10.1021/pr0603809 CCC: $37.00

 2007 American Chemical Society

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Oxidation of YADH-1 by H2O2

LC-MS/MS analysis of the proteolytic peptides of the oxidized protein together with measurements of enzyme activity, thiol/ thiolate content, and zinc release. The goals were to delineate the oxidative modification of this protein at the molecular level and to correlate the formation of the oxidation products with the enzyme activity loss.

Materials and Methods Reagents. Hydrogen peroxide (H2O2) and sodium phosphate were purchased from Fisher Scientific (Pittsburgh, PA). Alcohol dehydrogenase, catalase, glutathione, and iodoacetamide (IAM) were obtained from Sigma (St. Louis, MO). HPLC-grade CH3CN and ammonium bicarbonate were from EMD Chemicals (Gibbstown, NJ). H2O2 concentrations were determined spectrophotometrically (240 ) 39.4 M-1 cm-1). Oxidation and Reduction Conditions. YADH-1 (0.29 µg/µL) in 10 mM sodium phosphate buffer (pH 7.5, buffer A) was treated with different concentrations of H2O2 at 25 °C for 10 min, and the reactions were terminated by adding 5 mM L-methionine. The oxidized YADH-1 was subsequently alkylated with 55 mM IAM in 8 M urea in the dark for 30 min, and the buffer of the resulting YADH-1 solution was exchanged with 50 mM NH4HCO3 (pH 8.0, buffer B) by using a Microcon YM10 microcentrifuge filter (Millipore, Billerica, MA). The protein samples were then digested with chymotrypsin or trypsin (Roche Diagnostics, Indianapolis, IN) at an enzyme-to-substrate ratio of 1:40 (w/w) in a 100-µL NH4HCO3 buffer at 37 °C overnight. To map the interpeptide disulfide linkage, the chymotryptic digestion mixture was separated using HPLC, and the fraction containing the disulfide-linked peptide was reduced with 20 mM dithiothreitol (DTT) at 56 °C for 30 min and alkylated with 50 mM N-ethylmaleimide (NEM) for 30 min. The resulting reaction mixture was then subjected to matrix-assisted laser desorption/ionization (MALDI)-MS or liquid chromatographytandem MS (LC-MS/MS) analyses. YADH-1 Enzyme Activity Measurement. YADH-1 (0.29 µg/ µL) in buffer A was exposed to different concentrations of H2O2 at 25 °C for 10 min, and the reactions were terminated by the addition of excess amount of catalase. To measure YADH-1 activity, the oxidized protein was first diluted by a factor of 4000 in cold buffer A containing 0.1% BSA and then added to a 2-mL buffer containing 50 mM sodium phosphate, 7.5 mM β-NAD+, and 300 mM ethanol (pH 8.8). The formation of NADH was monitored at 340 nm (340 ) 6220 M-1 cm-1) at 25 °C for 10 min, and rates were determined based on the absorbance change in the first 6 min. Zn Release Measurement. Free Zn ion and 4-(2-pyridylazo)resorcinal (PAR) can readily form a complex, which has absorbance at 500 nm. The extinction coefficient was determined experimentally to be 69 900 M-1 cm-1 by the use of a calibration curve constructed from the measurements of known concentrations of ZnCl2. PAR (50 µM) was added to solutions containing the control or H2O2-treated YADH-1 in 50-mM sodium phosphate buffer (pH 7.5). A500 was monitored for approximately 200-300 s until no further increase in absorbance could be observed. Addition of an excess amount of EDTA (0.5 mM in final concentration), which binds zinc more tightly than PAR, led to a quick decrease in absorbance, and the difference in absorbance was taken as representing the ZnPAR complex. Ellman’s Assay. Ellman’s assay is based on a colorimetric reaction between the Ellman’s reagent [5,5′-dithiobis(2-ni-

trobenzoic acid), DTNB] and the free thiol groups of proteins. The control sample or H2O2-treated YADH-1 (10 µg) was mixed with buffer A containing 10 mM DTNB, 160 mM methanol, and 8 M urea. The thiol content was determined by taking the absorbance at 412 nm. LC-MS/MS for the Identification of Oxidized Cys and Met Residues. LC-MS/MS experiments were performed on an LCQ Deca XP ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA). MS/MS experiments were carried out in datadependent scan mode by selecting the most abundant protonated ions observed in MS mode for collisional activation with a relative collision energy of 35%. A 0.30 × 150 mm capillary C18 column (Micro-Tech Scientific, Vista, CA) was used for the separation, and the flow rate was ∼5 µL/min, which was obtained from a 120 µL/min pump flow after precolumn splitting. The mobile phases were 0.6% acetic acid in water (mobile phase A) and 0.6% of acetic acid in acetonitrile (mobile phase B). A linear gradient of 2-42% mobile phase B in A in 63 min was employed for the separation. The resulting MS/ MS data were searched against a yeast protein database by using SEQUEST (ThermoFinnigan) or against the sequence of YADH-1 by using MassAnalyzer 1.03,22 which was kindly provided by Dr. Zhongqi Zhang at Amgen, Inc. To carry out quantitative measurement of specific peptides, LC-MS/MS experiments were also performed in selective-ion monitoring (SIM) mode. The chymotryptic digests were also analyzed on a QSTAR XL hybrid quadrupole /time-of-flight mass spectrometer equipped with an o-MALDI ion source (Applied Biosystems, Foster City, CA). The peptide-containing samples were dissolved in an aqueous solution of 0.1% trifluoroacetic acid (TFA), and the sample aliquots were mixed with an equal volume of matrix solution, which was a saturated solution of R-cyano-4hydroxycinnamic acid in a solvent mixture of CH3CN, H2O, and TFA (50/50/0.1, v/v).

Results Primary Sequence of YADH-1. To date, the primary structure of YADH-1 from baker’s yeast has been studied by several research groups.23-25 Jo¨rnall24 utilized the commercially available preparation of the protein from S. cerevisiae and first determined the sequence of YADH-1 by using Edman sequencing analysis of proteolytic peptides. Later, Bennetzen et al.23 carried out nucleotide sequence analysis of the entire YADH-1 gene. However, the protein sequences determined from the two different approaches disagree for 5 out of the 347 amino acid residues. Some of these differences were attributed to ADH-1 protein heterogeneity in different yeast strains and not to sequencing errors.23 We searched our LC-MS/MS data against both sequences, and the LC-MS/MS data allowed us to confirm approximately 98% of the amino acid sequence of YADH-1 and our results are in full agreement with those from Jo¨rnall (Figure 1). Therefore, we applied the sequence published by Jo¨rnall for the assessment of the protein modification induced by H2O2. Yeast Alcohol Dehydrogenase Is Inactivated by H2O2. To examine whether oxidants inactivate YADH-1 in vitro, we exposed 7.7 µM enzyme to H2O2 at different molar ratios (control, 1:10, 1:40, 1:160, 1:400, and 1:1600 mol/mol, protein subunit/oxidant) at 25 °C for 10 min. After adding excess amount of catalase to decompose any residual H2O2, we measured the alcohol dehydrogenase activity of YADH-1 following the procedures described in Materials and Methods. As Journal of Proteome Research • Vol. 6, No. 1, 2007 217

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Figure 1. The primary sequence of YADH-1. Oxidized Cys and Met residues, identified in this study, are depicted in bold and italic letters. “∧” and “*” on Cys residues represent the formation of Cys-SO2H and Cys-SO3H, respectively. Disulfide bonds in H2O2-treated YADH-1 are shown in black lines. The first amino acid, serine, is partially acetylated. The letters in bold only indicate the conflicted amino acids compared with the YADH-1 protein sequence reported by Bennetzen et al.23

of 1:400 (subunit/H2O2), and the activity was almost completely abolished when the ratio reached 1:1600. We also examined the reversibility of inactivation induced by H2O2 exposure. To this end, we carried out the enzyme activity measurement after the H2O2-exposed protein had been treated with DTT. The results shown in Figure 2a revealed that the enzyme activity could be partially rescued upon 1 mM DTT treatment; the incubation with DTT recovered approximately 15, 19, 17, and 12% of the activity loss induced by H2O2 at molar ratios of 1:160, 1:400, 1:800, and 1:1600 (protein subunit/H2O2), respectively. Therefore, the loss in enzyme activity upon H2O2 treatment could not be completely reversed by DTT treatment, supporting the idea that the formation of disulfide bonds does not contribute significantly to the enzyme activity loss. The deactivation of the enzyme is, therefore, induced mainly from irreversible oxidations.

Figure 2. H2O2-induced thiol oxidation and YADH-1 activity loss (a) and H2O2-induced zinc release (b). Results represent the means and standard deviations of duplicate determinations of three independent experiments.

depicted in Figure 2a, YADH-1 gradually lost its activity upon incubation with increasing concentrations of H2O2; the enzyme activity was inhibited by approximately 50% at a molar ratio 218

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Thiol/Thiolate Oxidation and Zn Loss upon H2O2 Treatment. To investigate the correlation of the loss of free thiol residues in YADH-1 and the loss of enzyme activity, we performed the Ellman’s assay to quantify the free thiol residues in YADH-1 upon H2O2 treatment. Clearly, exposure to H2O2 results in a significant loss of free thiols (Figure 2a), which coincides with the activity loss. In addition, the loss of zinc, determined by PAR assay, strongly correlates with the activity loss induced by H2O2. In this respect, an equivalent of 0.55 zinc ion per subunit was released from the untreated YADH-1, whereas an equivalent of 1.8 zinc ions per subunit was released from the protein that had been treated with H2O2 at a molar ratio of 1:1000 (protein subunit/H2O2), which resulted in 80% inhibition of the enzyme activity (Figure 2b). Therefore, H2O2 treatment at this molar ratio leads to the release of an equivalent of approximately 1.2 zinc ions per subunit of the protein. These results suggested that zinc release was strongly associated with the activity loss. LC-MS/MS for the Identification of the Oxidized Cys and Met Residues. To locate the oxidized residues in YADH-1 upon H2O2 treatment, intact and H2O2-oxidized YADH-1 were carboxyamidomethylated (CAM) with IAM and subsequently cleaved with chymotrypsin. The resulting peptide mixtures were analyzed using LC-MS/MS. To exclude false-positive identi-

Oxidation of YADH-1 by H2O2

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Figure 3. LC-MS/MS analysis of chymotryptic peptide with AA 40-50 from YADH-1 upon H2O2 oxidation. Shown are MS/MS of the [M + 2H]2+ ion of the (a) CAM-modified peptide (m/z 641.8), (b) Cys43-SO2H-bearing peptide (m/z 629.5), and (c) Cys43-SO3H-bearing peptide (m/z 637.5).

fication, all product-ion spectra were manually inspected to confirm the sequence assignments. First, in the untreated YADH-1 sample, we identified five peptides (with amino acids 40-50, 93-102, 103-119, 129-159, and 268-281) carrying CAM-modified cysteine residues, which include all 8 Cys residues in this protein. Peptide (AA 40-50), containing Cys43, was detected as a doubly charged ion of m/z 641.8, which is consistent with the calculated m/z for the CAMmodified peptide (AA 40-50). In addition, the product-ion spectrum of the ion of m/z 641.8 showed a number of N- and C-terminal fragments (y1-y9 and b7-b10) whose m/z values are in line with the peptide sequence (Figure 3a). Furthermore, the mass difference between the y7 (m/z 879.4) and y8 (m/z 1039.4)

ions was 160 Da, matching the mass of a CAM-modified cysteine and supporting the alkylation of Cys43 by IAM. Next, Cys153-bearing peptide (AA 129-159) was detected at m/z 1042.1 as a triply charged ion, which is in keeping with the calculated mass for the peptide (3123 Da) containing a CAM-modified Cys. The product-ion spectrum of the m/z1042.1 ion (Figure S1, Supporting Information) confirmed the identity of the peptide. Similarly, we detected doubly charged ions of m/z 602.2 and 750.1 for two peptides containing two Cys residues individually: Cys97 and Cys100 in one peptide (AA 93-102), and Cys276 and Cys277 in the other (AA 268-281). MS/ MS analysis also confirmed that the above four Cys residues were alkylated by IAM (data not shown). These results suggest Journal of Proteome Research • Vol. 6, No. 1, 2007 219

research articles that all 8 cysteine residues are present in reduced form in the control YADH-1 sample, though we observed a minute amount of oxidation product of Cys43 in the control sample (vide infra). To determine whether H2O2 modifies YADH-1 at specific sites, we exposed YADH-1 to H2O2 at a molar ratio of 400:1 (oxidant/subunit), which can give rise to about 50% inhibition of the enzyme, and characterized the chymotryptic digestion mixture of YADH-1 using LC-MS/MS. The MS results revealed two peptides as doubly charged ions of m/z 629.5 and 637.5, corresponding to the peptide (AA 40-50) with Cys43 being oxidized to Cys-SO2H and Cys-SO3H, respectively. The MS/MS of the ion of m/z 629.5 (Figure 3b) showed facile neutral losses of H2O and H2SO2 from the precursor ion. The H2SO2 was lost from the side chain of the oxidized Cys43, supporting that Cys43 is oxidized to Cys-SO2H.26,27 In addition, we observed a mass difference of 135 Da between b3 and b4 ions, which is consistent with the mass of a Cys-SO2H and confirms again the presence of a Cys43-SO2H in this peptide. In the MS/MS of the ion of m/z 637.5 (Figure 3c), a number of abundant N- and C-terminal fragments, that is, b7-b10 and y1-y8, are observed. The mass difference between y7 and y8 ions supports the presence of a Cys43-SO3H. The oxidized peptide (AA 129-159) was detected as triply charged ions of m/z 1033.7 and 1039.0, corresponding to the expected masses of the peptide with the Cys153 being modified to Cys-SO2H and Cys-SO3H, respectively. The MS/MS of the ion of m/z 1033.7 showed mainly doubly charged ions (Figure S1b, Supporting Information), for instance, b132+, b152+ and b172+-b242+ ions. Any b ion, larger than b24, could not be detected, except b282+; however, a series of doubly charged b ions accompanied with water loss were observed, such as b26H2O2+, b27-H2O2+, b28-H2O2+, b29-H2O2+ and b30-H2O2+ ions, whose masses were increased by 32 Da individually relative to their calculated masses. The 32-Da mass increase again supports the modification of the Cys residue to a Cys-SO2H. The MS/MS of the triply charged ion of m/z 1039.0 (Figure S1c, Supporting Information) was dominated with doubly charged N-terminal fragments (b132+, b152+-b212+, and b232+b302+), which confirmed the sequence of the peptide (AA 129159). Two signature ions, b242+ and b252+, demonstrated the presence of a Cys153-SO3H in this sequence. Other than the oxidation of Cys43 and Cys153 residues, a number of Met residues are susceptible to H2O2 oxidation. For instance, the product-ion spectra shown in Figures S2 and S3 of Supporting Information supported the presence of both unmodified and oxidized Met-bearing peptides (AA 63-82 and AA330-341). We also found peptides (AA 168-172, 190-195, and 268-281) containing both intact and Met sulfoxide at positions 168, 193, and 270, respectively (data not shown). Collectively, oxidation of Cys43 and Cys153 led to the formation of Cys-SO2H and Cys-SO3H. Such oxidation products, however, were not found for the other 6 cysteines present in YADH-1 under the same oxidation conditions. In addition, all 8 cysteine residues could be observed in CAM-modified forms. Moreover, all methionine residues except Met98 were found to be modified to Met sulfoxides. Determination of Cysteine Pairs That Form Disulfide Bonds upon Oxidation. As revealed by X-ray structure analysis (2HCY.pdb), among the eight Cys residues in the primary sequence of YADH-1, Cys43 and Cys153 are bound to the same zinc atom at the catalytic center. Likewise, Cys97, Cys100, Cys103, and Cys111 are all linked with the second zinc atom in YADH1. Moreover, Cys276 and Cys277 are adjacent to each other in 220

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the primary sequence. Thus, we anticipate to observe the formation of disulfide bonds between some of those closely positioned Cys residues upon ROS treatment. To investigate the possible disulfide bonds induced by H2O2 treatment, the MS/MS data for the analysis of chymotryptic digest from the oxidized YADH-1 were searched against the sequence of YADH-1 by using MassAnalyzer 1.03, which can facilitate the assignment of disulfide bond-bearing peptides by searching, based on MS and MS/MS results, all possible disulfide configurations in a given protein. First, peptide (AA 103-119) containing Cys103 and Cys111 was detected at m/z 904.3 as a doubly charged ion, matching the calculated mass for the peptide (1807.3 Da) carrying a disulfide bond. MS/MS of this ion was dominated with abundant b and y ions, such as b9-b16 and y3-y8 (Figure 4a). The masses of the detected y ions were consistent with the expected ones. However, the masses of b ions were reduced by 2 Da individually, which were consistent with the presence of an intramolecular disulfide bond formed between Cys103 and Cys111. It is worth noting that we did not observe y or b ions resulting from cleavage of amide bonds between the two Cys residues that have been linked. This can be attributed to the fact that the formation of these y or b ions would require the cleavages of both an amide bond and the disulfide linkage, which is expected to be less facile than the cleavage of an amide bond alone. Another peptide (AA 268-281) was identified at m/z 691.8 as a doubly charged ion, matching the theoretical mass of this peptide (1382.6 Da) carrying an intramolecular disulfide bond. The MS/MS of this precursor ion (Figure 4b) showed a number of b and y ions, confirming the peptide sequence with the Cys276 and Cys277 being disulfide-bonded. A disulfide-linked pair of peptide that contains AA 40-50 and AA 129-159 was also identified in the chymotryptic digestion mixture of YADH-1. Figure 5 showed the tandem mass spectrum of quadruply charged ion (m/z 1073.2) for this disulfide-linked pair of peptides. We observed abundant fragment ions (i.e., b162+-b212+), generated from the cleavages of peptide (AA 129-159), and a series of Y11yn2+ ions (n ) 1020), which resulted from fragments containing the C-terminal portion of the peptide (AA 129-159) incorporated with the peptide (AA 40-50) by a disulfide bond linkage, Cys43-Cys153. To further confirm the disulfide bond assignment, we followed the previously reported procedures, which include stepwise alkylation, reduction, and alkylation, for disulfide mapping.28 In this respect, the chymotryptic digest was separated by HPLC, and the fraction containing this disulfide-linked peptide, which exhibits as a peak at m/z 4290 in MALDI-MS, was reduced with DTT and alkylated by NEM. The resulting mixture was then analyzed using LC-MS/MS. If the component exhibiting m/z 4290 in MALDI-MS was truly a disulfidebonded conjugate formed between AA 40-50 and 129-159, it will be converted, upon DTT reduction and NEM alkylation, into two NEM-modified peptides. Therefore, we selectively monitored three precursor ions of m/z 1430.3, 675.8, and 1596.2, corresponding to the disulfide-bonded peptide (triply charged), NEM-labeled peptide (AA 40-50, doubly charged), and NEM-labeled peptide (AA 129-159, doubly charged), respectively, for the HPLC fraction prior to and after the DTT and NEM treatment. Prior to the DTT and NEM treatment, only the disulfide bond-linked peptide was present at m/z 1430 at 56 min (Figure 6a) and none of the NEM-alkylated peptides at m/z 675.8 and

Oxidation of YADH-1 by H2O2

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Figure 4. LC-MS/MS analysis of chymotryptic peptides (AA103-119 and 268-281) containing an intramolecular disulfide bond generated upon H2O2 oxidation. (a) MS/MS of the [M + 2H]2+ ion (m/z 904.3) of the peptide (AA 103-119) containing an intramolecular disulfide bond between Cys103 and Cys111; (b) MS/MS of the [M + 2H]2+ ion (m/z 691.8) of the peptide (AA 268-281) containing an intramolecular disulfide bond between Cys276 and Cys277.

1596.2 could be detected (Figure 6b,c). In contrast, after DTT and NEM treatment, the relative ion intensity of the disulfide bond-containing peptide decreased substantially (Figure 6d). Concomitantly, two NEM-labeled peptides became apparent, supporting that the disulfide bond was reduced and converted into two separate peptides, AA 40-50 and 129-159 (Figure 6e,f). The MS/MS analysis of these two peptides confirmed the alkylation of Cys43 and Cys153 in these two peptides by NEM (Figure S4, Supporting Information). The above results, therefore, support the formation of a disulfide bond between these two cysteines.

Cys97 and Cys100 identified in this study were in CAMmodified form in both the control and oxidized protein samples, and we failed to detect the formation of a disulfide between these two Cys residues upon H2O2 treatment. Quantification of Oxidation Products Induced by H2O2 Treatment. So far, we had identified a number of Cys and Met oxidation products upon H2O2 treatment. Nevertheless, it is unclear which amino acid’s oxidation is directly correlated with the activity loss or which Cys or Met residue is more preferentially oxidized over others. To answer these questions, we carried out a series of quantitative measurements using LCJournal of Proteome Research • Vol. 6, No. 1, 2007 221

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Figure 5. MS/MS of the [M + 3H]3+ ion (m/z 1430.3) of chymotryptic peptide containing intermolecular disulfide bond between Cys43 (AA 40-50) and Cys153 (AA 129-159).

Figure 6. Selective-ion monitoring analysis of disulfide-bonded peptide (Cys43-Cys153), and its corresponding reduced and alkylated peptides (AA 40-50 and 129-159). (a and d) SIM analysis of triply charged precursor ion of disulfide-bonded peptide of m/z 1430; and (b and e) doubly charged precursor ions of NEM-modified peptides of m/z 675.8 (AA 40-50), and (c and f) m/z 1596.2 (AA 129-159). (a-c) Before DTT and NEM treatment; (d-f) after DTT and NEM treatment.

MS/MS in SIM mode. To this end, we estimated the relative occurrence of oxidized Cys or Met by measuring ion counts of oxidized Cys- or Met-bearing peptides and the corresponding CAM-modified Cys- or intact Met-containing peptides. In this context, it is worth emphasizing that the ionization efficiency 222

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is likely to be different for the oxidized and unoxidized peptides; therefore, the amounts of oxidized peptides estimated by this method are likely to be different from the actual amounts. The comparison of the oxidation products formed in different peptides, however, may allow us to draw meaningful conclu-

Oxidation of YADH-1 by H2O2

Figure 7. Quantification of peptides (AA 40-50 and 129-159) and their corresponding oxidized products in a chymotryptic digest of YADH-1 upon H2O2 oxidation. (a) The ion currents of CAM-modified peptide (AA 40-50), Cys43-SO2H-bearing peptide (AA 40-50), Cys43-SO3H-bearing peptide (AA 40-50), and disulfide-linked peptide (Cys43-Cys,153 AA 40-50 and 129-159), which were selectively monitored at m/z 641.8, 629.5, 637.5, and 1073.6, respectively. (b) The ion currents of CAM-modified peptide (AA 129-159), Cys153-SO2H-bearing peptide (AA 129-159), Cys153SO3H-bearing peptide (AA 129-159), and disulfide-linked peptide (Cys43-Cys153, AA 40-50 and 129-159), which were selectively monitored at m/z 1042.1, 1033.7, 1039.0, and 1073.6, respectively. The results represent the means and standard deviations of duplicate determinations from three independent experiments.

sions about the susceptibilities of different Cys or Met residues toward H2O2 oxidation. Oxidized Cys43 was detected in three different forms: Cys43SO2H, Cys43-SO3H, and disulfide-bonded Cys43. Thus, the relative percentage of each form can be expressed by its ion current value divided by the sum of those contributed from all possible forms. As shown in Figure 7a, approximately 32 and 22% of Cys43 were oxidized to Cys-SO2H and Cys-SO3H, respectively, when the molar ratio of H2O2 to YADH-1 reached 400. In contrast, less than 5% of Cys43 was converted to the disulfide form under the same oxidation conditions. We did observe some oxidized Cys43 in the control sample, which might

research articles be due to oxidative damage present in the commercial preparation of the enzyme or generated during sample handling processes. However, the advantage of this method is that only the net increase in relative percentages of the oxidized cysteines or methionines between the control and the oxidized sample was considered and correlated with enzyme activity. All other factors, which are not related with the oxidation treatment, can be canceled out. Overall, approximately 78% of Cys43 was oxidized at the highest concentration of H2O2 (molar ratio of H2O2/YADH-1 ) 1600). The oxidation of Cys153 occurs at a much slower rate than that of Cys43. As shown in Figure 7b, the conversion of Cys153 to the oxidized form only reached 27% at the highest oxidant concentration. Two intramolecular disulfide-bond bearing peptides (AA 103-119 and 268-282) are the only oxidized form detected for these two peptides. The quantification was carried out by monitoring selectively their intact and disulfide-bonded forms. The results showed that 30% of AA 103-119 and 14% of AA 268-282 were oxidized at the highest dose of oxidant that we applied (molar ratio of H2O2/YADH-1 ) 1600, data not shown). By employing a similar strategy, we estimated the occurrence of the oxidation of Met75, Met270, and Met332, which, at the highest H2O2 concentration, are converted to Met sulfoxide at 28, 35, 31%, respectively (Figure S5 in Supporting Information showed the results for the former two Met residues). In addition, low ion current in the SIM for the analyses of peptides (AA 93-102, 168-172, and 190-195) could not allow us to have reliable quantification of the oxidation of Met residues in these three peptides. Because the difference in ionization efficiency for the modified and unmodified peptides may introduce some error in the estimation of the extent of cysteine or methionine oxidation, we further assessed the levels of cysteine oxidation by monitoring the losses of unmodified cysteine-bearing peptides. To this end, we analyzed the tryptic digestion mixture of untreated or H2O2-oxidized YADH-1 by monitoring selectively the fragmentation of all cysteine-bearing peptides (AA 39-59, AA 276-286, and AA 92-160; MS/MS shown in Figures S6-S8, Supporting Information) and three control peptides (AA 1-7, AA 8-17, and AA 341-347; MS/MS not shown). The latter three peptides are not susceptible to oxidative modification, and they serve as internal references for determining the losses of the unmodified cysteine-containing peptides. In this regard, we measured the areas for peaks observed in the selected-ion chromatograms (SICs) for monitoring the formation of an abundant fragment ion from the reference and cysteine-bearing peptides (Figure S9, Supporting Information). We then calculated the ratio for the peak area for the cysteine-bearing peptide over that for the reference peptide and normalized the ratios determined for the oxidized samples against that for the control sample. Such normalized ratios based on the three reference peptides were then averaged and plotted (Figure S10, Supporting Information). The results illustrated that the disappearance of the unmodified Cys43-containing peptide (AA 39-59) occurred much more rapidly than those of the peptides (AA 276-286, which contains both Cys276 and Cys277) and (AA 92-160, which carries five Cys residues including Cys 97, Cys100, Cys103, Cys111, and Cys153). This observation is consistent with the results shown in Figure 7. The above quantitative measurement results, together with the fact that Cys43 is involved in coordinating with Zn ion at the catalytic site, demonstrate that the oxidation of Cys43 plays a major role in the H2O2-induced deactivation of the enzyme. Journal of Proteome Research • Vol. 6, No. 1, 2007 223

research articles Discussion Our enzymatic activity measurements showed that H2O2 treatment could lead to the pronounced loss of the dehydrogenase activity of YADH-1. In this respect, YADH-1 was inhibited by approximately 50% at an oxidant/protein subunit ratio of 400, and the enzyme lost its activity almost completely when the molar ratio reached 1600. On the other hand, the inactivated enzyme could be partially reversed upon DTT treatment. The majority of enzyme activity loss, however, could not be rescued by DTT reduction, suggesting that the formation of disulfide bond is not the major oxidation pathway leading to the activity loss. In addition, zinc release is closely associated with H2O2-induced enzyme activity loss; approximately 1.2 zinc ions and 3 thiols were lost when the enzyme was completely inactivated. Using on-line LC-MS/MS, we identified and quantified the in vitro modifications that were generated upon the exposure of YADH-1 to H2O2. Among the eight cysteine residues in YADH-1, Cys43 and Cys153, residing at the catalytic center, were selectively oxidized to Cys-SO2H and Cys-SO3H. The similar oxidation, however, did not occur on the other 6 Cys residues. Quantitative measurement results showed that Cys43 is the most susceptible to oxidative modification (78% at the highest concentration of H2O2). In contrast, Cys153 reached the highest conversion of only 27%. The conversion of Cys thiolates to the Cys-SO2H or Cys-SO3H obstructs their bindings toward zinc ion, and ligation by His66 alone, or together with Cys153, is inadequate to sustain the zinc ion associated with the protein. Thus, the oxidation of cysteine thiol and the resulting cleavage of the Zn-S bond could lead to the permanent loss of Zn ion from the catalytic center and disable the catalytic function of YADH1. We were also able to identify three pairs of disulfide bonds, that is, Cys43-Cys153, Cys103-Cys111, and Cys276-Cys277, in the H2O2-oxidized YADH-1. Our quantitative measurements by SIM illustrated that all three pairs of disulfide-bonded adducts were in relatively low abundance, which is consistent with the results obtained from the enzymatic activity measurement. The oxidation of Cys103 and Cys111, coordinated with the second zinc center, did not exhibit strong correlation with enzyme activity loss. This result is in line with a previous study showing that the second zinc ion assumes only a structural role.7 In this regard, complete removal of zinc ion from the noncatalytic zinc site of YADH-1 by excess DTT had no effect on the zinc located at the active site and enzyme activity.7 Therefore, the oxidative damage occurring at the second zinc center is not the major contributor to enzyme inactivation. In addition to Cys oxidation, we also demonstrated that a number of Met residues were susceptible to H2O2 oxidation (Figure 1). We assumed that the oxidation of Met was of less importance in association with the activity loss because the enzyme activity loss correlates well with the decrease of free thiol group and the release of zinc ion from the enzyme. In summary, treatment of YADH-1 with H2O2 gives rise to the conversion of Cys43 and Cys153 to Cys-SO2H and Cys-SO3H, which lead to zinc loss and enzyme inactivation. In addition, Cys43 is more susceptible to oxidation than Cys153 upon H2O2 treatment. Furthermore, results from this study supply, for the first time, spectroscopic evidence supporting that the oxidation of cysteine residues in the zinc-binding domain of proteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO2H and Cys-SO3H is also possible. 224

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Determining the nature of cysteine oxidation products in proteins is important for understanding the repair of cysteine oxidation products in proteins. In this context, disulfides can be reverted to unmodified cysteines in the presence of intracellular reducing agents. On the other hand, the formation of Cys-SO2H and Cys-SO3H were previously thought to be irreversible, though recent studies revealed that Cys-SO2H in human peroxiredoxin could be reduced to unmodified cysteine in vivo.29 Moreover, a yeast enzyme catalyzing this conversion was identified.30 It remains unknown whether a Cys-SO3H in proteins can also be reduced to unmodified cysteine by some unknown enzymes in vivo. In addition, the formation of disulfides and higher stage oxidation products, namely, CysSO2H and Cys-SO3H, may both result in the release of zinc from zinc-containing proteins, but the resulting conformational and functional changes elicited by different cysteine oxidation products might be different. This may have important implications in the oxidative damage of zinc-binding proteins in general. Abbreviations: ROS, reactive oxygen species; YADH, yeast alcohol dehydrogenase;Cys-SO2H, cysteine sulfinic acid; CysSO3H, cysteine sulfonic acid; PAR, 4-(2-pyridylazo)resorcinol; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); IAM, iodoacetamide; NEM, N-ethylmaleimide; CAM,carboxyamidomethylation; DTT, dithiothreitol; β-NAD+, β-nicotinamide adenine dinucleotide (oxidized form); β-NADH, β-nicotinamide adenine dinucleotide (reduced form); LC-MS/MS, liquid chromatography-tandem MS; MALDI, matrix-assisted laser desorption/ionization; SIM, selective-ion monitoring.

Acknowledgment. The authors thank Dr. Songqin Pan at the W. M. Keck Proteomics Laboratory, Center of Plant Cell Biology, University of California at Riverside, for assistance with MALDI-MS/MS measurements. Supporting Information Available: MS/MS data for unmodified and oxidized peptides. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Sun, H. W.; Plapp, B. V. Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. J. Mol. Evol. 1992, 34, 522-535. (2) Leskovac, V.; Trivic, S.; Pericin, D. The three zinc-containing alcohol dehydrogenases from baker’s yeast, Saccharomyces cerevisiae. FEMS Yeast Res. 2002, 2, 481-494. (3) Veillon, C.; Sytkowski, A. J. The intrinsic zinc atoms of yeast alcohol dehydrogenase. Biochem. Biophys. Res. Commun. 1975, 67, 1494-1500. (4) Klinman, J. P.; Welsh, K. The zinc content of yeast alcohol dehydrogenase. Biochem. Biophys. Res. Commun. 1976, 70, 878884. (5) Vallee, B. L.; Auld, D. S. Active-site zinc ligands and activated H2O of zinc enzymes. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 220224. (6) Vallee, B. L.; Auld, D. S. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 1990, 29, 56475659. (7) Magonet, E.; Hayen, P.; Delforge, D.; Delaive, E.; Remacle, J. Importance of the structural zinc atom for the stability of yeast alcohol dehydrogenase. Biochem. J. 1992, 287, 361-365. (8) Chen, K. C.; Zhou, Y.; Xing, K.; Krysan, K.; Lou, M. F. Platelet derived growth factor (PDGF)-induced reactive oxygen species in the lens epithelial cells: the redox signaling. Exp. Eye Res. 2004, 78, 1057-1067. (9) Finkel, T.; Holbrook, N. J. Oxidants, oxidative stress and the biology of ageing. Nature 2000, 408, 239-247. (10) McCord, J. M. The evolution of free radicals and oxidative stress. Am. J. Med. 2000, 108, 652-659.

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