the non-flavin redox center of the streptococcal nadh peroxidase

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 21, Issue of July 25, pp. 12330-12338, 1989 Printed in U S. A.

The Non-flavin Redox Center of the Streptococcal NADH Peroxidase 11. EVIDENCE FOR A STABILIZED CYSTEINE-SULFENIC ACID*

(Received for publication, January 18, 1989)

Leslie B. PooleS and A1 Claiborne From the Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103

Incubation of the streptococcal NADH peroxidase with 5-thio-2-nitrobenzoate under anaerobic denaturing conditions leads to the rapid incorporation of 1 eq/FAD of the aromatic thiol. Addition of dithiothreitol to the resulting conjugate, following ultrafiltration, demonstrates that a mixed disulfide has been formed. Analysis of the denatured NADH peroxidase by isoelectric focusing reveals the presence of two predomi- nant species differing in isoelectric point by -0.1 units. Preincubation with 20 mM hydrogen peroxide gives essentially complete and irreversible conversion to the more acidic species. Treatment of the native peroxidase with low concentrations of hydrogen peroxide also leads to irreversible enzyme inactivation; the low extinction long wavelength absorbance associated with the enzyme as purified is lost in the process. Anaerobic dithionite and NADH titrations of the peroxide-inactivated enzyme indicate that, while the cysteinyl redox center is nonfunctional, the enzyme is still capable of forming a binary complex with NADH. We propose that the redox-active cysteinyl derivative which serves as the second redox center in the native peroxidase is a stabilized cysteine-sulfenic acid derivative of Cys4’. This determination is consistent with the covalent modifications observed with both 5-thio-2-nitrobenzoate and with Hz02 and is supported by mass spectrometric analysis of a chymotryptic cysteinyl peptide derived from the unmodified peroxidase.

The streptococcal NADH peroxidase contains a single redox-active cysteinyl residuelsubunit, in addition to the bound FAD coenzyme (1, 2). Reduction of the enzyme with 1 eq/ FAD of dithionite or NADH leads to the appearance of a charge-transfer absorbance band centered at 540 nm and generates a single DTNB’-reactive thiol. Although this nas- cent thiol is reactive toward DTNB and iodoacetamide under

* This work was supported by National Institutes of Health Grant GM-35394 and by American Heart Association Established Investi- gatorship Award 88-0258 (to A. C.). Peptide composition and sequence analyses were performed by the Wake Forest University Cancer Center protein sequence analysis laboratory, directed by Dr. Mark Lively of this department and supported by National Institutes of Health Grant 12197 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Dept. of Chemistry and Biochemistry, Univer- sity of Maryland, College Park, MD 20742.

The abbreviations used are: DTNB, 5,5’-dithiobis-(2-nitrobenzoate); EH,, 2-electron-reduced NADH peroxidase; E , native oxidized peroxidase; E,,,,,ive, peroxide-modified NADH peroxidase; HPLC, high performance liquid chromatography; FAB, fast atom bombard- ment; TNB, 5-thio-2-nitrobenzoate; PTH, phenylthiohydantoin.

strong denaturing conditions (2,3), it is relatively inaccessible to most sulfhydryl reagents even in the presence of low concentrations of denaturants. As shown in the previous report (3), only phenylmercuric acetate reacts readily with the 2-electron-reduced peroxidase (EHJ in the presence of 1.3 M urea. The ensuing loss of charge-transfer absorbance and of peroxidatic activity, combined with our previous analysis (2) of the single cysteinyl peptide isolated from tryptic digests, demonstrates conclusively that Cys4* of the streptococcal peroxidase serves as the charge-transfer donor in the EH, species.

Our previous analysis of the 35S-labeled NADH peroxidase (2) eliminates a number of possible structures for the oxidized cysteinyl derivative found in the native enzyme. The possible presence of either mixed or cystine disulfides can be ruled out, based on a combination of amino acid analyses and direct disulfide and thiol assays of the purified enzyme (1,2) and on the observed appearance of a single DTNB-reactive thiol on NADH reduction. Performic acid oxidation of the 35S-labeled peroxidase should also have produced the 35S-labeled sulfonic acid derivative of any low molecular weight cysteine metabo- lite present in a putative mixed disulfide (4). However, trypsin digestion followed by HPLC analysis (omitting any dialysis or gel filtration steps) clearly shows a single 35S-labeled peak corresponding to the active-site cysteinyl peptide. This analysis eliminates several organic thiols (e.g. glutathione, cystea- mine, coenzyme A, methanethiol (4, 5)) from consideration as possible components of any mixed disulfide. The terminal sulfur of an enzyme persulfide (Cys-SSH) would also be derived metabolically from cysteine ( 5 ) , and this cysteinyl derivative can similarly be discounted.

We have shown that hydrogen peroxide titration of the EH, form of the peroxidase leads to its stoichiometric conversion to oxidized enzyme ( l ) , therefore suggesting a possible catalytic role for this reduced species. In order to provide the necessary structural basis for further mechanistic studies, however, it is essential that the identity of the resulting oxidized cysteinyl derivative be established. We have ap- proached this question by combining covalent modification protocols for the oxidized enzyme with FAB-mass spectrometric analysis of the unmodified cysteinyl peptide. On the basis of these studies, a structure for the cysteinyl derivative in the native peroxidase is presented.

MATERIALS AND METHODS

Both the unlabeled and [35S]cysteine-labeled NADH peroxidases were purified from Streptococcus faecalis lOCl (ATCC 11700) as previously described (1, 2). NADH was purchased from Pharmacia LKB Biotechnology Inc., methylamine and DTNB were from Aldrich, and dithiothreitol was from Sigma. 5-Thio-2-nitrobenzoate (TNB) was prepared from DTNB by titration with dithiothreitol; a slight excess of DTNB ensured against the presence of any excess dithiol, and the resulting TNB was quantitated by its 6412 values of 14,150 M” cm” and 13,700 M” cm” in the absence and presence of 6 M

12330

Cysteine-Sulfenic Acid in Streptococcal NADH Peroxidase 12331

guanidine HC1, respectively (6). Ultrapure guanidine HC1 and urea were purchased from Schwarz/Mann Biotech, and hydrogen peroxide (30%) was from Mallinckrodt Chemical Works. Isoelectric focusing reagents, including ampholytes, were from Bio-Rad. Chymotrypsin and L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin were from Worthington. HPLC solvents were from Pierce Chemical Co. All other reagents were of the best grade commercially available.

Isoelectric focusing gels were prepared and electrophoresed using the Bio-Rad Protean I1 cell essentially as described by the manufacturer (7, 8). Five to ten mg of solid urea was added to 5 @I of the protein sample and 1 pl of the sample concentrate as described, but without the 2-mercaptoethanol, Tube gels (5 X 250 mm) were cast from a solution of 5.5 g of urea, 1.3 ml of 30% acrylamide, 0.8% bisacrylamide, 2.3 mi of 10% Triton X-100, 0.45 ml of pH 4-6 ampholytes, 0.05 ml of pH 3-10 ampholytes, and 1.97 ml of HZO. Polymerization was initiated after degassing by the addition of am- monium persulfate and tetramethylethylenediamine as previously described (8). Two hundred gl of an overlay buffer prepared according to the manufacturer's suggestions was added to each tube prior to sample application. Following completion of the suggested electro- phoresis protocol, gels were extruded and soaked in destaining solution (methanol/acetic acid/H20 (3:1:6)) to remove urea; protein bands were then visualized with Coomassie Blue R-250 after destaining with the Bio-Rad model 556 destainer.

The TNB conjugate of the NADH peroxidase was prepared by mixing the enzyme (25 nmol) from the side arm of an anaerobic cuvette with 0.9 ml of 0.28 mM TNB in 4 M guanidine HC1 buffered at pH 7. The ensuing decrease in A412 was complete within 30 s. The sample was dialyzed exhaustively by repeated ultrafiltration in 4 M guanidine HCl with a CM-30 microconcentrator (Amicon). Net protein recovery was 63% based on an c280 value of 52,000 M" cm" for the deflavoperoxidase determined with a sample of [35S]cysteine- labeled protein.

The [3sSS]cysteine NADH peroxidase (11 nmol) was inactivated by 20 min of treatment with 20 mM Hz02 to allow isolation of the modified cysteinyl peptide. Following repeated ultrafiltration at 4 "C to remove excess HZOs, the concentrated protein was diluted directly into the trypsin incubation buffer and digested as previously described (2, 3) but without further chemical modification. HPLC purification of the modified [35S]cysteine-containing peptide was performed by methods previously established in this laboratory (2, 3). Amino acid analyses of total protein hydrolysates were performed by Dr. Jan Enghild of the Department of Pathology, Duke University. Samples of the native and peroxide-modified NADH peroxidase were dialyzed against several changes of deionized water prior to lyophilization and HC1 vapor-phase hydrolysis at 110 "C for 24 h.

Preliminary digests of the unmodified [35S]cysteine NADH peroxidase with chymotrypsin ( l%, w/w, with respect to peroxidase protein) revealed the presence of two major 35S-labeled peaks in the resulting HPLC profiles. However, careful time course studies showed that the peptide eluting at -48 min was a more stable secondary cleavage product of the cysteinyl peptide eluting at -72 min. The HPLC gradient conditions for these peptide maps were identical to those previously reported (2, 3). Since there is only 1 cysteinyl residue/subunit in the NADH peroxidase (2), we focused on the earlier eluting peptide for FAB-mass spectral analysis. The unlabeled peroxidase (12.5 mg) was digested with chymotrypsin at 37 "C in the 2 M urea/NH,HCO, buffer described previously (2, 3). A second 1% (w/w) aliquot of chymotrypsin was added halfway through the 10-h incubation. Following partial purification of the peptide using a modified HPLC protocol (3.5-35% acetonitrile gradient in 0.1% trifluoroacetic acid, over 100 min), the pooled sample was rechromato- graphed with a Zorbax-ODS column (6.2 X 80 mm; 3-pm particle size) and the following solvent system: Solvent A, 0.05% trifluoroacetic acid; Solvent B, 80% acetonitrile and 0.05% trifluoroacetic acid. HPLC-grade water (Pierce Chemical Co.) was used at this stage to minimize chemical contaminants, and only volatile solvents were employed (ie. no guanidine or urea) to dissolve peptide samples. Following amino acid composition and sequence determination, FAB- mass spectral analysis was performed by Dr. Blair Fraser of the Center for Biologics Evaluation and Research, Food and Drug Ad- ministration, as previously described (4, 9). Briefly, the peptide Sam- ple was delivered in a trifluoroacetic acid/acetonitrile solution (2-10 pl) to an equal volume of a glycerol matrix on a gold-plated copper probe tip. The sample film produced in uucuo was introduced directly into the ion source. Spectra were recorded with a Kratos MS-50 instrument calibrated with cesium iodide/dithiothreitol.

Neutron activation analysis of the native NADH peroxidase was

performed by Dr. Jack Weaver of the Department of Nuclear Engi- neering at North Carolina State University. One ml of a 9 PM sample of the enzyme was dialyzed exhaustively against 50 mM Tris.SO!-, pH 7.4, containing 2 g/liter Chelex 100 (Bio-Rad) and analyzed for copper, manganese, iron, zinc, selenium, and vanadium.

All UV-visible spectral experiments, anaerobic titrations, and enzyme assays were performed at 25°C as previously described (11, using either an HP 8451A diode array spectrophotometer or a Gilford 260 recording spectrophotometer. Unless otherwise indicated, all buffers for spectral titrations included 0.6 mM EDTA.

RESULTS

Metal Cofactor and Thioester Analyses-The earlier neutron activation analyses of the NADH peroxidase reported by Dolin (10) showed selenium to be absent, and we have recently repeated this analysis with an identical result. In addition, our analyses show iron, manganese, and vanadium, all of which are cofactors in other peroxidatic enzymes (11-13), to be absent as well. One possible structure for the cysteinyl derivative in the enzyme might involve a protein thioester. However, DTNB assays of the denatured peroxidase in the presence of 150 mM methylamine, which would be expected to convert any putative thioester to free thiol plus N-meth- ylcarboxamide (14, 15), showed sulfhydryls to be absent.

TNB Reactivity of the Streptococcal Peroxidase-The possibility remained that the active-site cysteinyl derivative might be an organic sulfur oxyacid (i.e. sulfenic, sulfinic, or sulfonic acid (16)). The Cys-S02H and Cys-SOaH species can easily be eliminated, since it is generally recognized that neither is reducible (15). T h e oxidized cysteinyl derivative in the peroxidase must be reduced by substrate during the catalytic redox cycle. Alkyl-sulfenic acids such as cysteine-sulfenic acid (Cys-SOH), while reducible to the thiol form on addition of 2 electrons, are notoriously unstable in solution (17). How- ever, these oxidized derivatives of cysteine are stabilized in the chemically oxidized forms of papain and glyceraldehyde- 3-phosphate dehydrogenase (18). Low concentrations of hydrogen peroxide inactivate the thiol protease by selective reversible oxidation of CysZ5 (19); the crystal structure of the fully 9xidized cysteic acid derivative of papain is available at 1.65-A resolution (20). Due primarily to their relative instability, however, as has been pointed out by Allison (18), the existence of protein sulfenic acids rests largely on indirect evidence. Since nucleophiles react with protein sulfenic acids t o yield, for example, sulfenamides (R-S-NHR' (18)), radio- labeled amines have been employed to measure the cysteine- sulfenic acid content of oxidized glyceraldehyde-3-phosphate dehydrogenase. Incorporation of l4C-1abeled benzylamine under nondenaturing conditions is stoichiometric and commen- surate with the loss of the acylphosphatase activity intrinsic t o t h e oxidized enzyme (18). Since methylamine is a relatively small isoelectronic substitute for hydrogen peroxide, we rea- soned that this nucleophile might present a good choice for probing the proposed cysteine-sulfenic acid of the peroxidase. However, exposure of the native enzyme to 150 mM CH3NHz at p H 7.5 has no effect on peroxidase activity even after a 6- h incubation. The presence of 1.3 M urea (3) had no effect in facilitating any methylamine inactivation at incubation times up to 45 min, and the inclusion of methylamine in the standard peroxidase assay had no effect on initial rates. Failure to inactivate may, however, be due to the relatively high pK, (10.6 (21)) for the primary amine function of methylamine.

Another well characterized reaction of sulfenic acids is their rapid condensation with thiols to produce disulfides (16). Given the relative inaccessibility indicated for the thiol of the peroxidase EH, species (3), we decided to probe the oxidized

12332 Cysteine-Sulfenic Acid in Streptococcal NADH Peroxidase

enzyme with TNB under anaerobic denaturing conditions. The native peroxidase was mixed anaerobically with a 10-fold excess of TNB in buffered 4 M guanidine HCl. The decrease in AdI2, due to reaction of the aromatic thiol, was rapid and corresponded to conversion of 0.9 mol/enzyme monomer (corrected for changes due to dilution and free FAD contribution). In order to confirm that the covalent coupling of TNB had occurred, the enzyme sample was freed of excess reagent by repeated ultrafiltration on a CM-30 microconcentrator in 4 M guanidine HC1. The concentrated protein was diluted in buffered guanidine, and the resulting spectrum is shown in Fig. 1 (curue 1 ). From the t2m determined for the peroxidase apoen- zyme on a separate sample of “S-labeled protein of known specific activity, recovery of the protein after repeated ultrafiltration in the presence of guanidine HCl was measured at 63%. In addition to the characteristic near-UV absorbance maximum (-335 nm) of the protein-thiol adduct (6), the quantitative release of TNB on addition of dithiothreitol confirms the identity of the conjugate as a mixed protein- TNB disulfide. We conclude that this mixed disulfide is formed by the reaction of the aromatic thiol with the Cys4*- sulfenic acid of the peroxidase as follows.

CYS-SOH + TNB-SH -, CYS-SS-TNB + Hz0

Hydrogen Peroxide Inactivation-Hydrogen peroxide is a commonly employed chemical modification reagent for methionine and cysteine residues (22). For example, the nones- sential Cys”‘ of the flavoprotein p-hydroxybenzoate hydroxylase has been shown to react with H202; oxidation is coincident with the generation of several more acidic species of the protein which are thought to correspond to the sulfenic, sulfinic, and sulfonic acid oxidation states of the cysteinyl sulfur (23). In early analyses of the isoelectric focusing behavior of the streptococcal peroxidase, two major protein bands separated by -0.1 units in PI were observed (Fig. 2, lane A) . Preincubation with dithiothreitol prior to sample preparation was shown to favor the less acidic band somewhat (Fig. 2, lane B ) , suggesting that an oxidative process, perhaps facili- tated by the denaturing conditions involved in the isoelectric

.3

8.2 z < lY 0 cn <

m

m . 1

TIME. m l n

400 500 600 WAVELENGTH. nm

FIG. 1. Reduction of NADH peroxidase-TNB conjugate with dithiothreitol. The enzyme-TNB conjugate (curue I , in 0.6 ml of phosphate-buffered 4 M guanidine HCI, pH 7.0) was introduced into an anaerobic cuvette. 220 nmol of dithiothreitol was then added from the side arm; spectra 2-5 represent scans taken 1, 2, 3, and 30 min, respectively, after mixing. The inset shows the semilogarithmic plot derived from the change in uersus time. values were converted to equivalents of TNB/subunit, taking the value of 13,700 M” cm-I (6) and a subunit concentration of 26 PM, as corrected for losses during repeated ultrafiltration in 4 M guanidine. Further details of the protocol are given under “Materials and Methods.”

FIG. 2. Isoelectric focusing of purified NADH peroxidase. Samples were prepared in urea as described under “Materials and Methods” and electrophoresed with pH 4-6 ampholytes. Gels A-D represent: A, enzyme without preincubation; B, enzyme preincubated for 1 h with 35 mM dithiothreitol; C, enzyme preincubated for 1 h with 20 mM H202; and D, a mixture of the samples applied to gels B and C. The cathode is located above the gels as shown.

focusing protocol, might be responsible for the more acidic species. In order to test this hypothesis, it was shown that preincubations of the native enzyme with hydrogen peroxide prior to sample preparation gave essentially complete conversion to the more acidic band (Fig. 2, lane C). Dithiothreitol had no effect on the mobility of the resulting species, suggesting that an irreversible oxidation had taken place. Since Van Berkel and Muller (23) had shown that peroxide oxidation of Cys”‘ of p-hydroxybenzoate hydroxylase gave rise to the more acidic sulfinic and sulfonic acid derivatives, we tentatively concluded, in view of the results of our TNB- coupling experiment, that oxidation of the proposed C Y S ~ ~ - sulfenic acid of the NADH peroxidase might be responsible for these observations.

Hydrogen peroxide treatment of the enzyme a t concentrations thought to lead exclusively to methionine and/or cysteine oxidations (24) causes an irreversible loss of peroxidatic activity; semilogarithmic plots of residual activity uersus time are linear over four half-lives. A direct plot of koba (pseudo first-order rate of inactivation) uersus peroxide concentration is linear with a zero intercept. The second-order rate constant of 8.1 M-’ min” is consistent with the following simple kinetic scheme.

E + HZOZ + Einanive

When the peroxide-inactivation process was followed spec- trally a t neutral pH, visible absorbance changes in the enzyme were observed which corresponded to the expected rate of activity loss (Fig. 3). Although little spectral change occurs in the vicinity of the 450-nm absorbance maximum, the near- UV maximum a t 380 nm decreases in extinction and is slightly blue-shifted. The low extinction long wavelength band previously observed with the purified peroxidase (1) essentially disappears during the incubation.

Redox Properties of Peroxide-inuctiuated Enzyme-In order to test the effects of peroxide inactivation on the redox properties of the peroxidase, the enzyme was inactivated with 10 mM H202 to a level of about 10% residual activity. After removal of excess peroxide by CM-30 ultrafiltration, the sample was subjected to an anaerobic dithionite titration. This experiment (data not shown) showed a lag of approximately 0.5 eq/FAD of reductant, after which the flavin peaks began to disappear. At no point in the titration, however, was there any hint of the EH2 intermediate seen at -1 eq/FAD of dithionite with the native enzyme (1). Full reduction (includ-


I

I . 400 1 . L- 500 600 700

...............,.

WAVELENGTH. n m

FIG. 3. Spectral changes accompanying peroxide inactivation of the streptococcal peroxidase. The enzyme (-, 21 nmol) in 0.55 ml of pH 7.0 phosphate buffer was mixed with 10 mM hydrogen peroxide, and the spectra shown were taken 8.5 (. . . .) and 34 min (---), respectively, after addition. The inset gives the semilogarithmic plot of the change in Asao uersus time and yields a tlh of 9 min, which agrees well with the corresponding kobs for activity loss.

ing the initial lag) required 3.5 eq/FAD of dithionite. Since full reduction of native enzyme requires only 2 eq/FAD of dithionite (1) and since the EH2 intermediate characteristic of preferential reduction of the cysteinyl redox center was absent, it occurred to us that reversible methionine oxidations (yielding the methionine sulfoxide derivative (25)) might account for those reducing equivalents in excess of the 1 es/ FAD stoichiometry expected for the bound flavin. It is known that organic thiols will reduce methionine sulfoxide to the thioether level under, in some cases, gentle conditions (22, 25); dithionite is a considerably more powerful reductant than are most thiols (26). In order to test the hypothesis that reversible oxidations involving methionine (there are 10-11 methionines/FAD in the native peroxidase, including Met44 only 2 residues away from the active-site cysteine (1, 3)) had occurred during peroxide modification, the dithionite-reduced sample described above was air-oxidized and dialyzed to remove sulfite generated in the original reductive titration. A second dithionite titration was then carried out with the prereduced sample; the results are shown in Fig. 4. In this case the lag in flavin reduction is essentially abolished, there is still none of the EH, charge-transfer intermediate, and the overall titration requires only 1.3 eq/FAD of dithionite. I t should be emphasized that prior reduction and air oxidation have no effect on the low level (-10%) of residual peroxidase activity nor do the spectral properties of the native oxidized enzyme reappear. At no point does significant flavin loss occur with the modified enzyme, indicating that the FAD- binding site is intact. The data of Fig. 4, given the absence of other reducible sites on the enzyme apart from the flavin, lead us to the conclusion that peroxide inactivation has led to irreversible oxidation of the cysteinyl redox center.

NADH titrations of the native peroxidase lead successively to EH, and EH2. NADH forms; each phase of the titration requires 1 eq/FAD of the pyridine nucleotide (1). The dithionite titration of Fig. 4 shows that the EH2 form does not appear with the peroxide-inactivated enzyme. Therefore, an anaerobic NADH titration was performed with a freshly inactivated peroxidase sample. The results shown in Fig. 5 confirm the absence of an EH2 intermediate in the NADH titration. Instead a spectral form very similar to the EH,. NADH species seen with the native enzyme appears, except

W U Z m a rr 0 ul m a OITHIONITE. oq i

400 500 600 700

WAVELENGTH. n m

FIG. 4. Dithionite titration of peroxide-inactivated NADH peroxidase. The starting enzyme had previously been inactivated by treatment with 10 mM H20, as in Fig. 3, freed of excess Hz02 by ultrafiltration, and prereduced with 3.5 eq/FAD of dithionite as described in the text. Following ultrafiltration, the sample (curue 1, 19 nmol) in 0.55 ml of 50 mM phosphate, pH 7.0, was titrated with dithionite; spectra shown represent the enzyme after addition of 0.48 (---), 0.93 (. . . .), and 1.59 (--) eq/FAD of dithionite, as corrected for the slight lag due to residual oxygen. The inset shows the change in Also versu.s added dithionite, which gives a corrected end point of 1.25 eq/FAD.

W

z U

4

(z 0

0 ul

4

m .02

1.0 2 . 0 1 t

"

, . .-, . . ' --- """" . . , . . . . . , . . . . .

500 600 700 400

WAVELENGTH. n m

FIG. 5. Anaerobic NADH titration of peroxide-inactivated streptococcal peroxidase. The enzyme (-, 20 nmol), previously inactivated as in Fig. 3 and freed of excess H 2 0 2 , was titrated with an anaerobic solution of 2.4 mM NADH; the sample volume was 0.55 ml in 50 mM phosphate buffer, pH 7.0. Spectra shown represent the additions of 0.61 (. . . .) and 1.68 (---) eq/FAD of NADH. The inset shows the change in AS4o versus added NADH and gives an end point of 1.14 eq/FAD.

for the presence of a resolved shoulder at 470 nm and a higher c450 value. The titration is complete with 1.1 eq/FAD of reductant; the presence of the reduced pyridine nucleotide is easily monitored by its absorbance a t 340 nm. Particularly significant is the fact that the 540-nm absorbance of this Ei,,,,,,,.NADH species is virtually identical to that of the native EH2. NADH complex. Opening the cuvette to air leads to the gradual disappearance of the reduced pyridine nucleotide; this effect could be due to an intrinsic oxidase activity of the modified enzyme (which seems unlikely given that flavin reduction appears minimal) or to the -10% residual, presumably unmodified enzyme (which has an intrinsic NADH oxidase activity (l)), in the peroxide-treated sample. In any event, the sample showed no change in peroxidase activity after NADH titration.

Analysis of the Cysteinyl Peptide from Inactivated Peroxi-


dase-As stated previously, peroxide treatment of the enzyme followed by denaturation and isoelectric focusing shows complete conversion to a more acidic protein species. Amino acid analyses of control and Hp02-inactivated peroxidase samples showed no change in methionine content; this is consistent with the results of previous studies which show methionine sulfoxide to be reduced under the conditions of acid hydrolysis (22, 25). Hydrogen peroxide, especially at the relatively low concentrations employed, is not thought to oxidize methionine to the irreversible sulfone level (22). The conversion of the proposed Cy~~~-sulfenic acid on peroxide treatment to the sulfinic or sulfonic acid levels is consistent with the observed loss of the cysteinyl redox center and with the more acidic PI of the oxidized apoprotein. Amino acid analyses also demonstrated the presence of cysteic acid in hydrolysates of the peroxide-inactivated enzyme; this derivative was not detected in control samples of the peroxidase. However, since quanti- tation was less than stoichiometric (-0.2 cysteic acidlsub- unit), the cysteinyl peptide was isolated from the peroxide- inactivated [35S]cysteine-labeled protein.

Fig. 6B shows the peptide map resulting from a tryptic digest of the modified protein, which is virtually superimpos- able with the map of an unmodified control enzyme sample (Fig. 6A). The only peptide peak showing a significant change in mobility is observed at -101 min; this peptide cannot be resolved from the more pronounced peak at 100 min in control samples. Rechromatography of both peaks from the modified peptide map confirms that the later eluting peptide is radioactive; this indicates that at least 90% of the cysteinyl peptide has been modified by hydrogen peroxide. The purified [35S] cysteine-containing peptide was analyzed by automated Ed- man degradation and gave the same sequence as that previ-

U u Z 4

[r 0 cn D 4

m

0 20 40 60 __ 80

l o o 600

400

200

20 100 1

cn I

TI a 3

"

TIME. min

FIG. 6. Comparative tryptic maps of the unmodified and peroxide-inactivated NADH peroxidase. A , 20 nmol of a tryptic digest of the unmodified enzyme; B, 5 nmol of a parallel tryptic digest of the [35S]cysteine enzyme inactivated with 20 mM H202. Conditions for digestions and HPLC analyses are described under "Materials and Methods." Peptide absorbance a t 206 nm (-) is plotted versus retention time, and the radioactivity determined for each of the 2- min fractions in B ( . . . .) is also presented.

ously reported for the active-site cysteinyl peptide (2, 3): Gly- Asp-Phe-Ile-Ser-Phe-Leu-Ser-Cys-Gly-Met-Gln-Leu-Tyr- Leu-Glu-Gly-Lys. The presence of an acidic cysteinyl derivative (probably cysteic acid) was suggested by the amino acid composition of this peptide. This was confirmed by coelution of the PTH-derivative in cycle 9 with an authentic PTH- cysteic acid standard and by counting all 18 sequenator cycles for radioactivity. In addition, comparison of the PTH-derivative in cycle 11 with an authentic PTH-methionine sulfone standard showed this oxidized derivative to account for 4 0 % of the methionine in this cycle. Taken together with the analyses of methionine and cysteine contents, isoelectric focusing behavior, and redox properties of the peroxide-inactivated enzyme, these data support the conclusion that peroxide treatment leads to irreversible oxidation of the Cy~~~-sulfenic acid.

FAB-Mass Spectral Analysis of the Unmodified Cysteinyl Peptide-In attempting to establish the chemical identity of the cysteinyl derivative in the NADH peroxidase without prior covalent modification, we sought to isolate the unmodified cysteinyl peptide from tryptic digests of 35S-labeled peroxidase. However, HPLC purification of the single 35S-labeled peptide peak from these digests, followed by sequence analysis, consistently indicated the presence of a contaminating peptide in roughly equal proportions. Since this contaminant could not be resolved using a number of other columns and/ or elution conditions, we turned to chymotryptic digestion of the unmodified peroxidase. Although two major radioactive peaks are present in the peptide map resulting from a 10-h digest of the peroxidase in the presence of 1% (w/w) chymotrypsin, we focused on the 35S-labeled peptide eluting at 48 min (see "Materials and Methods"). Automated Edman analysis of this material after purification by rechromatography gave the sequence Leu-Ser-Cys-Gly-Met-Gln-Leu-Tyr, which corresponds to residues 7-14 of the tryptic active-site peptide previously described (2, 3). The presence of phenylalanine in position 6 of the tryptic peptide is consistent with the cleavage by chymotrypsin, as is the cleavage following tyrosine at position 14. The PTH-derivative corresponding to cycle 3 of the chymotryptic peptide eluted at or near the solvent break- through on HPLC analysis; this is consistent with the presence of an acidic derivative and is identical to the elution position of authentic PTH-cysteic acid. The presence of a [35S]cysteinyl derivative in this cycle was confirmed by scin- tillation counting. This assignment is consistent with the corresponding amino acid composition, which also indicates the presence of an additional acidrc residue (probably cysteic acid).

The molecular ion calculated for this peptide (M + HI+, containing the Cys4'-sulfenic acid as proposed, is of mass 930. Further oxidations at Cys4' and/or at Met44 could lead to derivatives containing up to four additional oxygen atoms (each of atomic mass 16). The FAB-mass spectrum for this peptide displayed a major intensity at mass 978, corresponding to that mass expected for the molecular ion plus three additional oxygen atoms. A small intensity at m/z 914 indicated the presence of the cysteine form of the peptide. The amino acid composition and sequence analyses cited above indicated the presence of cysteic acid in the chymotryptic peptide. The ion at m/z 978 perhaps represents the fully oxidized Cys4' (cysteic acid; Cys-SOsH) and the partially oxidized Met44 (methionine sulfoxide) form of the peptide. The 914 ion, if interpreted correctly, could represent the product of disproportionation between two sulfenic acid moie- ties, yielding thiol plus sulfinic acid (16).


DISCUSSION

On the basis of the analyses presented in this report, we conclude that the redox-active cysteinyl derivative of the streptococcal NADH peroxidase is a stabilized cysteine-sulfenic acid (Cys-SOH). In reaching this conclusion numerous alternative structures, including disulfides, persulfides, and thioesters, have been eliminated experimentally. Selenocys- teine is absent as well, based on neutron activation analysis of the intact protein. The sulfenic acid structure of Cys4' in the native peroxidase is most consistent with a number of chemical properties of the enzyme. TNB reacts readily with the denatured enzyme under anaerobic conditions to give the mixed Cys4'-S-S-TNB disulfide. A similar reaction has been observed previously between TNB and the CysZ5-SOH form of oxidized papain (19). The absence of reaction observed with methylamine under nondenaturing conditions is probably due to the relative inaccessibility of Cys4' (3) combined with the predominance of the methylammonium cation (CH3NHz) at pH < 9.5 (21). Although the reversible sulfenic acid oxidation state of the peroxidase allows reduction by 1 eq/FAD of NADH or dithionite, further oxidations by hydrogen peroxide lead to the irreversible sulfinic (-S02H) and/or sulfonic (-S03H) acid oxidation states (15).

Cys4'--SOH - C~S~~-SOZH - C ~ S ~ ~ - - S O ~ H HzOz &Oz

Thus, NADH or dithionite titrations of the peroxide-inactivated enzyme do not reduce the cysteinyl redox center and do not give the characteristic 540-nm absorbance attributed to thiol + FAD charge-transfer. The -S02H pK, value of cysteine-sulfinic acid is 1.50 (27); the aliphatic sulfonic acids, including cysteic acid, are considerably more acidic (pK, - -6 for ethanesulfonic acid (16)). The combined effects of sulfenic acid autoxidation under the aerobic denaturing conditions employed in sample preparation and of peroxide oxidation during preincubation with H202, both of which lead to the appearance of a more acidic species on isoelectric focusing of the peroxidase, can thus be attributed to the increased acidity of the oxidized cysteine. Similar observations were made during peroxide oxidation of Cysl" of p-hydroxybenzoate hydroxylase (23). Finally, FAB-mass spectrometric analysis of the chymotryptic cysteinyl peptide, isolated from the NADH peroxidase without prior chemical modification, gives a molecular ion of m/z 978, which is consistent with the following structure.

0 T

I I S03H SCH3

NH2-Leu-Ser-Cys-Gly-Met-Gln-Leu-Tyr-OH

The assignment of the cysteic acid structure for the cysteinyl derivative in the chymotryptic peptide derived from the unmodified peroxidase is in agreement with the results of both amino acid composition and sequence analyses of this peptide. This assignment is also identical to that proposed for the cysteinyl derivative present in the active-site tryptic peptide of the peroxide-inactivated enzyme. It would appear that oxidation of the cysteine-sulfenic acid present in the native peroxidase has occurred during proteolytic digestion in the presence of urea and/or during subsequent purification of the chymotryptic peptide by HPLC. Our amino acid analysis of the peroxide-modified NADH peroxidase, which showed only -0.2 cysteic acid/subunit, might appear to contradict the structural assignment for the tryptic peptide derived from

this protein sample. However, it must be emphasized that the hydrolysis and composition analysis was performed on a sample of the intact enzyme which had been maintained at -20 "C following inactivation and removal of excess peroxide. Cys- teine-sulfinic acid, the primary product of the reaction of hydrogen peroxide with the Cys4'-sulfenic acid, is heat-labile (28) and therefore would be recovered only in low yields on vapor-phase hydrolysis at 110°C. Atmospheric oxygen (16, 29) is likely to be responsible for oxidations at the peptide level; our combined mass spectrometric, sequence, and composition data suggest that the cysteine-sulfonic acid is the ultimate product of this process. The oxidation of methionine to the sulfoxide is also commonly encountered in protein chemistry (25), particularly in the purification of methionyl peptides, and we attribute the oxidation of Met44 in the chymotryptic peptide from the unmodified peroxidase to this source. In the previous report (3) we indicated that alkylations of the peroxidase, following incubation under nitrogen with buffered 8 M guanidine HC1 for 3.5-4 h in the presence of dithiothreitol, gave only substoichiometric incorporation of i~do['~C]acetamide. In fact, the unlabeled cysteinyl peptide was easily identified by sequence analysis of the corresponding HPLC peak. We attribute the relatively poor alkylation to the instability of the C ~ S ~ ~ - S O H derivative under the denaturing conditions applied. The nitrogen atmosphere employed is not strictly anaerobic, and thiols such as dithiothreitol are known to catalyze oxidations of some protein residues in the presence of oxygen (15,30). We conclude that such oxidations during the 4-h denaturation lead to sulfinate and/or sulfonate derivatives of which are not reactive with iodoacetamide.

In discussing the sulfenic acid oxidation state of Cys4', it is important to stress that the solution behavior of alkyl-sulfenic acids differs considerably from that observed with protein derivatives (18). The aliphatic sulfenic acids are extremely reactive in solution (17), generally yielding thiosulfinates

0 T

(RSSR) in the absence of other reagents. On proteins, however, such reactions can be eliminated entirely, and reactivity with solvent-borne nucleophiles can be controlled by limited accessibility. Several additional stabilizing factors which have been described for these derivatives include ionization to the conjugate sulfenate base (R-S-0- (17)); the pKa values of aromatic sulfenic acids are roughly equivalent to those of the corresponding phenols (16). The extensive hydrogen bond contacts involving the oxygen atoms in the CysZ5-SO3H derivative of papain (20) and those protein residues which constitute the oxyanion binding pocket, in addition to their electrostatic interactions with the N terminus of the papain L1 helix, give detailed insights into the molecular forces likely to be involved in cysteine-sulfenate stabilization by elements of protein structure. Although the detailed analysis of the Cys4'-sulfenic acid environment of the peroxidase must await a complete structural determination, two active-site features which would lend stabilization to this derivative can be de- duced from available information. The previous report (3) shows that the EH2 thiol of the peroxidase is extremely unreactive toward alkylating reagents, methyl methanethiolsulfonate, and mercurials in the absence of denaturants. Only phenylmercuric acetate reacts rapidly even in the presence of 1.3 M urea. These data indicate that the Cys4' thiol of the 2- electron-reduced enzyme is relatively inaccessible to solvent. Barring major conformational shifts on reduction (the charge- transfer of glutathione reductase moves -0.1 A from its position in the oxidized enzyme (31)), the Cy~~~-su l f en ic acid


of the oxidized peroxidase should similarly be protected from solvent.

The 340-fold rate enhancement observed (3) for the reaction of mercurial with the peroxidase EH, thiol in the presence of low concentrations of urea also suggests that the environment of the essential Cys4' thiol is somewhat hydrophobic. This is consistent with the fact that the mercurial is considerably more effective than are alkylating agents or methyl methanethiolsulfonate in reacting with the peroxidase thiol, since mercury itself possesses markedly hydrophobic character (32). Liu (15) has suggested that in addition to limited solvent accessibility, the stabilization of protein sulfenic acids may involve hydrophobic interactions, consistent with the preferential association generally observed for cysteine thiols with apolar regions of protein structure. We have also shown, as in the case of lipoamide dehydrogenase, that the pK, for the charge-transfer thiol of the peroxidase EH, form is probably 5 5 , based on the absence of changes in e540

over the pH range 5.4-8.5 (1). From the high resolution structure of glutathione reductase (33, 34), this dramatic lowering of thiol pK, has been attributed to ion pair formation with His467'. In the case of the reduced NADH peroxidase, ion pair formation would also serve to allow charge complemen- tation in the relatively apolar environment indicated. A similar stabilization of the anionic cysteine-sulfenate form of the oxidized enzyme might have significance with respect to its intrinsic long wavelength absorbance. The high polarity of the sulfur-oxygen bond (15) strongly suggests that the electron-rich sulfenate oxygen could serve as a charge-transfer donor to oxidized flavin in the native enzyme. The electron- rich oxygens of bound phenolate ligands serve as charge- transfer donors in the flavoprotein Old Yellow Enzyme, for example (35). The catalytic reduction of the sulfenic acid presumably mediated by the transiently reduced flavin in the NADH peroxidase requires an intermolecular distance of at most a few angstroms, consistent with the CYS~~:FAD sepa- ration in glutathione reductase (31). The disappearance of the peroxidase long wavelength absorbance band on peroxide inactivation, associated with irreversible oxidation of the cysteinyl redox center, also appears to support such a conclusion. The structural elucidation of the native peroxidase will, however, provide the ultimate experimental test of this hypothesis.

Our identification of the redox-active Cys4* derivative of the streptococcal peroxidase as a sulfenic acid is entirely consistent with the 2-electron reduction and oxidation stoi- chiometries observed in the interconversion of the peroxidase E and EH2 redox states. The reversible formation of the cysteine-sulfenic acid is ensured by the fact that no other protein thiols exist; Cys4' is the only cysteine identified in tryptic digests of the 35S-labeled peroxidase. Thus, intramo- lecular disulfide formation is prohibited. Given this situation, the formation of the stabilized sulfenic acid by the oxidizing substrate hydrogen peroxide is in fact predicted. Although we have previously demonstrated that the peroxidase EH2 form is oxidized on stoichiometric addition of Hz02 (l), this is not necessarily the preferred catalytic mechanism. We have also shown that the peroxidase EH2 redox state binds NADH stoichiometrically to yield EH2.NADH; the very low Kd of -0.3 PM suggests a probable role in catalysis for this complex. The NADH peroxidase may thus be similar to the flavoprotein mercuric reductase, which is thought to cycle through a catalytically competent EH2.NADPH complex (36, 37). This characteristic distinguishes the latter enzyme from glutathione reductase and the other disulfide reductases, which cycle

between E and EH, redox states (38). Our working hypothesis for the catalytic cycle of the streptococcal peroxidase can therefore be represented as follows.

E e!, EH;NADH

I/HOOH

4 D I r N A D H

In this model, the oxidized enzyme is initially "primed" by 1 eq/FAD of NADH; subsequent catalytic cycles involve the EH, and EH2.NADH intermediates. There are two significant aspects to this proposed catalytic mechanism which should be stressed in view of the oxidations of both E and EH, species observed with hydrogen peroxide. In this report we have shown that the peroxidase in its native oxidized state ( E ) is susceptible to irreversible inactivation by hydrogen peroxide. With the EH2. NADH complex, however, the sulfenic acid formed initially should be rapidly reduced back to thiol, thus diminishing the possibility of irreversible oxidation of C ~ S ~ ~ - S O H t o t h e sulfinic acid level.

A second consideration of the EH2.NADH complex as a postulated catalytic intermediate concerns thiol reactivity with hydrogen peroxide. Based on the chemistry of alkyl sulfide reactions with peroxides, we would expect the reaction of Cys4* with Hz02 to proceed via nucleophilic attack on peroxide oxygen (39, 40).

+ H H:B - \

cys4,-s . . . a o . . . . 0. - \

CyS4,-SOH + H,O + :B

'H

Since the nucleophilic sulfur must provide both bonding electrons in forming the S-0 bond, the accessibility of one of the electron pairs of the cysteine S, atom to peroxide oxygen is a critical factor in the reaction coordinate. In the E H 2 form, however, the cysteine sulfur is sharing electron density with the oxidized flavin; in addition, the charge-transfer interaction in the peroxidase EH, form restricts the orientation of the sulfur and may further limit its role as a nucleophile toward peroxide. In the EH,.NADH complex, however, our data suggest that the charge-transfer absorbance is primarily due to an NADH -+ FAD interaction. The Cys4' thiolate of the peroxidase EH2.NADH complex may thus be further activated for nucleophilic attack on peroxide by displacement from its charge-transfer interaction with the flavin. A thor- ough analysis of the rates of peroxide reaction with EH, and EH2. NADH forms will, however, be required to test the validity of this hypothesis.

We have shown that thiol reagents in general, including mercurials and the relatively small neutral reagent methyl


methanethiolsulfonate, react very poorly with the peroxidase EH2 sulfhydryl (3). Hydrogen peroxide, in sharp contrast, reacts rapidly with the 2-electron-reduced enzyme at even substoichiometric concentrations. From the steady-state kinetic constants reported earlier (41) for the peroxidase, we can calculate2 k3, the second-order rate constant for the reaction of HzO2 with the reduced peroxidase, to be 5.6 x lo7 M-' min". Although the smaller size and neutral character of the peroxide molecule undoubtedly contribute to its accessibility to the active-site thiol, it would appear that other factors, perhaps specifically involved in stabilization of the transition state during peroxide reduction, must be operative in order to allow this enhancement of thiol reactivity. The calculated value for k , can also be compared directly with the second-order rate constant for peroxide inactivation (8.1 M" min"). There is a kinetic bias of -7 X 106-fold favoring the reaction of H202 with reduced versus oxidized peroxidase; intrinsic reaction rate factors undoubtedly contribute to this enhancement and may reflect an evolutionary influence in designing a more efficient protein catalyst which is also less sensitive to oxidative inactivation.

The streptococcal peroxidase, to our knowledge, represents the only enzyme thought to utilize the cysteine-sulfenic acid oxidation state in its natural catalytic function. An artificial peroxide-modified glyceraldehyde-3-phosphate dehydrogenase has intrinsic acylphosphatase activity which is absent, however, in the native enzyme (18). Mechanistically, the NADH peroxidase resembles the selenium-dependent glutathione peroxidase (43), which catalyzes peroxide reduction via the selenenic acid (Cys-SeOH) form of the selenocysteine derivative. The rate of peroxide reaction with the glutathione peroxidase selenolate center (Cys-Se-) is 1.8 X 10' M" s" at pH 7.0 and 37 "C (43); the reduced enzyme thus reacts with H2O2 approximately 100-fold faster than does the 2-electron- reduced NADH peroxidase at 25 "C. We have previously noted (3) that the active-site Cys4' of the streptococcal enzyme is located immediately following a structural element which is likely to represent an ADP-binding Pa0 fold (44). The selenocysteine derivative of Cys45 in bovine erythrocyte glutathione peroxidase is similarly located at the N-terminal end of helix al, which is known to participat! in a (lab superse- condary structural element from the 2-A crystal structure (45). Epp et al. (45) have suggested that this location near the a-helical N terminus may stabilize the active-site selenolate and enhance its nucleophilic behavior toward H202. Similar features may contribute to stabilization of both the NADH peroxidase cysteine-sulfenate and of the charge-transfer thiolate in the reduced enzyme.

Finally, it is of interest that two streptococcal proteins have now been described which represent unique cases for post-

' Stoll and Blanchard (41) have recently determined steady-state kinetic constants at pH 7.5 and 25 "C with a commercially available NADH peroxidase preparation. A ping-pong kinetic mechanism is indicated, Km(H20z) is 12 pM, and V,,, is 14.7 pmol/min/mg (680 min", based on a subunit molecular weight of 46,00O/flavin). Apply- ing the Dalziel model for a ping-pong system (Type IV(i) (42)), the oxidative half-reaction of this enzyme can be represented as

k3 k;

k4 E' + H202 E'Y E + HZ0

and the kinetic coefficient 62 = (k4 + k;)/(k,k;). Our earlier titration data (1) show the apparent dissociation constant for peroxide reaction with EH, to be 510-@ M, thus kB >> k,. If k; Z k3, then @* 2. Ilks. From the data of Stoll and Blanchard, & = 1/VmaX= 1.5 x min, and @Z = &.Km(H2O~) = 1.8 X lo-@ M min. Thus k, = = 5.6 x IO7 M-' min".

translational modifications of cysteine sulfur. The streptococcal proteinase is synthesized as an S-thiomethyl conjugate of Cys47 which is reduced during extracellular activation of the catalytically essential sulfhydryl (4). Cysteine-sulfenic acids have been proposed as intermediates in protein disulfide formation (18); it remains an open question a t this point whether this modification of the peroxidase occurs coincident with folding or after flavin incorporation. The latter hypothesis may be favored, however, since the native protein in its reduced ( i e . CyC2-SH) form should still readily yield the sulfenic acid on reaction with H202.

Acknowledgments-We would like to thank Dr. Blair Fraser of the Center for Biologics Evaluation and Research, Food and Drug Ad- ministration, for performing the FAB-mass spectral analysis and for many valuable discussions. We would also like to thank Drs. Mark Lively and Jan Enghild for performing the amino acid sequence and composition analyses.

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