Active-site Structural Comparison of Streptococcal NADH

THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 267, No. 6, Issue of February 25, pp. 3832-3840,1992 Printed in U. S. A.

Active-site Structural Comparison of Streptococcal NADH Peroxidase and NADH Oxidase RECONSTITUTION WITH ARTIFICIAL FLAVINS*

(Received for publication, July 24, 1991)

S. Ashrafuddin AhmedS and A1 Claiborneg From the Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157-1016

The apoproteins of the streptococcal NADH peroxidase (HzOz + 2Hz0) and NADH oxidase ( 0 2 * 2Hz0) stabilize the neutral forms of 6-hydroxy- and 6-mercapto-FAD, respectively. The redox behavior of the 6- hydroxy-FAD peroxidase closely mimics that of the native enzyme with both dithionite and NADH. Both oxidase and peroxidase preferentially stabilize the N( 1)-protonated p-quinonoid species of 8-mercapto- FAD, and the 8-position of the bound flavin is accessible to solvent in both proteins. The 8-mercapto-FAD peroxidase yields an EH, spectrum on reduction virtually identical to that seen with 8-mercapto-FAD glutathione reductase, but no distinct EHpNADH form appears. The dramatic decreases in reactivity at the flavin 2- and 4-positions for both the peroxidase and the oxidase, assessed with the reconstituted 2- and 4- thio-FAD enzymes, suggest that these positions are buried by elements of both protein structures. Further- more, reconstitution of the peroxidase with the higher potential 2- and 4-thioflavins yields enzyme forms which are fully reducible with 1.4 eq of NADH/FAD, giving rise to stable thio-FADH,-NAD+ complexes. This behavior closely mimics that of the native NADH oxidase and provides further evidence supporting the hypothesis that a major functional distinction between the two structurally related proteins is determined by the redox potential and/or NADH reactivity of the bound flavin coenzyme.

The NADH peroxidase from Streptococcus faecalis 1OC1 is an FAD-containing enzyme which reduces hydrogen peroxide t o water at the expense of the pyridine nucleotide donor (1, 2). Studies in this laboratory over the last five years (3, 4) have outlined some of the detailed similarities between this enzyme and the flavoprotein disulfide reductases, encompass- ing both redox and spectroscopic properties as well as the essential involvement of a cysteinyl redox center. In the peroxidase, however, the oxidized non-flavin center is an unusual stabilized cysteine-sulfenic acid (Cys-SOH) capable of accepting two electrons on reduction to the Cys-SH state

* This work was supported by National Institutes of Health Grant GM-35394 and by a grant-in-aid from the American Heart Associa- tion. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Laboratory of Biochemical Pharmacology, NIDDK, NIH, Bethesda, MD 20892.

3 Established Investigator of the American Heart Association. To whom correspondence should be addressed Dept. of Biochemistry, Wake Forest University Medical Center, Medical Center Blvd., Win- ston-Salem, NC 27157-1016. Tel.: 919-748-3914; Fax: 919-748-7671.

( 5 ) ; each of the disulfide reductases contains a redox-active cystine disulfide. The npr structural gene encoding the peroxidase has recently been sequenced (6) and, in spite of the relatively weak homology with glutathione reductase (only 21% identity in amino acid sequences), the recently refined 2.16-A structure for the peroxidase (7) clearly demonstrates the existence of a four-domain structure similar to that of glutathione reductase. The chain folds of the two enzymes are quite similar as well, further reinforcing the existence of a distant evolutionary relationship.

Although functionally distinct from the peroxidase, the streptococcal NADH oxidase shares a number of structural and redox features with this “peroxide reductase.” The NADH oxidase, which reduces molecular oxygen to water with 2 mol of NADH, contains one FAD per subunit and a cysteinyl redox center that is probably also a cysteine-sulfenic acid (8). Unlike the peroxidase, the flavin center of the oxidase EH21 species is readily reduced with NADH (9), suggesting a higher FAD redox potential and presumably yielding a functional four-electron-reduced enzyme species during turnover with oxygen. Complete reduction in anaerobic titrations, however, requires only 1.5-1.6 equivalents of dithionite or NADH per FAD. This unusual redox stoichiometry has been attributed to active-site asymmetry involving the non-flavin redox centers (9). Alignments of limited peptide sequence data available from tryptic digests of purified oxidase with the full-length peroxidase sequence have located significant stretches of greater than 40% identity in two of the four peroxidase domains (6). Specifically, the presence of the highly conserved active-site cysteinyl peptide sequence (10 of 18 identical residues, including the heptapeptide I-S-F-L-S-C-G) further supports the evidence linking the cysteinyl redox centers of the two enzymes (8). Although the oxidase does not formally reduce exogenous hydrogen peroxide, we have proposed (9) that the reduced active-site Cys-SH may act either on nascent peroxide produced by FADH, + 02, or on a transient flavin- C(4a)-hydroperoxide. These structural and mechanistic similarities have been taken to suggest that oxidase and peroxidase may represent divergent progeny of some ancestral peroxide reductase gene.

In order to further probe the active-site structures of the two enzymes, artificial flavins have been employed with the corresponding apoproteins. In general, these results strongly support the notion of a structural relatedness between oxidase and peroxidase proteins. Furthermore, by replacing the native peroxidase flavin with higher potential flavins such as 2-thio- and 4-thio-FAD, a fully reducible peroxidase analog results

The abbreviations used are: EH2, two-electron reduced enzyme; MMTS, methyl methanethiolsulfonate; NPX, NADH peroxidase; E‘’, midpoint oxidation-reduction potential a t pH 7.

3832

Active-site Comparison of NADH Peroxidase and NADH Oxidase 3833

which closely mimics the redox properties of the native NADH oxidase.

EXPERIMENTAL PROCEDURES

Materials-NADH was from Pharmacia LKB Biotechnology, and hydrogen peroxide (30%) was from Mallinckrodt Chemical Works. Dithiothreitol and FAD were from Sigma, and potassium bromide and sodium sulfide were from Fisher. Ultrapure ammonium sulfate was from Schwarz/Mann Biotech, and methyl methanethiolsulfonate was from Aldrich. All other chemicals were of the best grades com- mercially available. 2-Thioriboflavin, 8-chlororiboflavin, 6-hydroxy- FAD, 6-thiocyanato-FAD, and 4-thio-FAD were generous gifts of Dr. Vincent Massey, University of Michigan. 8-Chloro-FAD and 2-thio- FAD were prepared from the corresponding riboflavins as previously described (10, l l ) , except that the 2-thio-FAD pool following DEAE- cellulose chromatography was freed of excess NaCl over a column (100 X 2.5 cm) of Bio-Gel P2 in 10 mM (NH4),COs (12). 8-Mercapto- FAD and 6-mercapto-FAD were prepared from 8-chloro-FAD and 6- thiocyanato-FAD, respectively, as previously described (10, 13) and were used directly.

Apoenzyme Preparations-NADH peroxidase was purified from S. faecalis 10Cl following the modified procedure described recently (7). The apo-peroxidase was prepared using the general protocol originally developed for microsomal cytochrome b5 reductase (14). Buffers contained 2 mM dithiothreitol and 0.5 mM EDTA unless otherwise noted, and Tris buffer pH values refer to a standard temperature of 25 “C. NADH peroxidase (-30 pM; 0.5 ml) in 50 mM potassium phosphate, pH 7.0, was mixed on an ice-salt bath with 0.25 ml of cold 3 M KBr (pH adjusted to 2.7 with acetic acid). After 30 s a second aliquot of KBr was added. Within 50 s 0.5 ml of cold saturated (NH4)&304, pH 3.0, was added and mixed briefly. Forty seconds later 5.5 ml of the (NH4),S04 solution was added, and the suspension was immediately centrifuged a t 30,600 X g for 1 min. The yellow supernatant was discarded, and 0.5 ml of 1.5 M KBr in 50 mM Tris-acetate, pH 8.1, plus 1 mM EDTA (no dithiothreitol) was added to resuspend the pellet. After 30 min on ice the suspension, including 0.75 ml of 1.5 M KBr used to rinse the sides of the tube, was dialyzed initially against 50 mM Tris-acetate, pH 7.5, plus 1-2 mM dithiothreitol (no EDTA) and finally against 50 mM potassium phosphate, pH 6.8, plus 0.5 mM EDTA and 2 mM dithiothreitol. The apoenzyme was reconstituted by incubating on ice with a 5-fold excess of the appropriate FAD derivative for 30 min; the excess flavin was then removed by repeated ultrafiltration on a CM-30 microconcentrator (Amicon). Dithiothre- itol was removed prior to reconstitution with 4-thio-FAD, which has been shown to react with thiols in solution (12).

While the purified NADH oxidase has previously been demonstrated to be quite labile in its holoenzyme form (8), partially purified preparations of apo-oxidase (following Dyematrex Blue A chromatography) were observed to maintain reconstitutable activity for long periods of time when stored in the presence of dithiothreitol. Given the stability of this apo-oxidase form, we decided to attempt further purification directly rather than subject the labile holo-oxidase to the somewhat harsh protocols involved generally in apoenzyme preparations. This purification procedure follows that previously reported (8) through the DEAE-cellulose chromatography step. The pooled, concentrated eluate, which is primarily present as apo-oxidase a t this stage, was applied in a volume of 25 ml to a Dyematrex Blue A (Amicon) column as described earlier (8). The 10 mM phosphate, pH 7.0, buffer contained 1 mM dithiothreitol and 0.5 mM EDTA in this and all subsequent steps. After loading, the column was washed with 600 ml of buffer before a 1.9-liter linear gradient from 0 to 0.65 M KCl, in the 10 mM phosphate buffer, was applied. Apo-oxidase fractions, assayed after reconstitution as described previously (8), were pooled and concentrated for gel filtration. Apoenzyme (<6 ml total volume) was applied to a column (100 X 2.5 cm) of Sephacryl S-300 (Pharmacia LKB Biotechnology) and eluted in 10 mM phosphate, pH 7.0, containing 0.1 M KC1 and 1 mM each of dithiothreitol and EDTA. The pooled apoenzyme from the gel filtration step was dialyzed by repeated ultrafiltration to remove KC1 before freezing in aliquots (0.1-0.3 ml; 40-60 p M apo-oxidase) a t -20 “C in 10 mM phosphate, pH 7.0, containing 1 mM dithiothreitol and EDTA. Apoenzyme was thawed briefly prior to addition of 60 mM dithiothreitol and FAD (2- 7-fold excess over apoenzyme); the reconstitution mixture was then incubated for 5 min at 37 “C before removal of excess FAD by repeated ultrafiltration in 50 mM phosphate buffer, pH 7.0, containing 2 mM dithiothreitol and 1 mM EDTA. Very similar reconstitution protocols were followed for each of the flavin analogs, except that excess

dithiothreitol was removed before reconstitution with 4-thio-FAD. Titrations-Reductive titrations and pH titrations, as well as

chemical modifications with MMTS and Hz02 were carried out as previously described (3, 11). Extinction coefficients for the bound flavin analogs were either determined directly by denaturation of the reconstituted proteins in 4 M guanidine-HCl or indirectly by conversion of bound thio-FAD derivatives to native FAD with H202. In one case, the enzyme-bound flavin extinction coefficient was determined by parallel reconstitution of the apoprotein stock with native FAD. Values thus determined for the reconstituted peroxidase are: 6-hydroxy-FAD enzyme, = 19.6 mM” cm”; 8-mercapto-FAD enzyme, t554 = 30 mM” cm”; 2-thio-FAD enzyme, c498 = 20.4 mM” cm”; and 4-thio-FAD enzyme, c498 = 13 mM” cm”. The corresponding extinction coefficients for the reconstituted NADH oxidase are: 6-mercapto- FAD enzyme, c438 = 19.4 mM” cm”; 8-mercapto-FAD enzyme, e570 = 30 mM” cm”; 2-thio-FAD enzyme, t492 = 20.5 mM” cm”; and 4- thio-FAD enzyme, c4% = 13.9 mM” cm”.

Molecular Graphics-Molecular graphics applications were carried out with an Evans & Sutherland ESV2O workstation running Sybyl and the Sybyl/Biopolymer module (Tripos Associates).

RESULTS

Properties of Peroxidase and Oxidase Apoproteins-The KBr-acid ammonium sulfate procedure used in the preparation of apo-NADH peroxidase generally gave 60% recovery of reconstitutable activity with 5% residual FAD and is quite suitable for spectral work with the reconstituted enzyme. The apo-peroxidase could be stored at concentrations of 10-15 pM at 4 “C in the dithiothreitol-containing buffer for up to 2 weeks with no loss of reconstitutable activity. The activity- to-flavin ratio for the FAD-reconstituted enzyme was 90% that of purified native peroxidase (7), the visible spectrum of the reconstituted enzyme was virtually identical to that of the holoenzyme stock, and redox properties as assessed by anaerobic NADH titrations of the FAD-reconstituted protein were indistinguishable from those of native enzyme (3).The elution profile for Sephacryl S-300 chromatography of the apo- NADH oxidase preparation is shown in Fig. 1. As demonstrated in the sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis analysis given in the inset, this procedure does not yield a homogeneous preparation of the apo-oxidase. However, we estimate that the oxidase (M, = 50,000) band accounts for -85% of the total protein in pool I. The detailed spectral and catalytic properties of the apo-oxidase reconstituted with native and artificial FAD derivatives will be de-

400

200

1.5

1 .o

0.5

B h) Q, 0 n

I Y

5 0 75 100 125

FRACTION NUMBER FIG. 1. Sephacryl 5-300 gel filtration chromatography of

apo-NADH oxidase. 3.3 ml of the deflavo enzyme from Dyematrex Blue A chromatography (62 mg, -230 units/mg) was taken in 20% (v/v) glycerol and applied to a column of Sephacryl S-300 as described under “Experimental Procedures.” Fractions of 3.4 ml were collected and assayed for protein (-;taking ti& = 10) and for reconstitutable activity (- - -). Inset, sodium dodecyl sulfate-polyacrylamide gel elec- trophoretic analysis of pools I (fractions 78-82) and I1 (fractions 76, 77, and 83). Lanes A and B contain 10 and 20 pg of pool I, and lanes C and D contain 10 and 20 pg of pool 11.

3834 Active-site Comparison of NADH Peroxidase and NADH Oxidase

' \

0 9 10

1

400 500 600 700 800

. 15

y . 10 z <

0 cn

m a

m < . 05 DITHIONITE, eq

400 500 600 700 801

WAVELENGTH. n m

FIG. 2. pH Titration of 6-hydroxy-FAD NADH peroxidase. 6-Hydroxy-FAD NPX (14 IM, in 50 mM phosphate plus EDTA) was titrated with 2 M Tris base. Spectra were corrected for dilution, and p H values were obtained from a parallel titration with buffer only. Spectra shown correspond to pH values of 7.12 (-) and 9.96 (- - -). Inset, plot of A606 uersus pH (D) and points calculated for pK, of 9.6 (A).

scribed in a later paper.' By assaying the enzyme directly from the reconstitution mixture as previously described (8), an activity-to-flavin ratio of 2800-3300 was determined for the FAD enzyme. Overall, the purification gives approximately 20 mg of protein (microbiuret assay) from 170 g (wet weight) of cell paste with a 30% yield in reconstitutable activity. Although the apo-oxidase preparation is clearly not homogeneous with respect to protein, it should be emphasized that this activity-to-flavin ratio is approximately twice that previously reported (8) for the pure holo-oxidase. The apo- oxidase purification and reconstitution procedure employed thus gives a higher activity enzyme which catalyzes the fully coupled tetravalent reduction of oxygen, can be employed directly in artificial flavin studies, and is more stable over time as well. Reconstitutable activity decreases by -15% on storage at -20 "C for 1 week but remains stable thereafter for at least 6 months.

6-Hydroxy-FAD NPX-When apo-NPX was reconstituted with 6-hydroxy-FAD, the characteristic green color of the anionic form of the flavin analog (ionization of 6-hydroxyl substituent; Ref. 15) changed to yellow. Fig. 2 gives the result of a pH titration of the NPX-bound 6-hydroxy-FAD. The spectral properties of the enzyme at pH 7.0 are entirely consistent with the preferential binding of the neutral form of the flavin (pK, = 7.1 for free 6-hydroxy-FAD); most nota- bly, both the near-UV peak at 320 nm and the long wavelength maximum at 600 nm are absent at neutral pH, and there are distinct shoulders at 412 and 475 nm. The titration shown is isosbestic at 360,413, 450, and 512 nm and corresponds to a pK, of 9.6 (Fig. 2, inset) for bound 6-hydroxy-FAD. The apo- peroxidase thus increases the pK, of the flavin by 2.5 units.

The redox potential reported for 6-hydroxy-FAD is -265 mV at pH 7.0 (16), almost 50 mV lower than that for the native coenzyme. Dithionite titration of 6-hydroxy-FAD NPX proceeds in two distinct phases as with native enzyme (3), as shown in Fig. 3. The first phase corresponds to a slight increase in long wavelength absorbance from 500-650 nm and loss of the sharp maximum at 426 nm; this process requires 0.85 eq of dithionite/FAD. The second phase leads to general bleaching of the flavin spectrum and accounts for an additional 1.3 eq of reductant/FAD. Overall, the titration requires 2.14 eq of dithionite/FAD, and the course of reduction is very

S. A. Ahmed and A. Claiborne, manuscript in preparation.

WAVELENGTH. n m

FIG. 3. Dithionite titration of 6-hydroxy-FAD NPX. Oxi- dized enzyme (7.9 IM, in pH 7.0 phosphate buffer plus EDTA) was reduced anaerobically with dithionite. Spectra shown represent oxidized enzyme (-) and enzyme after addition of 0.81 (- - -), 1.47

absorbance at 426 nm (0) and 560 nm (A) are plotted uers'sus added dithionite. The endpoints correspond to 0.85 and 2.14 eq/FAD, respectively.

(- - - -) , and 2.97 (-1 eq of dithionite/FAD. Inset, changes in

similar to that originally observed for native FAD enzyme, except that the reduction of flavin by dithionite as the titration nears completion becomes somewhat sluggish. The absence of a major increase in 600 nm absorbance for the 2- electron reduced enzyme, indicating retention of the neutral state of 6-hydroxy-FAD, demonstrates that the environment of the flavin 6-position is not dramatically changed for the EH, species. Furthermore, the separation in redox potentials for the two phases is still at least 86 mV as observed for native NPX (17).

Anaerobic NADH titration of 6-hydroxy-FAD NPX (data not shown) also nicely mimics the redox behavior of native enzyme toward substrate. The first phase of this titration, which requires 0.9 eq of NADH/FAD, gives an EH, spectrum virtually identical to that given in the dithionite titration (Fig. 3). However, addition of a 13-fold excess of NADH does not lead to flavin reduction, instead yielding a stable EH,. NADH complex with a distinct oxidized flavin maximum at 419 nm and low extinction long wavelength absorbance extending from 500 to 820 nm. Full formation of this complex requires 1.9 eq of NADH/FAD, and the presence of the reduced pyridine nucleotide is easily demonstrated by the increase in A3*,, in the second phase of the titration.

6-Mercapto-FAD NADH Oxidase-6-Mercapto-FAD exhibits spectral and ionization properties quite similar to 6- hydroxy-FAD (13) and in addition, by virtue of the reactivity of the sulfur substituent, can serve as a regiospecific active- site probe with thiol reagents (18). When the deflavo-oxidase was reconstituted with freshly prepared 6-mercapto-FAD at pH 7.0, the green color of the free flavin anionic form (pK, = 5.9) quickly changed to yellow, as indicated in Fig. 4A. Al- though a quantitative pK, determination for enzyme-bound flavin was not attempted, the apoprotein must shift this pK, to at least 8 or so, i.e. by 2 or more units. This dramatic stabilization of the neutral form of 6-mercapto-FAD is, of course, very similar to that observed for 6-hydroxy-FAD NPX. We have proposed (8 ) , on the basis of chemical and spectroscopic data, that the non-flavin redox center of NADH oxidase is a stabilized cysteine-sulfenic acid (Cys-SOH). Since these residues combine with reactive thiols to yield disulfides, reconstitution of apo-NADH oxidase with 6-mercapto-FAD could, given proper active-site orientations and favorable interatomic distances, yield the mixed flavin-protein disulfide.

Active-site Comparison of NADH Peroxidase and NADH Oxidase 3835

1 400 500 600 700

""

WAVELENGTH, nm

400 so0 600 700 eo0 WAVELENGTH. nm

FIG. 4. Spectral properties and reactivity of 6-mercapto- FAD NADH oxidase. A , spectra of free 6-mercapto-FAD (-) and 6-mercapto-FAD NADH oxidase (- - -) were measured in 50 mM phosphate, pH 7.0, containing 0.5 mM EDTA the free flavin sample also contained 2 mM dithiothreitol. Flavin concentration in both samples is 11.4 WM. B, reaction of 6-mercapto-FAD NADH oxidase with HzOz. Enzyme (-; 6 WM) was mixed with 16 mM HZOZ in pH 7.0 phosphate buffer, without dithiothreitol, at 23 "C. Additional spectra shown were taken 10 s (- - -) , 6.5 min (- - - -), and 55 min (-) after peroxide addition.

T o test this possibility, excess dithiothreitol present during the reconstitution of apo-NADH oxidase with 6-mercapto- FAD was removed by exhaustive ultrafiltration. However, there were no spectral changes over 2 h which might have indicated formation of the active-site mixed disulfide.

Hydrogen peroxide inactivates the native NADH oxidase and NADH peroxidase with rate constants of 0.23 M-' min" and 8.1 M-' min" (5, 8), respectively, and these inactivations have been attributed to irreversible oxidation of the Cys-SOH redox centers. Massey et al. (18) have demonstrated that protein-bound 6-mercaptoflavins can also react with H202 to give initially the corresponding flavin-6-sulfenic acids, followed by further oxidation to the sulfinic (-S02H) and sulfonic ( -S03H) acid oxidation states. Fig. 4B gives the spectral course of the reaction following addition of 16 mM HzOz to 6- mercapto-FAD NADH oxidase, in the absence of dithiothreitol. The reaction monitored at 438 nm is biphasic with kfaSt = 0.14 min-' and kslow = 0.02 min", respectively. Each phase accounts for approximately half the total absorbance change at that wavelength. Since the final spectrum does not show significant long wavelength absorbance characteristic of 6- mercaptoflavin-S-oxides (18), we conclude that the apo-oxidase stabilizes the corresponding flavin-6-sulfenic acid. The reaction shown in Fig. 4B proceeds with isosbestic points at 335,394, 453, and 504 nm; prolonged exposure to H202 leads t o gradual loss of the shoulder at 376 nm.

Although reductive titrations of 6-mercapto-FAD NADH oxidase were not pursued, the enzyme was readily reduced anaerobically when mixed with 6.4 eq of NADH/FAD.

8-Mercapto-FAD NPX-Reconstitution of apo-peroxidase with 8-mercapto-FAD results in a nearly 20-nm red-shift of the oxidized flavin absorbance maximum, which is consistent with protein stabilization of the N( 1)-protonatedp-quinonoid flavin species (10). Addition of 10 mM methyl methanethiol-

sulfonate to the reconstituted peroxidase immediately gave the 8-SSCHs-FAD derivative (X,,, = 454 nm), indicating that the flavin 8-position is accessible to solvent in NPX. As a further probe of the accessibility of the bound 8-mercapto- FAD, the reaction of reconstituted NPX with H202 was analyzed (Fig. 5). The overall reaction is biphasic and is characterized by primary formation of a stabilized blue intermediate (Xmax = 635 nm), which gradually decays to give a double-banded spectrum (Xmax = 370, 462 nm). The course of this peroxide modification (krast = 0.29 min-', kslow = 0.03 min" at 15 mM HZOz) is very similar to that with 2-thio-FAD p-hydroxybenzoate hydroxylase (20). The blue intermediate stabilized in the 8-mercapto-FAD NPX reaction should logi- cally represent the corresponding flavin-8-S-oxide.

In addition to its use as a probe of active-site accessibility, model studies with the 1,lO-bridged 8-mercaptoflavin have shown (10) that resolution of the main spectral transition results from decreases in solvent polarity. We have previously shown that 1.3 M urea has pronounced effects on NPX redox properties and on the reactivity of the EH2 thiol with phen- ylmercuric acetate (17). However, 1.3 M urea has very little effect on the spectral properties of 8-mercapto-FAD NPX, consistent with the results of previous visible CD analyses (17).

8-Mercapto-FAD has a redox potential of -290 mV at pH 7 (20). The result of a standard dithionite titration of 8- mercapto-FAD NPX is shown in Fig. 6. The first phase of the titration, which requires 0.9 eq of reductant/FAD, leads to a blue-shift of the main transition from 554 to 548 nm with a slight increase in extinction coefficient. The 450-nm shoulder of the oxidized spectrum is lost on addition of the initial

I 400 500 600 700 eo0

WAVELENGTH. n m

FIG. 5. Peroxide modification of 8-mercapto-FAD NPX. Enzyme (-; 4.8 p ~ ) in 50 mM phosphate, pH 7.0, plus EDTA was reacted with 15 mM Hz02 at 23 "C. Additional spectra shown were taken 2.5 min (- - -), 8.5 min (- - - -), 40 min (- - -), and 4.75 h (-) after peroxide addition.

I I .30 I

WAVELENGTH. n m

FIG. 6. Dithionite titration of 8-mercapto-FAD NPX. En- zyme (--; 11.7 WM) in 50 mM phosphate, pH 7.0, plus 0.5 mM EDTA was titrated anaerobically at 23 "C with a standardized dithionite solution. Additional spectra shown represent enzyme after addition of 0.68 (- - -), 1.52 (- - - -), 2.08 (- - -1, and 4.05 (-) eq of dithionite/FAD. The endpoints of the titration were 0.94 and 2.62 eq of dithionite/FAD, respectively, as corrected for the lag of 0.44 eq/FAD.

3836 Active-site Comparison of N A D H Peroxidase and N A D H Oxidase

0.44 eq of dithionite/FAD, and this lag has been subtracted from the overall titration. Absorbance changes during conversion of oxidized enzyme to EH2 are sluggish, requiring up to 30 min for completion, quite unlike native NPX. Further additions of dithionite eventually lead to a fully reduced enzyme form; however, the long times required for equilibra- tion with each aliquot of reductant prevented a careful determination of redox stoichiometry. When anaerobic titration of 8-mercapto-FAD NPX was performed with NADH, an EH, spectrum very similar to that seen in Fig. 6 was obtained with 0.7 eq of reductant/FAD. However, addition of 8 eq of NADH/ FAD led to no further significant spectral changes suggestive of either flavin reduction or EH2.NADH complex formation.

8-Mercupto-FAD NADH Oxidase-Reconstitution of apo- NADH oxidase with 8-mercapto-FAD, in the presence of dithiothreitol, also gives rise to an enzyme-bound flavin spectrum characteristic of the N( 1)-protonated p-quinonoid species (X,,, = 570 nm). In order to employ MMTS and Hz02 as accessibility probes, an enzyme sample which had been stored in 2 mM dithiothreitol for 24 h was freed of excess thiol by repeated ultrafiltration; this procedure did not appear to change the spectral properties of the reconstituted protein, which are shown in Fig. 7. In contrast to the peroxide reaction with 8-mercapto-FAD NPX, addition of 15 mM H202 to reconstituted oxidase immediately shifts the absorbance maximum to -580 nm, followed by gradual appearance of a two- component spectrum consisting of the flavin-8-S-oxide (X,,, - 650 nm) and a second chromophore characterized by a single absorbance maximum at 453 nm and a distinctive shoulder at 390 nm. Both components gradually decay to a final product with a double-banded spectrum (X,,, = 355,453 nm) similar to the final peroxide-modified 8-mercapto-FAD NPX. Analysis of the biphasic peroxide reaction at 580 nm yields kobs values of 2.4 min-l and 0.04 min", respectively, for the fast and slow phases. When the same reaction is carried out in the presence of 2 mM dithiothreitol, using a peroxide concentration of 160 mM, the spectral course is changed to one closely resembling that shown in Fig. 5 for 8-mercapto- FAD NPX. The single-banded component (X,,, = 453 nm) of the intermediate spectrum in Fig. 7 is now absent, consistent with at least a 2-fold increase in 640 nm absorbance associated with the S-oxide species. Subtraction of the near-UV component in the final oxidized product due to oxidized dithiothreitol (X,,, = 280 nm) gives values of 358 and 458 nm for the absorbance maxima of this species, very similar to that shown in Fig. 7, and this material is non-fluorescent. Kinetic analysis of the decay of the S-oxide intermediate at 640 nm yields a kobs value of 0.5 min-l at 160 mM Hz02. Under these conditions dithiothreitol is oxidized by H202 at an apparent

rate of 1 min", as monitored at 280 nm in control experi- ments.

2-Thio-FAD NPX-In contrast to the rapid reactions observed for 8-mercapto-FAD NPX with methyl methanethiolsulfonate and HzO,, the 2-thio-FAD reconstituted enzyme is quite refractile toward both reagents. In the presence of 1 mM MMTS only -10% of the 2-thio-FAD enzyme is converted to the 2-SSCH,-FAD form over 40 min, and a similar absorbance change at 520 nm was seen over 2.5 h with 15 mM H2O2. These results suggest that solvent access to the flavin 2- position is severely restricted in the peroxidase. In addition to its utility as a probe of active-site structure, the higher potential of -126 mV for the 2-thioflavin derivative (21) relative to FAD (E" = -219 mV) has been exploited in studies of redox behavior with a number of flavoproteins (22, 23). Dithionite titration of 2-thio-FAD peroxidase requires only 1.4 eq of reductant/FAD (data not shown). The first phase requires 0.4 eq and leads to a small increase in long wavelength absorbance beyond 580 nm and a 10% decrease in Ad9+ The second phase of the titration requires 1 eq of dithionite and yields the characteristic bleached spectrum (X,,, = 350, 425 nm) of the anionic form of reduced 2-thio-FAD. Furthermore, anaerobic addition of 2.6 eq of NAD' to the reduced 2-thio- FAD enzyme immediately gives rise to an impressive charge- transfer band (X,,, -750 nm) which is attributable to an FADH, + NAD+ interaction. I t is important to stress that NAD' titration of the fully reduced (with dithionite) native peroxidase yields the EH,.NADH complex directly (3). The 2-thio-FADH2 enzyme.NAD+ complex is quite stable under anaerobic conditions.

A major distinction between NADH peroxidase and NADH oxidase concerns the redox behavior of the flavin centers in the respective EH, species toward NADH. The peroxidase EH2 form, though it binds NADH tightly to give a spectro- scopically distinct EH2. NADH complex, is not reduced by excess pyridine nucleotide substrate (3). On the other hand, the EH, form of the native NADH oxidase is reduced directly with 1.1 eq of NADH/FAD to give a very stable FADH2. NAD+ complex with long wavelength absorbance (X,,, = 725 nm; Ref. 9). When 2-thio-FAD peroxidase is titrated anaerobically with NADH, redox behavior very similar to that of native NADH oxidase is observed (Fig. 8). As with the dithionite titration described previously, complete reduction requires 1.4 eq of NADH/FAD. The first phase requires 0.4 eq and leads to a small increase in long wavelength absorbance and a relatively small decrease in A494. The second phase requires 1.0 eq of NADH/FAD and differs from the dithionite titration only in that the FADH,. NAD+ charge-transfer com-

I ' A ' I .20 1 / \

WAVELENGTH. n m

FIG. 7. Peroxide modification of 8-mercapto-FAD NADH oxidase. Enzyme (-; 7.6 pM) in 50 mM phosphate, pH 7.0, plus 0.5 mM EDTA had been freed of excess dithiothreitol by repeated ultrafiltration. Additional spectra shown represent enzyme 10 s

H,O, at 23 "C. (- - -), 2.33 min (- - - -), and 2.7 h (-) after addition of 15 mM

WAVELENGTH. n m

FIG. 8. NADH titration of 2-thio-FAD NPX. Enzyme (-; 23.5 y ~ ) in 50 mM phosphate, pH 7.0, plus 0.5 mM EDTA was titrated anaerobically with NADH a t 23 "C. Additional spectra shown represent enzyme after addition of 0.48 (- - -), 0.95 (- - - -1, and 1.55 (-) eq of NADH/FAD. Inset, absorbance change at 494 nm uersus added NADH. Endpoints of the titration were 0.4 and 1.36 eq of NADH/FAD, respectively.

Active-site Comparison of N A D H Peroxidase and N A D H Oxidase 3837

plex appears as a stable reduced enzyme species. I t is important to stress that titrations of native NADH oxidase with dithionite and NADH require a total of 1.5-1.6 eq of reductant/FAD (8,9), similar to the redox stoichiometries observed with 2-thio-FAD peroxidase.

The spectral and redox properties of 2-thio-FAD NADH oxidase will be discussed in a later paper. Addition of 0.14 M H202 to the reconstituted enzyme led to a very slow loss of absorbance at 508 nm ( tl,x - 33 min) which became nonlinear before even three half-lives. Analysis of the sample after overnight incubation (21 h) showed that at least 90% of the product FAD was released from the enzyme. These results nonetheless demonstrate that the flavin 2-position is relatively inaccessible to solvent in the NADH oxidase.

4-Thio-FAD NPX-Since 4-thio-FAD reacts with thiols to generate the native (4-OXO) coenzyme (12), the apo-peroxidase preparation was freed of dithiothreitol prior to reconstitution. This had no adverse effect on flavin binding and allowed direct assessment of solvent accessibility with MMTS and H202. Incubation with 1 mM MMTS for over half an hour led t o virtually no change in the 4-thio-FAD enzyme spectrum. Similarly a 2-h incubation of the enzyme with 15 mM Hz02 led to only a 10% decrease in Asso. As with the 2-thio-FAD enzyme, these results indicate that solvent accessibility for the flavin 4-position is very restricted in the peroxidase.

The redox potential for 4-thio-FAD is -55 mV at pH 7 (12), 71 mV higher than that for 2-thioflavin. The result of a standard dithionite titration of 4-thio-FAD peroxidase is given in Fig. 9A. Full reduction requires 1.8 eq of dithionite and gives the spectrum of the anionic reduced 4-thio-FAD enzyme (Xmax = 326,430 nm). The first phase of the titration requires only 0.4-0.5 eq of reductant and leads to a slight increase in long wavelength absorbance beyond 570 nm. The oxidized peak at 498 nm is decreased by only lo%, and the

. 1 5

W y . 10 m 0 m

. 0 5

0.5 1 1 5 I DITHIONITE. eq

400 500 600 700 800 ~~~

WAVELENGTH. n m

WAVELENGTH. n m

FIG. 9. Dithionite titration of 4-thio-FAD NPX. A , enzyme (-; 12.2 PM) in 50 mM phosphate, pH 7.0, plus 0.5 mM EDTA was titrated anaerobically a t 23 "C with a standardized dithionite solution. Additional spectra shown represent enzyme after addition of 0.65 (- - -), 1.3 (- - - -), and 2.38 (-) eq of dithionite/FAD. Inset, absorbance change a t 498 nm uersus added dithionite. Endpoints of the titration were 0.44 and 1.81 eq of dithionite/FAD, respectively. E, reduced enzyme (-) was mixed anaerobically with 2.8 eq of NAD+/FAD (- - -). The resulting complex was then reacted with oxygen by opening the anaerobic cuvette to air (-). The reoxidized enzyme spectrum was essentially unchanged from that in panel A .

absorbance changes are isosbestic in the vicinity of 435 nm. The second phase appears to require 1.3-1.4 eq of dithionite/ FAD. The redox behavior of 4-thio-FAD peroxidase is thus very similar to that of the 2-thio-FAD enzyme. In addition, when 2.8 eq of NAD+ are added anaerobically, a long wavelength absorbance band (X,,, - 735 nm) corresponding to the charge-transfer complex of reduced 4-thio-FAD enzyme and NAD+ appears immediately (Fig. 9B). Air oxidation of the reduced enzyme. NAD' complex restores at least 96% of the original oxidized 4-thio-FAD enzyme; there appears to be no significant desulfurization of bound 4-thio-FAD (yielding native 4-oxo-FAD) as was observed with reduced 4-thio-FAD p - hydroxybenzoate hydroxylase (12,24). Direct anaerobic NADH titration of 4-thio-FAD peroxidase gave results very similar to those of the dithionite titration. The overall titration required 1.4 eq of NADH/FAD, and the second phase (1.0 eq/FAD) led directly to the stoichiometric appearance of the 735-nm charge-transfer complex of reduced enzyme and NAD'. Air oxidation again led to essentially full restoration of the original 4-thio-FAD enzyme absorbance.

The spectroscopic and redox properties of 4-thio-FAD NADH oxidase will be detailed in a later paper. In the absence of dithiothreitol, the 4-thio-FAD enzyme reacted with Hn02 in a biphasic process (kobs values -0.09 min-' and 0.007 min" at 16 mM H202) to yield the native flavin. Analysis of the filtrate following ultrafiltration showed that almost 80% of the flavin had been released from the enzyme during the 18- h incubation. It must be remembered that native NADH oxidase is inactivated by H202 (kobs = 0.004 min-' at 16 mM H202; Ref. 8); this process undoubtedly contributes to the observed flavin loss. Free 4-thio-FAD reacts with Hz02 under these conditions a t a rate of 0.1 min-'.

DISCUSSION

The recent 2.16-A crystal structure for S. faecalis NADH peroxidase (7) demonstrates that the overall domain structure and chain fold are rather similar to that of glutathione reductase, in spite of the limited sequence homology. The results of the present study with artificial flavins compare very favorably with those reported by Krauth-Siege1 et al. (25) for human glutathione reductase and, in light of the three-dimen- sional structure for the peroxidase, can be rationalized as follows. First, the sulfur atom of C y P , which is present as a stabilized sulfenic acid (Cys-SOH) in the native peroxidase, is located 4.4 and 4.9 A, respectively, from the O(4a)- and C(6)- positions of tbe isoalloxazine ring. If a C(6)-S(6a) single bond (length = 1.8 A) is constructed and interatomic distance! recalculated on this basis, the new S(Ga)-position is 5.7 A from the sulfur atom of Cys4'. These interatomic distances most likely explain the failure to observe mixed protein-flavin disulfides, which could have resulted from condensation of the active-site Cys-SOH and the thiol substituents of either 4-thio-FAD (with reconstituted NPX) or 6-mercapto-FAD (with reconstituted NADH oxidase). The spectral properties of 8-mercapto-FAD NADH peroxidase are most consistent with protein stabilization of the N( 1)-protonatedp-quinonoid form of the flavin, as originally described by Massey et al. (10). It is, however, unlikely that this tautomeric species can account quantitatively for the spectral properties observed, and the presence of some N(1)-anion is probable. The antic- ipated effects of hydrogen bond interaction betwee! N( 1) and the backbone nitrogen of Alazg9 (distance = 3.16 A) and the partial positive charge contributed by helix a 7 ( L e ~ ~ ~ ~ - A s n " ~ ) are consistent with this interpretation of the NPX:8-mercapto-FAD interaction. Connolly (26) analysis of the peroxidase structure confirms that the C(8a)-position is very acces-

3838 Active-site Comparison of NADH Peroxidase and NADH Oxidase

sible to solvent, explaining the facile reactions of 8-mercapto- FAD enzyme with MMTS and H202. Similar analyses show that the C(6)-, N(5)-, and 0(4a)-positions are completely inaccessible to solvent; the latter result in particular is consistent with the lack of reactivity of the 4-thio-FAD peroxidase with thiol reagents. Connolly analysis using HzO as a probe suggests there may be some limited accessibility to the O(2a)-oxygen; however, we find virtually no reaction of the 2-thio-FAD enzyme with MMTS or Hz02.

A major difference between the flavin environments in the peroxidase and in glutathione reductase concerns the results with 6-substituted flavins. Krauth-Siege1 et al. (25) observed that 6-hydroxy-FAD was only weakly bound by the apoenzyme of human glutathione reductase; this was attributed to unfavorable van der Waals contacts between the active-site residues Gly6*, C Y S ~ ~ , and Lys@, and the O(Ga)-oxygen of the modified flavin. A recent study, however, has demonstrated tight binding of the apoenzyme to several 6-substituted flavins, including 6-hydroxy- and 6-mercapto-FAD (27). In each of the latter cases the pK, of the bound flavin is lowered, reflecting in part stabilization of the partial negative charge at O(6a) and S(6a), respectively, by LyP. The pK, of 6- hydroxy-FAD glutathione reductase is 5.6 (a decrease of 1.5 units), and the pK, of the 6-mercapto-FAD enzyme is <5.0 (a decrease of at least 1 unit). Both derivatives were analyzed by x-ray crystallography at 3.0-A resolution. The C(6)-0(6a) bond length of 1.2 A in the 6-hydroxy-FAD enzyme corresponds to a double bond, strongly supporting stabilization of the o-quinonoid form of the anionic flavin, with the negative charge delocalized toward the N(1)-0(2a) locus where it is stabilized by the helix dipole contributed by amino acid residues Thr339-Phe354. The flavin o ( 6 a ) : N z - L ~ ~ ~ ~ distance is 2.9 A, and the strong hydrogen bond which results induces a tilt of about 5" in the isoalloxazine ring. Although side chain elements contributed by Cys4', TyrI5', Ilel6O, and G ~ u ' ~ ~ are found within a 5.0-A sphere about the flavin C(6)-position in NADH peroxidase, 6-hydroxy-FAD binds tightly to the apoenzyme. In contrast to glutathione reductase, however, the peroxidase strongly stabilizes the neutral form of 6-hydroxy- FAD as seen in the pK, shift from 7.1 (free flavin) to 9.6 (enzyme-bound flavin). While the hydrogen bonding and polar environments of the N(1)-0(2a) loci are similar in both proteins, the electrostatic environments about the respective flavin C(6)-positions appear to differ. Although both L y P and G1uZo1 in glutathione reductase are within the immediate vicinity of the oxygen substituent of 6-hydroxy-FAD, their respective side chains form a strong internal salt bridge which is preserved in the reconstituted enzyme, with only slight movements to accommodate the oxygen atom. However, in NADH peFoxidase the oxygen atoms of the G ~ u ' ~ ~ side chain are 4.6 A (OEl) and 4.0 A (OE2), respectively, from the C(6)-carbon. Furthermore, there is no internal salt bridge involving G ~ u ' ~ ~ ; the presence of this surface-accessible (and possibly charged) residue is likely to influence the pK, of the bound hydroxyflavin so as to stabilize the neutral form. As all three o-quinonoid forms of 6-hydroxyflavin are thought to be green in color (15), the yellow phenolic (-OH) tautomer may be further stabilized by a hydrogen bonding interaction with G ~ u ' ~ ~ , via the OEl and OE2 oxygens and and

Hydrogen peroxide reacts poorly, if at all, with both 2- and 4-thio-FAD NADH peroxidase. With 8-mercapto-FAD enzyme, however, hydrogen peroxide treatment proceeds in a distinctly biphasic reaction characterized by a high extinction, long wavelength intermediate. Massey et al. (12) first reported such blue flavin derivatives in peroxide oxidations with 8-

respectively.

mercaptoflavins, concluding that these species likely repre- sented the flavin-8-S-oxides by analogy to similar compounds formed with 2- (28) and 4-thioflavins (12). In the case of reconstituted NADH peroxidase, the S-oxide species gradually decays to a final product which might represent the enzyme-bound flavin-8-sulfonate, but the spectral properties (X,,, = 370, 462 nm) differ from those of the free 8-sulfonyl flavin (X,,, = 342,448 nm; Ref. 20) in that both maxima are red-shifted by 15-25 nm. With 2-thio-FAD p-hydroxybenzoate hydroxylase (19), peroxide oxidation yields the corresponding flavin-2-S-oxide as an intermediate which decays in a second peroxide-dependent reaction to give a covalent protein-flavin adduct. The spectral properties of the final product of Hz02 modification of 8-mercapto-FAD peroxidase do not resemble those of the 8-SR, %OR, or 8-NHR flavins (X,,, = 474, 436, and 492 nm, respectively; Ref. 29) that would be expected to result from nucleophilic attack at C(8), however. In the absence of dithiothreitol, the peroxide oxidation of 8- mercapto-FAD NADH oxidase seems to involve a more complex reaction scheme. The initial rapid shift in X,,, for the starting enzyme from 570 to 582 nm on peroxide addition appears to represent a perturbation of the bound flavin spectrum reflecting a change in the active-site environment. This is followed by the appearance of a two-component spectral mixture, and the identification of the long wavelength species (Xmax = 648 nm) as the 8-S-oxide is straightforward. However, only about 50% of the enzyme-bound flavin is accounted for by this intermediate, judging from the maximal A648. The second component has spectral properties (X,,, = 453 nm) resembling those of 8-SR flavin derivatives, except that their X,,, values generally range around 475 nm (20). The spectrum of the 8-mercaptoflavin disulfide dimer (20) resembles this second component closely, but we would not expect this product to appear in reactions with the enzyme-bound 8- mercapto-FAD. Massey et al. (18) have reported the stabilization of both sulfenic acid and S-oxide forms of 6-mercaptoflavins resulting from peroxide oxidations of the corresponding reconstituted enzymes. The 6-sulfenic acid (-SOH) form is characterized by a single absorbance maximum (X,,, = 440-448 nm) with a shoulder at 375 nm. The 6-S-oxide spectrum is distinguished by an additional long wavelength absorbance band extending beyond 800 nm, due to its benzo- quinoid structure. It is possible that NADH oxidase, which has been shown to exhibit active-site asymmetry with respect to its redox properties (9), can stabilize both sulfenic acid and sulfoxide forms of the 8-mercaptoflavin during peroxide oxidation. The observation that both spectral components are converted in a slower peroxide reaction to a final product ( Xmax = 355,453 nm) with spectral properties similar to those expected for the flavin-8-sulfonate (X,,, = 342, 448 nm) supports this conclusion. The presence of dithiothreitol reduces the complexity of this reaction with 8-mercapto-FAD oxidase, and the S-oxide intermediate is essentially fully formed immediately on addition of peroxide at the higher concentration. The spectral properties of the final product (Amax = 358,458 nm) are very similar to those for the product shown in Fig. 7 and also support the flavin-8-sulfonate structure. Finally, the peroxide modification of the 6-mercapto- FAD NADH oxidase, also carried out in the absence of dithiothreitol, provides evidence for stabilization of the enzyme-bound flavin-6-sulfenic acid, without further oxidation to the sulfinic or sulfonic acid forms. Similar stabilizations were observed with riboflavin binding protein, flavodoxin, and Old Yellow Enzyme (18). Both the NADH oxidase and riboflavin binding protein (13) preferentially stabilize the neutral 6-mercaptoflavin as well.

Active-site Comparison of N A D H Peroxidase and N A D H Oxidase 3839

The redox properties of the reconstituted NADH peroxidase forms studied compare very favorably with the results of artificial flavin studies with glutathione reductase as well. The EH, form of the 6-hydroxy-FAD peroxidase resembles the two-electron reduced 6-thiocyanato-FAD glutathione reductase, formed by addition of GSH (27). Reduction of the sulfenic acid in the peroxidase does not appear to affect the preferential stabilization of the neutral 6-hydroxy-FAD, and the separation of 286 mV between the sulfenic acid and flavin potentials found with native peroxidase is maintained. The EH, spectrum of the 8-mercapto-FAD peroxidase is very similar to that of two-electron-reduced 8-mercapto-FAD glutathione reductase (25), and the maximum in the peroxidase difference spectrum (EH2 minus oxidized) at 518 nm is very close to that observed for the native enzyme (AA,,, = 517 nm) despite the 82-mV difference in oxidized flavin potentials. Although there is no evidence for EH2.NADH complex formation in the 8-mercapto-FAD enzyme, this cannot be attributed simply to the presence of the bulky sulfur substituent at the 8a-position; the fact that the 6-hydroxy-FAD (E’’ = -265 mV) enzyme EH2.NADH complex is observed spectrally would seem to minimize the role played by the lower flavin redox potential in this observation. Krauth-Siege1 et al. (25) have obtained evidence for reduction of 8-mercapto-FAD glutathione reductase with a 20-fold excess of NADPH. Fi- nally, the redox behavior of the NADH peroxidase reconstituted with 2-thio-FAD (E” = -126 mV) and 4-thio-FAD (E” = -55 mV) is of particular interest. The stability of these N(l)-N(5)-reduced flavins demonstrates, as reported with glutathione reductase (25, 30), that there is space to accommodate -H at N(5) during substrate reduction of the enzyme. These titrations are complete on addition of only 1.4 eq of NADH/FAD, very similar to the redox stoichiometry observed with native NADH oxidase, which exhibits active-site asymmetry in the reduction of the non-flavin redox centers. Flavin reduction in the peroxidase, allowed now by the higher redox potentials, may induce this non-equivalence in redox behavior at the Cys-SOH centers in the tetrameric protein. In addition to the full flavin reductions and asymmetric redox behavior seen with the 2- and 4-thio-FAD peroxidases, these enzymes also tightly bind NAD’ in their reduced forms, yielding the corresponding reduced thio-FAD + NAD+ charge-transfer absorbance at long wavelengths. This prop- erty of the reconstituted peroxidases also nicely mimics the native NADH oxidase, where stable FADH, . NAD+ complexes have been observed in static titrations. As previously noted in discussions of the native oxidase (9), however, there is no clear correlation between flavin and/or bound pyridine nucleotide potentials and the wavelength maxima for these intermediates as would be expected. In the case of the 2-thio- FAD peroxidase, the X,,, for the reduced enzyme-NAD+ complex (750 nm) contrasts sharply with the spectral character- istics of the reduced 2-thio-FAD p-hydroxybenzoate hydroxylase-NADP+ (22) and 2-thio-FMN lactate oxidase-pyruvate (23) intermediates (X,,, - 500 nm). In addition to the similarities in redox behavior between the 2-thio- and 4-thio-FAD peroxidases and native NADH oxidase, virtually all of the features revealed in these artificial flavin studies pertaining to the peroxidase active-site environment are conserved in the reconstituted NADH oxidase forms studied. Solvent accessibility to the C(8a)-position is coupled with a rather inaccessible pyrimidine subnucleus (O(2a)- and O(4a)-positions) in both proteins. Both proteins stabilize the N(1)- protonated p-quinonoid form of 8-mercapto-FAD and the neutral phenolic (or thiophenolic) forms of 6-hydroxy- or 6- mercapto-FAD. From the high resolution crystal structure of

the peroxidase and the NADH oxidase gene sequence, we hope to soon be able to extend the similarities indicated in these artificial flavin studies to a detailed molecular analysis of the difference in catalytic redox functions of the two streptococcal enzymes. Our results with the reconstituted NADH oxidase also serve to further distinguish this unusual flavoprotein, which reduces O2 + 2Hz0, from those flavoprotein oxidases catalyzing the reduction of O2 + H202 (10). An analysis of the catalytic and redox properties of these reconstituted NADH oxidases will be presented in a later report.

Finally, these artificial flavin studies with the peroxidase contribute to our analysis of various mechanistic alternatives, particularly in the reaction of reduced enzyme with hydrogen peroxide. We have previously proposed (5) that H202 reacts directly with the thiolate form of Cys4’ to generate the Cys- SOH redox center and H20. This hypothesis is supported by the peroxidase crystal structure (7), which reveals the presence of a hydrogen peroxide access channel leading to C y ~ 4 ~ and the His” active-site base. The interior surface is rather polar, with contributions from a number of fixed water mol- ecules and from some elements of both active-site residues and the bound FAD coenzyme. The dimeric interface contact and the tight contact between the FAD-binding and central domains provided by the “domain zipper” effectively allow only one way for the substrate H202 to enter. Stoll and Blanchard (31) have recently proposed a chemical mechanism for the peroxidase involving nucleophilic attack by H202 on the C(4a)-position of the flavin, yielding the C(4a)-peroxyflavin intermediate. Nucleophilic attack by the Cys4’ thiolate generates Cys-SOH and the C(4a)-hydroxyflavin, which decays further by elimination of water. In addition to those elements of our earlier mechanistic proposal supported by the crystal structure, the present study clearly shows that the flavin-O(4a)-position is not accessible to H202, therefore greatly diminishing the possibility that H202 reacts at the adjacent C(4a)-position. Furthermore, it is important to stress that 4-thio-FAD has been used specifically as a mechanistic probe (12, 24) for those flavoproteins known to form the flavin-C(4a)-hydroperoxide in reactions of reduced enzyme plus 02. 4-Thio-FAD p-hydroxybenzoate hydroxylase, for ex- ample, undergoes desulfurization of 53% of the starting thioflavin (to native, 4-oxo-FAD) on single turnover with oxygen in the absence of substrate or effector. Up to 90% conversion occurred on single turnover in the presence of the effector 6- hydroxynicotinate; the loss of sulfur was attributed to a reaction involving oxygen transfer from the transient peroxyflavin. When the 4-thio-FAD NADH peroxidase is reduced and reacted with 02, however, essentially no conversion of thioflavin occurs. While this result alone does not disprove the hypothesis for a C(4a)-peroxyflavin intermediate in nor- mal peroxidatic turnover, it certainly adds to a number of independent observations which, taken together, argue strongly against such a mechanism.

Acknowledgments-We would like to thank Dr. Vincent Massey for providing the artificial flavins used in this study, Holly Miller for assistance with the Evans & Sutherland graphics workstation, and Thilo Stehle for critical reading of the manuscript.

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Documents

Active-site Structural Comparison of Streptococcal NADH