Multifunctionality of lipoamide dehydrogenase: Role of histidine residues in reductase reaction

ARCHIVES OF BIOCHEMISTRY ANDBIOPHYSICS Vol. 217, No. 2, September, pp. 721-729, 1982

Multifunctionality

of Lipoamide Dehydrogenase: Role of Histidine Residues in Reductase Reaction’

C. S. TSAI,2 A. J. WAND, Department

of

J. R. P. GODIN,

Chemistry and Institute of Biochemistry,

AND G. W. BUCHANAN

Carleton University, Ottawa KlS 5B6, Canada

Received Apri!. 20, 1982

Rose bengal sensitizes photoinactivation of lipoamide dehydrogenase from pig heart to a constant residual reductase activity resulting from specific destruction of histidine residues. The rate of sensitized photoinactivation is pH dependent and is associated with an ionizable group with pK 6.6 f 0.2. All steady-state kinetic parameters are markedly reduced by photooxidation. Spectroscopic studies indicate the contribution of oxidized flavinidithiol to the half-reduced form of the photooxidized enzyme. The proton magnetic resonance spectrum of lipoamide dehydrogenase shows resolved histidine C2 proton peak at 69.18 ppm and a shoulder at 69.23 ppm. The shoulder protons are eliminated by the sensitized photooxidation and shifted upfield on deprotonation. At high pH, the characteristic Faraday A term also disappears. These observations suggest that the essential histidine stabilizes the nascent thiolate via the ion pair formation to facilitate the reductase reaction catalyzed by lipoamide dehydrogenase.

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3), isolated from pig heart, is a dimeric flavoprotein containing one molecule of FAD and a disulfide linkage per subunit at the active site (1). The interaction between the reduced thiolate and the oxidized flavin has been proposed as the catalytic intermediate for the physiological reductase reaction (2, 3). The thiolate is thought to be stabilized by an essential base (4) which has been recently identified as histidine (5). Rose bengal sensitizes photooxidation of five histidine residues with a concomitant loss in the spectroscopic features characteristic of the reduced enzyme intermediate (6). However, considerable lipoamide reductase activity remains after photooxidation. It is, therefore, necessary to investigate the source of residual re‘Taken submitted Research, s Author addressed.

in part from the M.Sc. thesis of A.J.W. to the Faculty of Graduate Studies and Carleton University. to whom all correspondence should be 721

ductase activity of the photooxidized enzyme to assess the catalytic role of the histidine residues. Experimental results presented here implicate the ability of histidine residues to stabilize thiolate ion (‘7-9) of the catalytic intermediate in the reductase reaction mediated by lipoamide dehydrogenase. EXPERIMENTAL

PROCEDURES

Materials Pig heart lipoamide dehydrogenase was obtained from Sigma Chemical Company (type III) and checked for the purity as described (10). Enzyme concentrations were determined on the basis of flavin content, or where necessary, by Biuret assay based on dimeric structure having a molecular weight of 110,000. Thionicotinamide adenine dinucleotide, NAD+, NADH, D,L-lipoamide (LipSs),s and iodoacetic

3 Abbreviations used: EHs, half-reduced native lipoamide dehydrogenase; Ehr, photooxidized lipoamide dehydrogenase; E,,.Ha, half-reduced form of photooxidized lipoamide dehydrogenase; Lip&, D,L,-lipoamide; Lip(SH), dihydrolipoamide; MCD, magnetic circular dichroism; PMR, proton magnetic resonance. 0063-9861/82/100721-09$02.00/O

Copyright Q1982 byAcademic Press, Inc. All rightsofreproduction inmyformreserved.

722

TSAI ET AL.

acid were products of Sigma Chemical Company. Rose bengal and methylene blue were obtained from Fisher Chemical Company. 5,5’-Dithiobis(2-nitrobenzoic acid) was purchased from Aldrich Chemical Company. Deuterated compounds were obtained from Stohler Isotope Chemicals. Dihydrolipoamide was prepared from lipoamide according to the procedure of Reed et al (11). Deuterated buffers were prepared by dissolving the appropriate amount of NazDP04 and NaDz.P04 in DzO. The pD values were estimated by adding 0.44 units to pH meter readings (12). Kinetic studies. Kinetic studies of lipoamide reductase were carried out as described previously (10) in a Perkin-Elmer spectrophotometer (Coleman Model 124) equipped with a variable output recorder (Coleman Model 165) or a Beckman Model 25 spectrophotometer. All reagents were prepared in 50 mM sodium acetate, potassium phosphate of the desired pH except for lipoamide which was prepared in absolute ethanol. All buffers contained 0.10 mM EDTA. Initial rates in the asymptotic region were analyzed for parallel reciprocal plots, VAB ’ = AB i-

KbA i- K,B ’

Dl

where V, K,, and Kb are maximum velocity and Michaelis constants for NADH (A) and lipoamide (B), respectively. Product and rose bengal inhibition studies were analyzed according to linear competitive inhibition or noncompetitive inhibition as described by vs ’ = Ks(l

+

J./Kis) + S ’

’ = Ks(l

+

I/K,,) + S(l + J./Kii) ’

vs

PI r31

where Ki, and Kii are inhibition constants for inhibitor, I, which affects the slope and intercept, respectively, of reciprocal plots of initial rates 2)ervarying substrate, S(A or B). Photooxidation. The dye-sensitized photooxidations were studies under pseudo-first-order conditions with respect to sensitizer and molecular oxygen. An enzymatic solution (0.41 PM lipoamide dehydrogenase) in 52 PM rose bengal or methylene blue and 9.0 ppm Oz was illuminated isothermally at 25°C at a suitable distance from a 200-W photoflood lamp. Reductase activity was measured during illumination. Control experiments indicate that enzyme activity is not affected without illumination. For preparative photooxidation, the enzyme concentration was increased and illumination continued without stirring until a constant residual reductase activity was obtained. Rose bengal was removed by Cellex T anionexchange resin at pH 6.5 and methylene blue by Cellex CM cation exchanges at pH 6.6. Spectroscopic studies. Absorption spectra were recorded on a Cary 14 spectrophotometer. Magnetic

circular dichroism (MCD) spectra were obtained as described (6). Fluorescence emission spectra were acquired on a Perkin-Elmer fluorescence spectrometer (Model 204s) equipped with a Perkin-Elmer xenon lamp power supply and a Beckman variable output recorder. Correction curves were obtained with rhodamine B (13). To record proton magnetic resonance (PMR) spectra, deuterated lipoamide dehydrogenase was dissolved in DzO and the solution was lyophilized. This procedure was repeated three times to obtain deuterated enzyme which was dissolved in 0.10 M deuterated phosphate buffer at pD 6.94 or 8.64. The deuterated photooxidized enzyme was prepared by illuminating lipoamide dehydrogenase to 28% residual reductase activity in protonic buffer followed by deuteration. The reduced enzyme was prepared by an anaerobic reduction of the deuterated lipoamide dehydrogenase with NADD. Proton magnetic resonance spectra were obtained with a Varian XLlOO-12 NMR spectrometer equipped with Nicolet Fourier transform and TT-100 Data systems. The proton signal from Hz0 was used as an internal standard which was checked using tetramethylsilane as the external standard. The spectral acquisition at room temperature for enzyme solutions of 10 mg/ml took lo-15 min. No detectable change in enzymatic activities and flavin spectra was observed before and after PMR experiments. Other analytical methods. Multifunctional activities of lipoamide dehydrogenase’ were assayed as described (14,15). Cysteine and cystine residues were determined with 5,5’-dithiobis(2-nitrobenzoic acid) and iodoacetic acid as described by Matthew et al. (16). Amino acid analyses of proteins were carried out in a Beckman 119BL amino acid analyzer following in vocuo hydrolysis in 6.0 N HCl at 110°C for 22 h. Percentage DzO/HzO in photooxidation experiments was measured by the dioxane proton resonance peak integration method (1’7) using a Varian T-60 NMR spectrometer. RESULTS

Table I shows that both methylene blue and rose bengal sensitize lipoamide dehydrogenase to photoinactivation. Although the thiazine dye is a more effective sensitizer, it induces photooxidation of a number of amino acids nonselectively. The fluorescein derivative, on the other hand, * Multifunctional activities of lipoamide dehydrogenase refer to reductase assayed by NADH + Lip(SH)z, transhydrogenase assayed by NADH + thio-NAD+, electron transferase assayed by NADH + Fe(CN)i-, and disphorase assayed by NADH + 2,6dichloroindophenol.

HISTIDINE TABLE DYE SENSITIZED LIPOAMIDE

RESIDUES

OF LIPOAMIDE

I PHOTOOXIDATION DEHYDROGENASE

OF

Photosensitizer Methylene blue at pH 6.5 Percentage reductase activity of control remained (30 min) Rate of inactivation X l@ (mine’) Amino acid residues per FAD photooxidized

5fl

k

6.26 + 1.25 Histidine (10) Methionine (1) Phenylalanine (5) Tyrosine (4)

Rose bengal at pH 7.0 28 f 3

1.32 + 0.14 Histidine

(5)

Note. Occasional agitation of methylene blue sample prevents its bleaching and ensures effective photooxidation. An anaerobic ([OJ < 0.01 PM) photoreaction (30 min) retains 97% of the reductase activity (k = 0.16 X lo-’ min-I). The active-site disulfide is not affected in the protonic buffer. In the control sample, none of the amino acid residues, His(ll), Met(S), phe(l5), and Tyr(8) per FAD, was oxidized. Other multifunctional activities remained: transhydrogenase, 64%; electron transferase, 106%; and diaphorase, 125%.

photooxidizes histidine residues specifically and reproducibly to yield an enzyme derivative with a constant (28 k 3%) reductase activity with minimal effects on other activities (Table I, Note) and protein conformation as revealed by infrared spectroscopy (14). A further photooxidation resulted in a large enhancement of diaphorase activity indicative of a deleterious structural change. Lipoamide protects the enzyme from the sensitized photoinactivation whereas NAD+ fails to offer such protection (Fig. 1). Excess rose bengal quenches the 517-nm fluorescence band of bound FAD (X,, = 350 nm) to approximately 72% of initial intensity. Assuming the limiting value of FAD fluorescence quenching to represent saturation of rose bengal binding sites, regression analysis of the Stockel plot (18) as shown in Fig. 2 gave a linear function with n (binding sites) = 1.8 and dissociation constant (&) of 31.7 pM for the enzyme-rose bengal complex. Figure 3 shows that rose bengal inhibits lipoamide dehydrogenase competitively versus lipoam-

723

DEHYDROGENASE

ide (inhibition constant, Ki = 80.0 pM) but noncompetitively with respect to NAD+. These results suggest that rose bengal interacts with the lipoamide binding site of the reductase. Therefore, rose bengal was used as the photosensitizer to investigate the role of histidines in lipoamide dehydrogenase catalysis. The photoinactivation of lipoamide reductase by rose bengal follows first-order kinetics (Fig. 4, inset) in the initial phase with an oxidation of five histidine residues per FAD (Table I). To corroborate the nature and state of the amino acid residues which are responsible for the observed inactivation upon photooxidation, rates of sensitized photoinactivations were determined over a pH range where rose bengal (pK = 4.5) remained anionic (19). The pHrate profile (Fig. 4) indicates the involvement, in the oxidized enzyme, of the ionizable group(s) with pK 6.6 + 0.2. Its protonated form enhanced the anionic rose bengal sensitization.

.lO_J TIME (m(n)

FIG. 1. Protection of lipoamide dehydrogenase to rose bengal-sensitized photoinactivation. Lipoamide dehydrogenase (1.0 PM) in 0.20 M phosphate buffer, pH 5.0, containing 20 @d rose bengal (0) was photoinactivated in the presence of 240 PM (-2 K,,, A) or 600 PM (-5 K,,, 0) of NAD+ and 240 pM lipoamide (-3 Kb, 0). To facilitate the rate and extent of photoinactivation, the experiments were carried out in a low pH with stirring.

724

TSAI

ET AL. TIME 0 I

IO I

6

9

I

-2.51

I 5

6

7

(min) 20 I

= *

IO

P” FIG. 4. pa-rate profile for rose bengal-sensitized photoinactivation of lipoamide dehydrogenase. The inset shows the first-order kinetic plot at pH 7.0.

(Et-P)-’

FIG. 2. Stoekell plot for rose bengal quenching of FAD fluorescence. Lipoamide dehydrogenase (20.6 M) in 0.10 M phosphate buffer, pH 7.0, was excited at 350 nm. The fluorescence intensities at 520 nm in the presence of rose bengal (O-35 M) were recorded. Data were fitted for n binding sites (18) according to [RB]/ P = n + l/Kd(Et - p), where [RB], Et are concentrations of rose bengal and enzyme, p is the fraction of binding sites filled, and Kd is the dissociation constant.

Figure 5 shows that sodium azide, a singlet oxygen quencher, prevents photoinactivation. However, the rate of photoinactivation in Da0 is 5.2 times faster than in HzO. Sensitized illumination of lipoamide

E

‘-I[ROSE

BENGAL]

(PM)

dehydrogenase in deuterated buffer effectively eliminates the reductase activity with an enhanced diaphorase activity. Other multifunctional activities are reduced to variable degrees. An extensive oxidation of histidines (nine histidines per FAD) with concurrent destruction of the disulfide was noted in D20. The destruction of the disulfide was deduced from the free and total thiol determinations (16), a complete inactivation of lipoamide reductase activity (30 min) and a bleaching of FAD by approximately one equivalent of dithionite.

/

?lizsza .02

A

04

.06

[ NAD+]-’

(pm’)

.DB

01

1.0 B

2 [ LIPOAMIDE]-’

4

6 (mM-’

I

6

)

FIG. 3. Rose bengal inhibitions of reductase reaction catalyzed by lipoamide dehydrogenase. Reductase reactions at pH 7.0 and 25°C were carried out with (A) varied [NAD+] at constant @p(SH)r] = 30 KM and (B) varied &ipspl at constant [NADH] = 25 PM. Concentrations of rose bengal are indicated.

HISTIDINE

RESIDUES

OF LIPOAMIDE

TABLE KINETIC

60

II

PARAMETERS FOR LIPOAMIDE REDWTASE REACTION

E 5 40 F Y 2 z

725

DEHYDROGENASE

Native (5.0 nM)

20

iii i IO

20 TIME

30

V (flbl mini) Ka (PM) Kb (PM) V/E, (min-‘)

(532&

154.5 43.8 920 30.9 x 103

56.2 11.2 251 1.05 x 103

(min 1

5. Effects on rate of rose bengal-sensitized photoinactivation on lipoamide dehydrogenase. Lipoamide hydrogenase was illuminated in a protonic (HsO) buffer (0), a protonic buffer containing 5.0 mM N; (A), and a deuterated (DaO) buffer (0). The firstorder rate constants of photoinactivation are 1.32 x 10-z min-’ (0), 0.13 X lo-’ min-’ (A), and 6.80 X 10-z min-’ (Cl), respectively, at pH(D) 7.0. The photooxidation (30 min) in the deuterated buffer destroys nine histidines per FAD and the active site disulfide. FIG.

The photooxidized lipoamide dehydrogenase (EAY)with constant residual reductase activity (28 f 3% of the control) was isolated for kinetic studies. Double-reciprocal plots of initial rates of Ehv catalyzed reductase reactions versus either substrate in the asymptotic region are parallel at pH 6.5 indicating an operation of a Ping-Pong kinetic scheme. Kinetic parameters are listed in Table II. Product inhibition studies are consistent with the Ping-Pong scheme (Table III). NAD+ produces noncompetitive inhibition patterns versus NADH but competitive inhibition patterns versus lipoamide. Whereas dihydrolipoamide is the competitive inhibitor with respect to NADH but the noncompetitive inhibitor with respect to lipoamide. Unlike the native lipoamide dehydrogenase, Ehu does not exhibit the 530-nm absorption band characteristic of the charge transfer complex upon reduction. Instead, an anaerobic titration of Ehu at pH 6.3 with dithionite displays biphasic fluorescence spectra (Fig. 6). One equivalent reduction in the first phase results in a decrease in fluorescence emission at 517 nm to approximately 49% of the oxidized Ehv. An

Note. Photooxidized enzyme (E&J with the constant residual activity was prepared as described in the text. Kinetic studies were carried out in 50 mM phosphate buffer, pH 6.5, at 25°C.

isosbestic point occurs at 496 nm in the flavin emission spectra indicating the presence of a spectrally different enzymatic form from the oxidized form of Ehv. The emission maximum of this half-reduced Ehu (E&H,) is blue shifted relative to oxidized enzyme. Further reduction (EhvHQ) results in bleaching of flavin fluorescence. Histidine residues of proteins are amenable to PMR studies (20). Figure ‘7 shows resonance peaks of histidine C2 protons of lipoamide dehydrogenase in deuterated buffer (pD 6.94) at 69.18 ppm with shoul-

TABLE PRODUCT REACTION

Product inhibitor NAD+ LidW2

INHIBITION CATALYZED

III

CONSTANTS FOR REDUCTASE BY PHOTOOXIDIZED ENZYME

Varied substrate

Kii (FM)

(2,

NADH Lip& NADH Lip&

667 65.8

1401 325 426 232

Note. Product inhibition studies at pH 6.5 were carried out at fixed iJipSzJ = 270 FM with varied [NADH] from 10 to 50 pM and fixed [NADH] = 25 pM with varied [Lip&j from 125 to 540 jtM. Inhibitor concentrations of @AD+] vary from 0 to 100 pM and [Lip( from 0 to 800 gM. An increase in the inhibition constants for Ehr as compared to the corresponding values of the native enzyme (10) is noted.

726

TSAI ET AL.

575

550 EMISSION

525 WAVELENGTH

500 ( nm 1

FIG. 6. Fluorescence spectra of reduced photooxidized lipoamide dehydrogenases. The photooxidized enzyme with 28% reductase residual activity was isolated and reduced at pH 6.3 with sodium dithionite to different reducing equivalents: (1) 0, (2) 0.64, (3) 0.83, (4) 0.93, (5) 1.21, (6) 1.42, and (7) 1.76 equivalents. Their fluorescence spectra (excitation at 460 nm) were recorded.

der(s) centered around 69.23 ppm consisting approximately 25% of the histidine CZ protons. The shoulder protons are eliminated by the rose bengal-sensitized photooxidation and shifted upfield to 69.09 ppm upon reduction, thus, providing spectroscopic evidence for the identity between the photooxidized histidines and those which are implicated in the reduced native enzyme. At pD 8.64 where the histidines are deprotonated, identical histidine C2 PMR spectra (Fig. 8) are obtained for the oxidized form of native lipoamide dehydrogenase and its reduced form which also lost its MCD A term (Fig. 9). DISCUSSION

In general, sensitized photooxidations proceed by two mechanisms; type I mechanism involving triplet sensitizer giving rise to reactive singlet oxygen or type II mechanism requiring direct reaction of sensitizer with substrate resulting in its decomposition via a radical intermediate (21). The observed quenchings of inacti-

vation by azide and depletion of dissolved oxygen suggest an operation of the type I mechanism (22). An enhanced rate of photoinactivation in DzO which is shown to stabilize singlet oxygen (23) further supports an involvement of the singlet oxygen. However, the photooxidation in DzO is complicated by inactivation due to a loss of the catalytic disulfide. The pH-rate profile indicates that the sensitized photoinactivation is associated with the amino acid residue(s) with a pK of 6.6 f 0.2. Since only histidines are oxidized in rose bengal sensitization, the ionization constant of the photooxidized histidines in the native (oxidized) form of lipoamide dehydrogenase is ascribed to pK 6.6 f 0.2. Under the experimental condition, rose bengal specifically sensitizes the photooxidation of histidine residues with preferential inactivation of lipoamide reductase to the constant residual activity. An exceedingly short lifetime of the singlet oxygen in aqueous solution (23) and the specificity of rose bengal sensitization necessitate a propinquity of the sensitizer

HISTIDINE

RESIDUES

OF LIPOAMIDE

727

DEHYDROGENASE

tooxidized. This in conjunction with pHdependent photoinactivation implicates that the anionic fluorescein derivative interacts with the protonated histidines at or near the substrate binding site. Initial rate and product inhibition studies indicate the Eh,-catalyzed reductase reaction proceeds via the Ping-Pong kinetic scheme at pH 6.5. The pronounced suppression in the catalytic efficiency (WE,) of Ehv implicates an involvement of the photooxidized histidines in the reductase catalysis. The half-reduced photoinactivated enzyme (E,,H,) lacks both the MCD Faraday A term and 530-nm absorption band which characterize the charge transfer complex of the half-reduced native enzyme (EH,). However, the Eh,H2 displays substantial flavin fluorescence in contrast to the EHz of native lipoamide dehydrogenase from pig heart (24). The half-reduced lipoamide dehydrogenase from Escherichia coli does exhibit appreciable flavin fluorescence arising from the EHz form composed of oxidized flavin and fully protonated reduced disulfide (25). The spectral charac-

A

LDHtEH,)

I

L

I

9.5

FIG. 7. Proton tidine C, protons 6.94. (a) Oxidized tooxidized enzyme der at 69.23 ppm. enzyme showing

1

9.1 btppm)

1

I

8.7

8.3

magnetic resonance spectra for hisof lipoamide dehydrogenases at pD form of the native enzyme. (b) Phoshowing the absence of the shoul(c) Half-reduced form of the native an upfield peak at 69.09 ppm.

and histidines which are photooxidized by the singlet oxygen generated at the site. The protection experiment, binding study, and kinetic inhibitory effect of rose bengal suggest that this photosensitizer interacts with the lipoamide binding site of the enzyme. Only histidine residues are pho-

9.5

9.1

,

8.7

L LDH

FIG. 8. Proton magnetic resonance tidine C, protons of oxidized (lower the half-reduced (upper spectrum) 8.64.

spectra for hisspectrum) and enzymes at pD

728

TSAI ET AL. cm-’ +1.0

FIG. 9. Expanded magnetic circular dichroism A term region of the half-reduced lipoamide dehydrogenase at pH 7.0. The inset shows the A term region of MCD spectrum of the half-reduced enzyme at pH 8.65.

teristics of EhuH2 presented here demonstrate the presence of the oxidized flavin and the fully protonated dithiol. The observed blue shift in the flavin emission maximum upon reduction to EJ& is consistent with a shift to a more polar flavin environment (26) accompanying relaxation of the active site disulfide. Therefore, the residual reductase activity of Ehv at pH 6.5 arises from the oxidized flavin/dithiol intermediate without the benefit of the charge transfer complex. Templeton et al. (6) have shown that the MCD A term at 18,100 cm-’ which is eliminated by the sensitized photooxidation, is due to a structure unique to the reduced enzymes. To accommodate the requirement of a degeneracy in the excited state which gives rise to the A term, a charge transfer between the thiolate and the active site histidines was considered, although an ion pair would presumably be sufficient to allow the spin forbidden singlet-to-triplet transition assignment to the Faraday A term. Both experimental and theoretical studies demonstrate the ion pair formation between the thiolate anion and the imidazolium cation at the active site of papain (7,827). An identical structure has been proposed for glyceraldehyde-3-phosphate dehydrogenase (9) and glutathione reductase (28), while a similar

structure involving an unidentified base has been stipulated for lipoamide dehydrogenase (29). Histidine residues in proteins are readily monitored by PMR spectroscopy because of the unique downfield resonance of their CZ protons in deuterated buffer. At 100 MHz, one sharp resonance peak with a distinct shoulder corresponding to the CZ protons of histidines is seen in the oxidized form of native lipoamide dehydrogenase. The ion pair formation of the histidines which are photooxidized is evidenced by elimination of the shoulder histidine protons in the Ehv and its shift upfield upon reduction of the native enzyme. The observed upfield shift is in agreement with deprotonation of histidines during PMR titration (19,30) or in the present case, a reduced cationic character of the imidazolium ring which interacts with the thiol anion in the EHz. The absence of the PMR shift and the MCD A term of the EH2 at pD(H) 8.64 which deprotonates the interacting histidines provide further evidence for the ion pair formation between the thiolate anion and the imidazolium cation of the histidines. Therefore, the stabilization of the thiolate anion by the histidines via the ion pair provides the favored intermediate in the efficient reductase reaction catalyzed by

HISTIDINE

RESIDUES

OF LIPOAMIDE

the native lipoamide dehydrogenase. The destruction of the active site histidines renders Ehv incapable of supporting the thiolate ion. In the absence of the ion pair formation, the photooxidized enzyme functions via the oxidized flavin/dithiol intermediate with a greatly reduced efficiency. ACKNOWLEDGMENTS

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This work was supported by a grant from Natural Sciences and Engineering Research Council of Canada. J.R.P.G. is a holder of NSERC graduate scholarship.

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