Eur. J. Biochem. 148,239-243 (1985) 0 FEBS 1985

NADPH oxidation catalyzed by the peroxidase/H,O, system Iodide-mediated oxidation of NADPH to iodinated NADP

Alain VIRION, Jean Luc MICHOT, Danitle DEME and Jacques POMMIER Unite de Recherche sur la Glande Thyroide et la Regulation Hormonale, Institut National de la Sante et de la Recherche Medicale, Le Kremlin-Bicktre

(Received November 9, 1984) - EJB 84 1179

Oxidation of NADPH catalyzed by the peroxidase/H202 system is known to require the presence of mediating molecules. Using either lactoperoxidase or horseradish peroxidase, we demonstrated that in the peroxidase/H202 system, NADPH oxidation was mediated by iodide. The oxidation product was the iodinated NADP. This product was shown to possess spectral characteristics different from those of NADP' and NADPH, since for iodinated NADP, increased absorbance was observed in the 280-nm region and was directly proportional to the rate of iodination. It is suggested that oxidation and iodination of NADPH proceed via a single reaction between the intermediary iodide oxidation species and NADPH. Experiments with different molecules of NADPH analogues indicated that iodination occurred in the nicotinamide part of the NADPH molecule.

In the thyroid, an NADPH-dependent H202-generating system has been suggested to be involved in iodination catalyzed by thyroid peroxidase [I - 31. Previous results showed that in an in vitro system containing lactoperoxidase, H202, NADPH and iodide, NADPH was iodinated even in the presence of normal iodination substrates [3]. This suggested that in thyroid, the NADPH-dependent H202- generating system and the peroxidase are compartmentalized. However, a linkage might exist between NADPH oxidation, iodide and the peroxidase reaction. On the one hand, NADPH oxidation is only catalyzed by peroxidase/H202 in the presence of mediating molecules like thyroxine [4, 51, hematoporphyrin [6], guaiacol or scopoletin [7]. On the other hand, iodide has been shown to be involved as a mediator in many peroxidase reactions such as thyroxine formation [8], drug oxidation [9], catalatic reactions of peroxidase [lo] or increased resistance to dissociation of thyroglobulin [I I]. In addition, De Groot observed that NADPH oxidation was greatly increased by iodide [12].

The experiments described here show that iodide acts as a mediator in NADPH oxidation catalyzed by lactoperoxidase or horseradish peroxidase. The oxidation product of NADPH is the iodinated NADP. Our data show that both NADPH oxidation and iodination result from a single reaction between the intermediary oxidized iodide species and NADPH, and

Correspondence to J. Pommier, Unite 96 de I'INSERM, HBpital- Hospice de Bicktre, 78 Avenue du Genkral-Leclerc, F-94270 Le Kremlin-Bicktre, Val-de-Marne, France

Ahhreviations. HRP, horseradish peroxidase; LPO, lactoperox- idase; TPO, thyroid peroxidase; Glc6P, glucose 6-phosphate; NMN, P-nicotinamide mononucleotide; NMNH, P-nicotinamide mono- nucleotide, reduced form.

Enzymes. Horseradish peroxidase and lactoperoxidase (EC 1.11.1.7); thyroid peroxidase (EC 1.11.1.8); Glc6P dehydrogenase (EC 1.1.1.49); glucose oxidase (EC 1.1.3.4).

that iodination occurs on the nicotinamide part of the NADPH molecule.

MATERIALS AND METHODS

Products

Chemicals used in the study were obtained from the following sources : NADPH and the different analogue molecules of NADPH, lactoperoxidase (LPO, Rz = 0.706) and type I1 ~-glucose-6-phosphate dehydrogenase from Sigma; grade I horseradish peroxidase (HRP) and grade I glucose oxidase from Boehringer; D-glucose and guaiacol from Prolabo; Perhydrol (30% H202) from Merck; Na12'I from the Commissariat a 1'Energie Atomique. Thyroglobulin was prepared as previously described [13].

Experimental procedures

NADPH oxidation assays were carried out in a standard incubation mixture including 50 mM phosphate buffer, pH 7.2. The NADPH oxidation was monitored at 340 nm, using a molar abserption coefficient of 6.2 x lo3 M-' cmpl , in a Cary 15 or a Zeiss PMQ2 spectrophotometer. Spectra were recorded with a Cary 15 spectrophotometer at room temperature. NADPH iodination was performed in the pres- ence of 1251-labelled iodide and was measured by paper chromatography. The detailed experimental conditions of in- cubations are given in the legends to the figures. Incubations were started by addition of H 2 0 2 or glucose oxidase. The reaction was stopped by depositing 2-3-1.11 aliquots of the incubation medium on Whatman no. 1 chromatography paper. These samples were analyzed by ascending chro- matography 1141 in acidic solvent (butanol/acetic acid/water, 8/2/2 or 78/5/17). Under these conditions, iodinated NADPH

240

0.6

E $ 0.5 m c 0

aJ u C 0

g 0.1 VI n 4

0.3

I I

O O 1 2 Time Imin)

Fig. 1. Kinetics of iodide-mediated NADPH oxidation catalyzed by LPO. Samples containing 0.1 mM NADPH, 6 mM glucose, 0.4 pg glucose oxidase were incubated with 5 pg LPO in the presence (0-0) or absence (0-0) of 0.1 mM iodide in 1 ml phosphate buffer, pH 7.2

remained at the origin whereas iodide migrated [3]. Experi- ments were done at least in duplicate.

RESULTS

Iodide-mediated NADPH oxidation

Fig. 1 represents the kinetics of NADPH oxidation catalyzed by lactoperoxidase in the presence of an H202- generating system. In the absence of iodide, no oxidation was observed, whereas in its presence the absorbance at 340 nm rapidly decreased. Under these conditions, production of HzOz was rate-limiting. When 0.1 mM HzOz was directly supplied to the system, the iodide-mediated oxidation of NADPH was almost complete in a few seconds. Addition of 5mM Glc6P and 5 pg/ml Glc6P dehydrogenase after complete NADPH oxidation did not allow the regeneration of NADPH (results not shown), indicating that the NADPH species oxidized was not NADP'.

Identical results were obtained when experiments were performed with 10 pg/ml HRP instead of LPO. However, the rate of iodide-mediated oxidation of NADPH catalyzed by HRP was very low at pH 7.2 and much higher at pH 5.7. These results are compatible with the requirement of an oxidized iodide species for catalysis of NADPH oxidation, since it is known that HRP cannot efficiently catalyze iodide oxidation at pH 7.2.

Further, with LPO at pH 7.2 and HRP at pH 5.7, the stoichiometry between H2O2 and oxidized NADPH was found to be 1 : 1.

Evidence for NADPH iodination during iodide-mediated NADPH oxidation

Previous results [3] showed that when NADPH was pre- sent in the peroxidase system comprising LPO, 'z51-labelled iodide and HzOz, it was iodinated. Iodinated NADPH constituted a non-migrating material when measured by paper chromatography in acidic solvent, whereas no iodination product could be detected by paper chromatography in

0- 0 1 2 3 1 5 6

Time (min)

Fig. 2. Kinetics of NADPH iodination catalyzed by the LPO/H202 system obtained with 25 pM (0-0) and 100 pM (0-0) iodide. Lactoperoxidase (2 pg/ml) was incubated in 50 mM phosphate buffer, pH 7.2, at 30°C in the presence of iodide labelled with '"INa, 0.1 mM NADPH and 6 mM glucose. Reactions were initiated by the addition of 0.4 pg/ml glucose oxidase. (A-A) Control with 100 pM iodide and without NADPH. Iodination was measured by paper chromatography as described in Materials and Methods

alkaline solvent, indicating that the iodinated NADPH mole- cule is unstable in alkaline medium. Fig. 2 shows the kinetics of NADPH iodination catalyzed by LPO for two iodide con- centrations. Under these conditions, about 60% of the lZ5I- labelled iodide was present in non-migrating material when chromatographed in acidic solvent. No iodination was ob- served when NADPH was omitted from the incubation medi- um. As the conditions used in this experiment were the same as those used for NADPH oxidation (Fig. l), it seems clear that the iodide-mediated NADPH oxidation product is an iodinated product.

The substitution of NADP' for NADPH in the exper- iment illustrated in Fig. 2 did not permit iodinated NADP formation, thus showing that the iodinated product of NADPH is only formed during iodide-mediated NADPH oxidation. Further, addition to the assays of free tyrosine or proteins (thyroglobulin or serum albumin), which are normal substrates for iodination, did not inhibit NADPH iodination (results not shown). In the presence of these substrates, NADPH was preferentially iodinated.

As Fig. 1 shows, iodide-mediated NADPH oxidation in- duced complete disappearance of absorption at the 340 nm wavelength. The experiments in Fig. 3 represent a comparison between the ultraviolet spectra of NADP' and NADPH on the one hand, and the spectra obtained after guaiacol and iodide-mediated NADPH oxidation with the same NADPH and NADP' concentrations, on the other. There was no difference between the spectra of NADPH, NADP' or the guaiacol-mediated NADPH oxidation product, whereas in the iodide-mediated NADPH oxidation, a large increase in absorbance was observed in the 260 - 300-nm region, with a maximum variation at 282 nm.

Evidence that iodination occurs in the nicotinamide part of the NADPH molecule

The iodination of the different NADPH analogue molecules in the LPO/H202 system is compared in Table 1. It

24 1

" 250 260 270 280 290 300 310 W a v e l e n g t h ( n m )

Fig. 3. Comparison of the ultraviolet spectra for NADP' ( 4 ) , NADPH ( 1 ) and the NADPH oxidation products obtained when oxidation is mediated byguaiacol(3) or iodide ( 2 ) . NADPH and NADP' concen- trations were 0.1 mM. In (2), the iodide concentration was 0.1 mM. In (3), the guaiacol concentration was 5 pM. In (2) and (3), 10 pg/ml LPO was present and oxidation reactions were initiated by addition of 0.1 mM HzOz. All experiments were performed in 50 mM phosphate buffer, pH 7.2

Table 1. Behaviour of the different NADPH analogue molecules in the iodination reaction ( A ) . Inhibition of thyroglobulin iodination by these molecules (B) (A) Iodine incorporation into nucleotides. 20 pM nucleotides were in- cubated with 2.5 pg/ml LPO in 50 mM phosphate buffer, pH 7.0, in the presence of 20 pM '251-labelled iodide and 10 pM HzOz. After 10 min of incubation at room temperature, aliquots of the incubation medium were analyzed by paper chromatography in butanol/acetic acid solvent. The percentages of non-migrating radioactive material were calculated and data are expressed as the amount of iodide in- corporated in this material. (B) Inhibition by nucleotides of thyroglobulin iodination. 1 pM noniodinated goiter thyroglobulin (TG) was incubated under the same conditions as (A). Thyroglobulin iodination was performed in the presence of 20 pM of each nucleotide, measured by paper chromatography in ethanol/ammonium carbonate solvent, and compared to the control, treated in the absence of nucleotide. Significant values are printed in bold-face type

Product (B) Inhibition of TG iodination

(A) Iodination

NADPH NADP+ NADH NAD' NMNH NMN' AMP Nicotinamide

nmol 4.71 0.42 5.47 0.41 2.71 0.35 0.43 0.35

%

99 3 99 4

99.5 1 7 0

shows that only the reduced form of the molecules containing nicotinamide (NADPH, NADH and NMNH) could be iodinated, and that this did not require the adenine part of the NADPH molecule. The ability of these molecules to undergo iodination is correlated with their ability to inhibit protein iodination. This indicates that only the reduced form of

I I I I I I I I I I

i 15 260 270 280 290 300 310 320 330 310 350

W a v e l e n g t h ( n m )

Fig. 4. Comparison of the ultraviolet spectra for NMNH, NMN' and the N M N H oxidation product obtained by iodide-mediated oxidation in the L P O / H 2 0 2 system. 50 pM PNMNH was incubated in 50 mM phosphate buffer, pH 7.0, in the presence of 2.5 pg/ml LPO at room temperature. Its spectrum was recorded (1) before and (2) 2.5 min after addition of 20 pM HzO2, (3) 2.5 min after addition of 50 pM KI and (4) 5 min after further addition of 40 pM H202. The spectrum of this final product was compared to that of 50 pM PNMN' in the same buffer [5]

nucleotide molecules can act as substrates in iodide-mediated oxidation catalyzed by the LPO/H202 system.

The iodination capacity of NMNH allowed us to study the spectral changes occurring in the native and iodinated molecules in the 260 - 340-nm region without interference by adenine absorption. The data in Fig. 4 show that similar spectra were obtained with 50 pM native NMNH and NMNH supplemented with the LPO/H202 system (spectra 1 and 2). No absorbance peak appeared in the 280-nm region, whereas the spectrum for oxidized NMNH (NMN') (spectrum 5) had an absorbance peak at 265 nm.

In contrast, spectra 3 and 4 show that after addition of iodide to the assays containing LPO, H202 and NMNH (spectrum 2), a large increase in absorbance appeared at 280 nm; it correlated with the decreased absorbance at 340 nm. The spectral characteristics of this oxidized NMNH molecule are different from those of the NMN' molecule. No change in absorbance at 280 nm could be observed when NMNH was replaced by molecules which cannot be iodinated (Table 1) such as nicotinamide or AMP, indicating that the increase in absorbance at 280 nm was not due to I; forma- tion.

As observed for iodide-mediated oxidation of NADPH, these results are consistent with the formation of an iodinated NMN molecule during the iodide-mediated NMNH oxida- tion. This suggests a relationship between the iodination of the nicotinamide part of NADPH, NADPH oxidation and the increased absorbance at 280 nm.

Comparison of NADPH iodination, NADPH oxidation and increased absorbance at 282 nm

In the experiment in Fig. 5A, the kinetics of NADPH iodination measured by paper chromatography, of NADPH

242

I I I I I I 0.9

0.8

2 0.7 -

E 5 0.6 m c o 0.5 m u 5 0.L n

0 0.3 n

L

Q

0.2

0.1

I

30 2 z c .- U a

20 ;;

"0 50 100 150 Hydrogen peroxide (nmol)

0

Hydrogen peroxide lnrnol)

Fig. 5. Comparison of NADPH iodination, NADPH oxidation and increased absorbance at 282 nm, catalyzed by LPO as a,function ojthe H 2 0 2 concentration. Increasing amounts of H 2 0 2 were added to samples containing 0.1 mM NADPH, 2 pg/ml LPO, 50 mM phosphate buffer, pH 7.2, in the presence of 0.1 mM (A) or 0.2 mM (B) iodide. NADPH iodination ( A-A) measured by paper chromatography (see Materials and Methods), NADPH oxidation (0-0) and the increased absorbance at 282 nm (0-0) were followed under the same experimental conditions

oxidation measured by the decreased absorbance at 340 nm, and of the increase in absorbance at 282 nm were simulta- neously followed in the LPO/HzOz system as a function of the HzOz concentration.

A strict parallel was observed between these three different measures, suggesting that oxidation and iodination of NADPH result from a single reaction between the in- termediary oxidized iodide species and NADPH, thus:

LPO + HzOz + 1- + LPO-I,, NADPH + LPO-I,, + iodinated NADP + LPO.

Since the iodide-mediated oxidation of NADPH produced iodinated NADP, the increase in absorbance at 282 nm might reflect iodination of NADPH. Fig. 5 B depicts the same exper- iment conducted with an iodide concentration of 200 pM, i.e. higher than that of NADPH. Similar results were obtained when the HzOZ concentration was lower than that of NADPH. However, when the HzOz concentration was higher than that of NADPH, a decrease in absorbance at 282 nm was seen concomitantly with deiodination of the iodinated NADPH. These observations again demonstrate the existence of a correlation between NADPH iodination and the increase in absorbance at 282 nm. In addition, comparison of the data in Fig. 5A and B shows that an excess of iodide and H202 probably mediated a subsequent oxidation of iodinated NADPH that produced a deiodination reaction of the iodinated product.

From these results, it can be concluded that in the peroxidase/H2O2 system, iodide-mediated oxidation of NADPH, NADH or NMNH probably proceeds by the same mechanism, and that the iodination occurs in the nicotinamide part of the nucleotide molecule.

DISCUSSION AND CONCLUSION

The experiments described in this study clearly show that iodide mediates the oxidation of NADPH catalyzed by the peroxidase/HzO2 system (HRP or LPO). Unlike NADPH oxidation mediated by thyroxine [ 5 ] , hematoporphyrin [6], guaiacol [7] or scopoletin [7], the product of iodide-mediated

oxidation is not the enzymatically active NADP', but the iodinated NADP. The inability of HRP to catalyze the iodide- mediated NADPH oxidation at pH 7.2, a pH at which iodide is not oxidized by HRP [15], indicates that NADPH oxidation is mediated by an intermediary iodide-oxidized species.

The similarity observed between the kinetics of NADPH iodination, of NADPH oxidation and of the increased absorbance at 282 nm suggests that NADPH iodination pro- ceeds via a single reaction between the oxidized iodide and NADPH, as follows:

I - + H ~ O ~ peroxidase[,,

NADPH + I,, + iodinated NADP.

Under these conditions, the iodide species oxidized would be necessarily It, as previously proposed for the mechanism of iodide oxidation catalyzed by peroxidases [16,17]. In addition, experiments with different NADPH analogue molecules show firstly that iodination occurs in the nicotinamide part of the NADPH molecule and, secondly, that only the reduced form of nucleotides can be iodinated.

In addition, NADPH iodination catalyzed by the peroxidase/HzO2 system constitutes a new method of measur- ing HzOz, since the amount of NADPH iodinated has been shown to be directly proportional to the amount of HzOZ present in the medium. This method is sensitive enough to permit determination of 0.1 KM H2O2 and to follow the kinetics of H 2 0 2 production.

Another interesting conclusion is based on the observation of preferential NADPH iodination in a peroxidase system containing thyroglobulin or other proteins. Such a system exists in vivo in the thyroid gland. De Groot and Davis [I21 reported that NADPH oxidation catalyzed by particulate pro- teins from sheep thyroid tissue required the presence of iodide. Further, iodination of free tyrosine catalyzed by this prepara- tion was competitively inhibited by reduced pyridine nucleotides. Although these data were obtained with a very crude thyroid peroxidase (TPO) preparation, they suggest that TPO and LPO oxidize NADPH in a similar manner. In that case the observations just reported imply that the two enzyme systems involved in the thyroglobulin iodination process,

243

(NADPH-dependent H 2 0 2 generation and thyroid per- 7. Michot, J. L., Virion, A,, Deme, D., De Prailaune, S. & Pommier, oxidase) must be strictly cornpartimentalized.

8. Deme, D., Pommier, J. & Nunez, J . (1976) Eur. J . Biochem. 70,

Bahloul for the preparation of the manuscript, and Mrs DreYfus for 9. Michot, J . L., Nunez, J., Johnson, M. L., Irace, C. & Edelhoch, English language editing. This work was supported by a grant from the Fondation pour la Recherche MPdicale. 10. Magnusson, R. P. & Taurog, A. (1983) Biochem. Biophys. Res.

J. (1985) Eur. J. Biochem., in the press.

The authors wish to thank Mrs C. Sais, A. Guedec and M. M. 435-440.

H. (1979) J . Biol. Chem. 254,2205-2209.

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