Role of Calcium in the Reaction between Pyrroloquinoline Quinone and Pyridine Nucleotides Monomers and Dimers

Archives of Biochemistry and Biophysics Vol. 368, No. 2, August 15, pp. 385–393, 1999 Article ID abbi.1999.1270, available online at http://www.idealibrary.com on

Role of Calcium in the Reaction between Pyrroloquinoline Quinone and Pyridine Nucleotides Monomers and Dimers 1 Antonio Casini,* ,2 Alessandro Finazzi-Agro`,† Stefania Sabatini,† El Said El-Sherbini,‡ Silvano Tortorella,* and Luigi Scipione* *Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita` “La Sapienza,” Rome, Italy; ‡Department of Biochemistry, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt; and †Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita` di Roma “Tor Vergata,” Rome, Italy

Received April 5, 1999

Redox reactions were carried out in aerobiosis and anaerobiosis between NAD(P) dimers or NAD(P)H and pyrroloquinoline quinone (PQQ) in different buffers. The buffer system and pH significantly affected the oxidation rates of nucleotides and the ESR signal intensity of the PQQ • radical formed in anaerobiosis by comproportion between the quinone and quinol forms. The relative reactivity of the four nucleotides toward PQQ was affected by pH and buffer nature. PQQ, which behaves as an electron shuttle from nucleotides to oxygen, was first converted to PQQH 2 and then rapidly reoxidized by oxygen, with formation of hydrogen peroxide. Both NAD(P) dimers and NAD(P)H consumed 1 mol of oxygen per mole of reacted molecule of pyridine nucleotide, yielding 1 or 2 mol of NAD(P) 1 from NAD(P)H or from NAD(P) dimers, respectively. Chelating agents such as EDTA and phytate strongly decreased the reaction rate and the PQQ • radical signal intensity. Kinetics carried out in the presence of metal ions showed instead an increased reaction rate in the order Ca 21 .. Mg 21 > Na 1 .. K 1. Spectrofluorimetric measurements of PQQ with increasing concentrations of Ca 21 showed a fluorescence quenching and shift of the maximum emission toward lower wavelengths, while other metal ions showed minor effects, if any. Therefore, it is demonstrated that Ca 21 binds to PQQ, probably forming a complex which is more reactive with both one-electron (NAD(P) dimers) or two-electron donors (NAD(P)H) in nonenzymic reactions. It is important to recall that Ca 21 was already found to play active role in PQQ-containing enzymes. © 1999 Academic Press

1 This work was supported by a 40% research fund from M.U.R.S.T. (Rome). 2 To whom correspondence should be addressed. Fax: 0039-0649913888. E-mail: [email protected].

0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

Key Words: redox reactions mechanism; electron transfer; Ca 21 binding to PQQ; one- and two-electron transfer.

Pyrroloquinoline quinone, 2,7,9-tricarboxy-1H-pyrrole[2,3,7]quinoline-4,5-dione (PQQ, Methoxatin), 3 the cofactor of some important bacterial alcohol and glucose dehydrogenases, has been extensively studied for the past two decades. There have been claims that it could represent a growth factor even for eucaryotic systems, also on the basis of several reports on the presence of PQQ in some plant amineoxidases (1) or mammalian amine-dehydrogenases (2, 3). More recently, however, the cofactor of these enzymes was actually shown to be another quinoid species, namely TPQ and TTQ (4, 5), lowering the interest in PQQ itself. Nevertheless, important work has still been produced on PQQ and PQQ-containing enzymes regarding structural, mechanistic, or nutritional aspects (6 –9). Our interest for PQQ stands in its redox properties and redox chemistry. PQQ can exist in three forms, namely, fully oxidized quinone (PQQ), half-reduced semiquinone radical (PQQ • or PQQH •), and fully reduced quinol (PQQH 2), implying that it may undergo either one-electron or two-electron reduction. In fact, several two-electron donors, such as NaBH 4, alcohols, b-mercaptoethanol, or hydrazine, can reduce PQQ to quinol form (10, 11). Some years ago, Sugioka et al. Abbreviations used: [(NAD(P)) 2 and NAD(P) dimers] (b)1, (b9)19,4,49-tetrahydro-3,39-dicarbamoyl-4(R or S), 49 (S or R)-bipyridine1,19-bis-diphosphoribosyl-adenosine ((NAD) 2 ) and 1,19-bis-diphosphoribosyl-adenosine-29-phosphate ((NADP) 2); [NAD(P) monomers] b-NADPH and b-NADH; PQQ, pyrroloquinoline quinone; PQQ • , PQQH • , pyrroloquinoline semiquinone radical; PQQH 2 , pyrroloquinoline quinol; TPQ, topaquinone; TTQ, tryptophan-tryptophylquinone; DMSO, dimethyl sulfoxide. 3

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SCHEME I

(12), studying the redox reaction between NAD(P)H and PQQ, suggested that a one-electron transfer was taking place from the pyridine coenzymes NAD(P)H to PQQ to form the PQQ • radical anion. We first extended the study on the reactivity of PQQ using the NAD(P) dimers (NADP) 2 and (NAD) 2 as reductants (18), prepared in our laboratory by one-electron electrochemical reduction of NAD(P) 1 (13, 14) (Scheme I). These tetrahydrobipyridine structures can act as one-electron donors with many biologically relevant oxidizing agents, such as horseradish peroxidase, cytochrome c (Fe 31), adriamycin, or synthetic models such as phenazine methosulfate. Moreover, they can be used as a tool for discriminating between one- or two-electron redox mechanisms (15–17). Previously (18) we have shown that (i) (NAD(P)) 2 can reduce PQQ to PQQH 2 with a two-step electron transfer mechanism under both aerobic and anaerobic conditions; (ii) the quinol is easily back oxidized to PQQ by oxygen and by hydrogen peroxide, whereas the PQQ • radical cannot be oxidized by oxygen; and (iii) PQQ • radical can be generated in a comproportionation reaction between PQQH 2 and PQQ itself in anaerobiosis, rather than from a direct one-electron transfer from the dimers, at variance with the report of Sugioka et al. (12). In the course of these experiments, evidence was obtained that the nature of reaction medium (buffer type, pH, metals, etc) could significantly influence the redox kinetics. Furthermore, the interaction between PQQ or PQQ-enzymes and some cations has been reported (19 –24). In the present paper, we have investigated the redox behavior of NAD(P) dimers or NAD(P) monomers toward PQQ under different reaction conditions, such as medium composition, buffers, pH, and in the presence of metals or metal chelators.

Beef liver catalase (65,000 U/mg) was obtained from Calbiochem. Yeast alcohol dehydrogenase (ADH, 230 U/mg) and yeast glucose-6phosphate dehydrogenase (25 U/mg) were from Boehringer. All other chemicals, unless otherwise specified, were of analytical purity grade. NAD(P) dimers showed the following UV absorbance maxima (M 21 cm 21): « 259 5 32,600 and « 338 5 6400 ((NAD) 2); and « 259 5 34,300 and « 338 5 6250 ((NADP) 2)). Their purity was also checked by chromatographic (HPLC) (14, 25) or enzymatic methods for the absence of NAD(P) 1 or NAD(P)H. The coenzymes NADH and NADPH were purchased from Boehringer and showed molar absorbancies at 340 nm (M 21 cm 21): 6200 (NADPH) and 6100 (NADH), respectively. The reactions between the NAD(P) dimers or NAD(P)H with PQQ were followed spectrophotometrically at 333 nm at room temperature. The chosen wavelength roughly corresponds to the absorption maximum of 1,4-dihydropyridine, and the absorbance decrease was routinely used to follow the oxidation of both NAD(P) dimers and monomers. Moreover, this wavelength is an isosbestic point between PQQ and PQQH 2. The molar absorbancies used at this wavelength were as already reported (18). The buffers used were prepared daily and checked by a Metrohm 654 pH-meter at 25°C before each experiment. Reactions in anaerobiosis were carried out in a Thunberg-type cuvette. Anaerobiosis was obtained by four or five cycles of vacuum-refilling with UPP Ar or nitrogen. The spectrophotometer was a Perkin–Elmer UV-VIS 555 Model. The metal salts (chlorides or nitrates) other than heavy metals, added to the reaction medium, were previously purified by passing through a Chelex X-100 filter. The absence of heavy metals was checked by atomic absorption spectrometry. Fluorescence spectra were recorded in a fluorimetric 1-cm-path length cuvette by a Jobin Yvon 3D Spectrofluorimeter, using an excitation wavelength of 337 nm. PQQ dissolved in 0.1 M Tris–HCl buffer, pH 7.0, showed an emission maximum at l 5 482 nm. ESR measurements were made in a Bruker ESP 300 X-band spectrometer as already described (18). Oxygen uptake experiments were carried out in a Gilson Oxygraph cell kept at 25°C by a Clark-type electrode. The formation of enzymatically active NADP 1 or NAD 1 was checked by enzymatic methods on aliquots of the reaction mixtures as already reported (18). The presence of PQQ did not interfere, as shown by control experiments.

RESULTS

MATERIALS AND METHODS

Oxidation of (NAD) 2 and (NADP) 2 by PQQ in Different Media

PQQ (Methoxatin), obtained from Fluka, showed the UV features already reported (18). The dimers (NAD) 2 and (NADP) 2 were prepared according to Carelli et al. (13) and Ragg et al. (14), respectively.

Figure 1 shows a typical time course of the aerobic oxidation of (NADP) 2 or (NAD) 2, in 0.1 M Tris–HCl

EFFECT OF METAL IONS ON PYRROLOQUINOLINE QUINONE

387

Tris–HCl or phosphate, showed an effect of the medium on the reaction rate. Although reproducibility was difficult in anaerobiosis, the greater (NADP) 2 reactivity vs PQQ, particularly in Tris–HCl buffer, with respect to (NAD) 2, was found also in the absence of air. ESR measurements, on anaerobic, equimolar, or slight quinone excess PQQ–NAD(P) dimers mixtures, allowed detection of the well-known PQQ • radical ESR signal. The respective hyperfine coupling constants were a 1N 5 0.75 G and a 6N 5 0.83 G, a 1H 5 0.99 G, a 3H 5 1.20 G, and a 8H 5 2.024 G, as previously found (18). The PQQ • radical ESR spectrum did not significantly change on varying the reaction medium, while its intensity was strongly affected. For example, the ESR signal intensity was much lower in phosphate buffer than that in Tris–HCl buffer, keeping constant the concentrations of reactants. The maximum signal intensity could be observed when the concentration ratio for PQQ–NAD(P) dimers was about 2. Furthermore, there was a roughly proportional relationship between the relative ESR signal intensity and the reaction rate. Interestingly, the addition of EDTA almost abolished this esr signal, prevented the back oxidation of PQQH 2 by air, and strongly reduced the dimers oxidation rate, mainly if added before the reaction was started (see below). Comparison of NAD(P)H and (NAD(P)) 2 Reactions with PQQ in Different Media

FIG. 1. Time course of aerobic oxidation of NAD(P) dimers in presence of 20 mM PQQ. The curves represent the time course of the optical density difference at 333 nm and at room temperature, between sample and reference quartz cuvettes containing the same NAD(P) dimer solutions, that is, 202.8 mM (NADP) 2 (curve 1) or 208.1 mM (NAD) 2 (curve 2) in 0.1 M Tris–HCl buffer (pH 7.0) or 217.7 mM (NADP) 2 (curve 3) or 193.0 mM (NAD) 2 (curve 4) in 0.1 M phosphate buffer (pH 7.0). Reactions were started by adding 20 mM PQQ (final concentration) to the sample cuvette.

buffer (curves 1 and 2), or in 0.1 M phosphate buffer (curves 3 and 4), pH 7.0, in the presence of catalytic amounts of PQQ. Apparently, (NADP) 2 (curves 1 and 3) is always oxidized faster than (NAD) 2 (curves 2 and 4) and both dimers are more readily oxidized in Tris–HCl than in phosphate buffer. All curves have been corrected for the spontaneous slow autooxidation of NAD(P) dimers. The PQQ was not consumed, as demonstrated by the restored oxidation rate obtained after further addition of dimer to the reaction mixture (not shown). It should be recalled that anaerobic, nearly equimolar, mixtures of NAD(P) dimers and PQQ gave rise to formation of the quinol form PQQH 2, which was rapidly back oxidized to the starting PQQ upon air equilibration of the reaction mixture (18). These reactions, when carried out in different buffer systems like

The aerobic and anaerobic oxidation of natural coenzymes NADH and NADPH by PQQ was previously studied, but only in Tris–HCl buffer, pH 7.0 (12). Our experiments gave results in good agreement with the previous ones. However, when the same reactions were carried out in phosphate buffer, we observed again a somewhat decreased reaction rate. Moreover, the ESR signal intensity of the PQQ • radical, observed in anaerobiosis, was strongly lowered on changing from Tris– HCl to phosphate buffer, all other conditions being identical (see below). Therefore, we reinvestigated the overall reactivity of PQQ toward these four nucleotides, under various reaction conditions, with the aim of better understanding the redox chemistry of this cofactor. Keeping in mind the above results, the effect of the nucleotide concentration on the reaction rate with PQQ and the stoichiometry of NAD(P) dimers and monomers aerobic oxidation were studied, both in Tris–HCl and phosphate buffer, at pH 7.0. A second-order kinetics in the reaction with PQQ was always observed with every nucleotide used, as shown by the almost linear relationship between the initial reaction rate and the concentration of each nucleotide, keeping constant the concentration of PQQ (Fig. 2). Pseudo-first-order velocity constants, when [PQQ] ,, [Nucleotide], were as follows (10 24 s 21 6 SE):

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FIG. 2. Effect of nucleotides concentration on oxidation rate carried out in air in the presence of 20 mM PQQ. Each value is the mean of triplicate experiments (SD within 6%); the velocity of the reaction (V) was determined spectrophotometrically at 333 nm in the first 2 min after addition of PQQ: (a) in 0.1 M Tris–HCl buffer (pH 7.0) or (b) in 0.1 M phosphate (pH 7.0). Symbols: (NADP) 2, empty circles; (NAD) 2, full circles; NADH, triangles; and NADPH, squares.

while the nucleotides oxidation rate was not significantly affected. In the absence of catalase, the stoichiometry of NAD(P) 1 production vs the oxygen consumed was 2:1 in the case of NAD(P) dimers and 1:1 in the case of NAD(P)H. Again, the addition of EDTA strongly lowered the oxygen uptake rate. The stoichiometry of oxidation was the same also under anaerobic conditions, either for NAD(P) dimers (18) or for NAD(P)H. When the reaction was carried out in a DMSO–aqueous 0.1 M NH 4 HCO 3 mixture in presence of air, as already described for NAD(P) dimers (18), we observed the formation of an absorption at 302 nm, indicating the formation of PQQH 2 , and a concomitant decrease of the absorbance at 340 nm, due to the disappearance of both NAD(P)H and PQQ (Fig. 3). The ESR signal of the PQQ • radical was not detected under these conditions, as already found for NAD(P) dimers. DMSO also lowered the oxygen uptake rate, without affecting the stoichiometry of nucleotides oxidation. The PQQ • radical ESR spectrum was easily detected in anaerobic mixtures of NAD(P)H and PQQ, when Tris–HCl buffer was used as the solvent, whereas it was hardly observed in phosphate. Again, EDTA almost suppressed the ESR signal and inhibited the back oxidation of PQQH 2 by oxygen. It is worth noting that the nature of the buffer used, and, more generally, the composition of the reaction medium, affected the relative reactivity of the nucleotides toward PQQ. Therefore, the following experiments were further carried out, in which the pH influence on the reaction rate was studied, using a different TABLE I

(a) in Tris–HCl buffer, pH 7.0, 6.49 6 0.24 ((NADP) 2), 2.90 6 0.50 ((NAD) 2), 2.68 6 0.15 (NADH), and 2.53 6 0.42 (NADPH); (b) in phosphate buffer, pH 7.0, 1.73 6 0.15 ((NADP) 2), 1.33 6 0.04 ((NAD) 2), 2.47 6 0.13 (NADH), and 2.07 6 0.92 (NADPH). The effect of EDTA addition in aerobiosis on the PQQ-catalyzed oxidation of NAD(P) dimers and monomers is shown in Table I. The lowering of the initial reaction rate by EDTA is more more evident in Tris– HCl than in phosphate buffer. The stoichiometry of oxidation of the four nucleotides is shown in Table II, both in Tris–HCl and phosphate buffers (pH 7.0). Two moles of NADP 1 or of NAD 1 were produced per mole of dimer oxidized, whereas from monomers 1 mol of enzymatically active pyridine coenzyme was formed. Polarographically detected oxygen uptake by solutions of NAD(P)H or (NAD(P)) 2 in Tris–HCl or phosphate buffers, pH 7.0, is shown in Table III. The reduction product of oxygen is hydrogen peroxide, as demonstrated by the addition of catalase in the reaction medium, which almost halved the oxygen uptake,

Effect of EDTA on NAD(P) Dimer and Monomer PQQ-catalyzed Oxidation Rate a V 0 (mM min 21) c Buffer b

Nucleotide

No EDTA

Plus EDTA d

V 0EDTA/V 0 (%)

1 1 1 1 2 2 2 2

(NADP) 2 (NAD) 2 NADPH NADH (NADP) 2 (NAD) 2 NADPH NADH

7.76 3.48 2.96 3.50 2.08 1.60 2.13 2.81

0.66 0.64 0.49 0.52 0.50 0.97 0.52 0.61

8.5 18.4 16.7 14.8 24.0 60.5 24.2 21.9

a Reactions were followed by absorbance decrease at 333 nm, when 200 mM NAD(P) dimers or monomers were incubated at room temperature and in air in the presence of 20 mM PQQ (final concentration). Each experimental value is the mean of duplicate experiments, with variation coefficients ranging between about 5 and 25%. b 1, 0.1 M Tris–HCl buffer, pH 7.0; 2, 0.1 M phosphate buffer, pH 7.0. c Determined in the first 2 min after the addition of 20 mM PQQ. d [EDTA] 5 1 mM (final concentration), added previously in the buffer.

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EFFECT OF METAL IONS ON PYRROLOQUINOLINE QUINONE TABLE II

Stoichiometry of Aerobic Oxidation of NAD(P) Dimers and Monomers Catalyzed by 20 mM PQQ a [Nu] 09 c

Nucleotide and buffer b

[Nu] 209 c

[NAD(P) 1] d

Oxidation (%)

Yield (%)

169.9 69.9 32.4 34.3

39.9 e 16.0 e 15.1 16.1

95.0 e 82.0 e 92.3 96.1

36.1 28.7 18.7 24.4

8.3 e 7.4 e 9.4 11.5

94.9 e 95.6 e 90.8 92.4

(1) (NADP) 2 (NAD) 2 NADPH NADH

212.8 218.1 214.3 205.8

123.4 175.5 179.2 170.1 (2)

(NADP) 2 (NAD) 2 NADPH NADH

217.7 192.9 198.0 212.7

198.7 177.3 177.4 186.3

All concentrations were mM and are mean values of triplicate experiments, with SD within 8%. The reactions were carried out at room temperature in 0.1 M Tris–HCl buffer (pH 7.0) (1) and 0.1 M phosphate buffer (pH 7.0) (2). c Determined by absorbance at 333 nm, after subtraction of PQQ contribute, or by HPLC (dimers). d Measured enzymatically on aliquots of reaction mixtures. e By assuming a stoichiometry of 2 mol of NAD(P) 1 formed per each mole of NAD(P) dimer oxidized. a b

buffer (borate), and a different strong chelator, i.e., myo-inositol hexaphosphoric acid (phytic acid). The results further showed that the reactivity of the various NAD(P) derivatives is dependent on buffer type and on the pH value, generally being lower oxidation rates at higher pH. The reactivity order at pH 7.0

TABLE III

Oxygen Uptake from Nucleotides in the Presence of 20 mM PQQ a O 2 uptake (mM) Nucleotide b

No catalase

Plus catalase c

[NAD(P)1] d (mM) (No catalase)

[NAD(P) 1]/O 2 (No catalase)

0.1 M Tris–HCl (NADP) 2 (NAD) 2 NADH NADPH

72.5 34.5 28.0 27.7

(NADP) 2 (NAD) 2 NADH NADPH

16.8 12.9 20.6 15.9

50.7 24.1 20.6 19.0

133.4 61.4 26.9 25.5

1.84 1.78 0.96 0.92

0.1 M phosphate

a

12.6 9.0 13.4 11.1

30.2 23.1 19.0 14.3

1.80 1.79 0.92 0.90

The reactions were carried out for 10 min from addition of PQQ, at pH 7.0, in a Gilson Oxygraph at 25°C and at an initial [O 2] 5 250 mM. The solution volume was 3 ml. Each value is the mean of three measurements, with SD within 5% (Tris–HCl) or within 8% (phosphate). b All nucleotides had 200 mM starting concentrations. c Catalase added from start (10 U/mL, final concentration). d Enzymatically determined after 10 min of incubation on aliquots of samples.

was NADH . NADPH . (NADP) 2 . (NAD) 2 (phosphate), (NADP) 2 .. (NAD) 2 . NADH . NADPH (TrisHCl), and (NADP) 2 . NADPH . (NAD) 2 . NADH (borate). At pH 8.0 the reactivity order was NADH . NADPH . (NAD) 2 . (NADP) 2 (phosphate) and (NADP) 2 . NADPH $ (NAD) 2 . NADH (Tris–HCl). The phytate, like EDTA, significantly lowered the reaction rates in Tris–HCl more efficiently at pH 9.0 (Table IV). While the lack of a fixed order of reactivity of the four nucleotides with PQQ could be indicative of an overall similar reaction mechanism (see Discussion), the presence of chelating anions (phosphate, EDTA, and phytate) had a strong negative influence on both the reaction rate and the PQQ • radical detection. Therefore, the effect of the presence of metal cations on the reaction rates was investigated, recalling that PQQcontaining dehydrogenases showed an enhanced enzyme activity in the presence of various alkaline hearth ions, particularly Ca 21 (19 –24). Figure 4 shows the effect of Ca 21 concentration on the aerobic oxidation of NAD(P)H and (NAD(P)) 2 by PQQ in Tris–HCl, pH 7.0. The oxidation rate of all nucleotides was significantly enhanced at increasing Ca 21 concentrations, mainly above 0.1 mM, the effect being larger for NAD(P)H. We carried out also control experiments with other metal ions, namely, Mg 21 , Na 1 , and K 1 . Even when the concentration of these ion was at least 2 or 3 orders of magnitude greater than those tested for Ca 2 1 , the rateenhancing effect was minor for Na 1 and negligible for K 1 . Magnesium was effective only with the monomers, increasing the rate by about 50% in the case of NADH and about 80% in the case of NADPH at a

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PQQ in fact also showed a well-defined emission fluorescence spectrum with a maximum intensity (l exc 5 337 nm) at 482 nm in 0.1 M Tris–HCl buffer (pH 7.0) at 25°C. Figures 5 and 6 report the change in fluorescence of PQQ upon addition of increasing amounts of cations (Ca 21, Mg 21, Na 1, and K 1) added as their chloride salts in the concentration range from 0 to 100 mM. All the cations induced a more or less marked shift toward shorter wavelengths (Fig. 5). The order of the effectivity was Ca 21 .. Mg 21 . Na 1 . K 1. A quenching of the PQQ fluorescence was observed, upon addition of Ca 21 (about 20%) and K 1 (3.9%), while a slight increase occurred in the presence of Mg 21 and Na 1 (Fig. 6). The addition of stoichiometric EDTA to PQQ solution fully reversed the effect of Ca 21 on the PQQ fluorescence. DISCUSSION

FIG. 3. Changes in the time of the UV spectrum of an aerobic mixture of NADH and PQQ in DMSO–aqueous 0.1 M NH 4HCO 3. The reaction mixture contained 100 mM NADH and 60 mM PQQ in 0.1 M aqueous NH 4HCO 3: DMSO (1:4, v/v). The curves were recorded every 60 min in the sequence indicated by the arrows.

concentration of 50 mM. None of the above metals, including Ca 2 1 , stimulated the oxidation of ((NADP)) 2 , nor of NAD(P)H, when tested in the absence of PQQ. When EDTA or phytate, at least equimolecular with respect to the cation concentration, was added, the enhancing effect of Ca 21 and Mg 21 was abolished. Other metals such as Fe 31 , Mn 21 , or Cu 21 showed a very poor rate-enhancing effect, but it was always independent from the PQQ presence. Ultraviolet and Fluorescence Spectroscopy As a whole, the above results seem to indicate a direct interaction between the alkaline earth metals, notably Ca 21, and PQQ. To test this hypothesis the spectroscopic properties of PQQ in the presence of Ca 21 were studied. The UV absorption maximum of PQQ at 330 nm undergoes a small red shift (about 10 nm) in the presence of Ca 21 (rather than 30 nm as reported (19)), while its fluorescence shows a blue shift and a quenching.

The present work is part of a thorough study from our laboratory on the reactivity of the NAD(P) dimers with various electron acceptors compared to NAD(P)H coenzymes. These studies have always shown significant differences of reactivity between NAD(P) dimers and NAD(P)H monomers, mostly depending on the nature of the acceptor (i.e., a oneelectron or a two-electron acceptor) (15–17) or on its redox potential (26). NAD(P) dimers, at variance with NAD(P)H, can easily react in a one-electrontransfer-type mechanism, yielding NAD(P) • radicals, which are strongly reducing agents. Thus, they can provide useful information on the operating redox mechanism of the electron acceptor, especially if tested in comparison with the monomers NADH and NADPH. Our previous study on PQQ (18) substantially showed that the redox chemistry of this quinone may follow either one-electron or two-electron transfer pathways. We also found that the quinol form of PQQ is crucial for the reaction with oxygen. The two-step reduction of PQQ by (NAD(P) dimers to give PQQH 2 (18) required, however, further investigation concerning the reducing species involved. For that reason we investigated the reactivity of PQQ with both NAD(P) dimers and monomers. We observed that the reaction rate was dependent on the buffer used (Figs. 1 and 2 and Tables II and IV) as it was for the intensity of the PQQ • radical ESR signal. The overall similarity in oxidation rates of NAD(P) dimers and monomers with PQQ rules out significant differences in the redox mechanisms. This is well supported by the data shown in Table IV, which clearly demonstrates that the kinetics of the redox process toward PQQ is practically the same with both NAD(P) dimers and NAD(P)H.

391

EFFECT OF METAL IONS ON PYRROLOQUINOLINE QUINONE TABLE IV

Comparison of Aerobic Oxidation Rates of NAD(P) Dimers and Monomers in the Presence of 20 mM PQQ in Different Buffers as a Function of pH a Buffer type b

Borate (mM)

Phosphate (mM)

Tris–HCl 1 phytate d (mM)

Tris–HCl (mM)

Nucleotide c (pH):

7.0

8.0

9.0

6.0

7.0

8.0

9.0

7.0

8.0

9.0

7.0

9.0

(NADP) 2 (NAD) 2 NADPH NADH

78.2 41.6 73.5 28.9

75.6 25.2 67.2 17.4

11.8 7.8 10.8 7.8

90.0 38.4 54.0 49.2

31.2 24.0 37.2 44.4

7.8 19.2 31.2 43.2

6.0 18.8 25.2 40.2

103.2 46.8 40.8 43.2

53.2 33.6 34.8 25.2

49.0 28.6 6.0 20.4

56.4 42.0 33.6 31.2

6.0 9.6 4.0 8.4

a The reactions were followed spectrophotometrically at 333 nm; the reported values are means of three distinct measurements, with SD within 7%, and represent the amounts of reacted nucleotide after 10 min of incubation in the presence of PQQ. b All buffers used were 0.1 M. c All nucleotides, 200 mM starting concentration. d In the presence of 400 mM phytate.

Also, the oxygen consumption and stoichiometry, together with the aerobic formation of PQQH 2, using either NAD(P) dimers (18) or NAD(P)H (Fig. 3) as reductants, are in agreement with the above statement. Thus, we propose the following reaction mechanism for the aerobic reaction between NAD(P)H or (NAD(P)) 2 and PQQ (see Scheme II). Reaction 49 is a very rapid dimerization of NAD(P) • radicals (25). Thus, while the possible re-

action between the NAD(P) • radical and PQQ or PQQ • does not occur in the presence of air, because its reaction with oxygen is diffusion controlled, under anaerobic conditions, the NAD(P) • radical most likely reacts with itself to form back the dimer (18). Finally, also the spontaneous dismutation of superoxide (reaction 5) is well known to occur at fair rate at pH 7.0. Thus, PQQH 2 (and products therein) represents a common key intermediate, arising either

FIG. 4. Enhancing effect of Ca 21 on the oxidation of nucleotides in the presence of 20 mM PQQ. All reactions were carried out in 0.1 M Tris–HCl buffer (pH 7.0) at the same initial concentrations as those listed in Table IV. Each experimental point represents the ratio between the oxidized nucleotide in the presence of the corresponding [Ca 21] (mM) and that obtained in the absence of the cation. The values at [Ca 21] $ 1 mM, were obtained on the basis of only a 5-min incubation. V O values (5100) are the same as those in Table IV (0.1 M Tris–HCl, pH 7.0). Symbols: (NADP) 2, empty circles; (NAD) 2, full circles; NADH, triangles; and NADPH, squares.

FIG. 5. Effect of metallic ions on the PQQ maximum fluorescence wavelength. Each point represents the maximum wavelength value (nm) determined by the fluorescence spectrum of 10 mM PQQ recorded in 0.1 M Tris–HCl buffer (pH 7.0) in the presence of increasing concentrations of Ca 21 (circles), Mg 21 (squares), Na 1 (crosses), or K 1 (diamonds).

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FIG. 6. PQQ fluorescence quenching induced by metallic ions. Each point represents the fluorescence maximum intensity of a 10 mM PQQ solution in 0.1 M Tris–HCl buffer, pH 7.0, as recorded in the presence of increasing concentrations of Ca 21, Mg 21, Na 1, or K 1. Symbols as described in the legend to Fig. 5.

by the two-step one-electron transfer by NAD(P) dimers (reactions 19 and 2) (18) or from a one-step two-electron transfer by NAD(P)H (reaction 1) to PQQ. This fact can explain the small difference in reactivity of both NAD(P) dimers and monomers with PQQ, further supporting the reaction mechanism that we previously proposed for NAD(P) dimers (18). The mechanism of the oxidation of PQQH 2 by oxygen was already investigated (11, 12) in terms of a two-step one-electron transfer mechanism with no conclusive evidence. However, the rate-limiting step of the whole process cannot be, in any case, the reaction of quinol with oxygen (18). Instead, any difference in the overall reaction rate among the nucleotides must be attributed to the reduction of PQQ (reaction 1 or 19). The lower reaction rate observed in phosphate, which we attributed to its chelating properties, induced us to test the effect of two other strongly metalsequestering agents, namely, EDTA and phytate. As expected, they produced (Tables I and IV) a marked reduction in the reaction rate and a drastic drop in the PQQ • radical signal intensity. These results suggest that the presence of metallic ions may accelerate the reaction. Several recent papers dealing with the role of Ca 21 (19 –22), Mg 21, Zn 21 (23), or even Ba 21 (24) in the activity and stability of various PQQ-dependent enzymes, and in particular methanol dehydrogenases, are also in keeping with this hypothesis. Therefore, we tested the reactivity of both NAD(P) dimers and NAD(P)H with PQQ in a nonchelating buffer such as Tris–HCl in the presence of increasing

amounts of Ca 21 (Fig. 4) or in the presence of Mg 21, K 1, and Na 1. A strong effect of Ca 21 on the kinetics of the reaction between both NAD(P) dimers and monomers with PQQ was apparent. Instead, Mg 21 and Na 1 are much less effective, and K 1 practically did not have any effect. The role of Ca 21 may be explained by the results obtained by observing the effect of metal ions on the fluorescence of PQQ in Tris–HCl buffer (Figs. 5 and 6). Both fluorescence quenching and spectral changes of PQQ were observed, mainly with Ca 21. They showed, in fact, that Ca 21 binds to PQQ, inducing a 20% quenching of fluorescence and a significant blue shift of its fluorescence maximum. Thus, Ca 21 binds to PQQ in solution, as occurs in some PQQ-dependent enzymes, and this binding affects the reactivity of the quinone. Obviously, the concentration of Ca 21 to saturate the binding with PQQ is much higher than that required to bind it in an active site of an enzyme. It is likely that calcium ions bind to PQQ in a way similar to that found in quinoproteins by X-ray measurements (27) or by molecular orbital theory calculations performed on MDH active-site models (28), that is, to the pyridine ring of PQQ, through tight interactions with the ring nitrogen and the carboxylate group or even with the nearest carbonyl group. Whether Ca 21 acts in the reaction between the nucleotides and PQQ, or between PQQH 2 and oxygen, or even in the equilibration with PQQ itself to give PQQ • radical is still matter of investigation. The data in our hands allow us only to hypothesize that the observed strong rate-enhancing effect of Ca 21 is dependent on the formation of a complex with PQQ, which is effective in a rate-limiting step of the redox

SCHEME II

EFFECT OF METAL IONS ON PYRROLOQUINOLINE QUINONE

process and thus most probably in the formation of PQQH 2 . As a matter of fact, the suppression of at least one negative charge of PQQ by Ca 21 might facilitate the reaction with the negatively charged nucleotides. Further work is in progress in our laboratories to investigate these latter aspects with NMR and electrochemical techniques. ACKNOWLEDGMENTS The assistance of A. Desideri and F. Polizio (University of Tor Vergata, Rome) for ESR measurements is gratefully acknowledged.

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