Photosynthesis Research 30: 123-130, 1991. (~) 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

Interaction of Z n 2+ with the donor side of Photosystem II

Abdur Rashid, Michelle Bernier, Leroy Pazdernick & Robert Carpentier ~ Centre de recherche en photobiophysique, UniversitO du QuObec ~ Trois-RiviOres, C.P. 500, Trois-RiviOres (Quebec), Canada G9A 5H7; IAuthor to whom all correspondence should be addressed

Received 13 May 1991; accepted 19 September 1991

Key words: oxygen evolution, calcium, chloride, electron transport, photosynthetic inhibitors

Abstract

The inhibitory effect of Zn 2+ on photosynthetic electron transport was investigated in native and CaC12-treated (depleted in extrinsic polypeptides) Photosystem II (PS II) submembrane preparations. Inhibition of 2,6-dichlorophenolindophenol photoreduction by Zn 2+ was much stronger in protein- depleted preparations in comparison to the native form. It was found that Ca 2+ significantly reduced the inhibition in the native PS II preparations, as did Mn 2+ in a combination with H20 2 in the protein- depleted counterparts. No other tested monovalent or divalent cations could replace Ca 2+ or Mn 2+ in the respective experiments. Diphenylcarbazide could partially relieve (40-45%) the inhibition in both types of preparations. The above indicates the presence of an active Zn 2+ inhibitory site on the donor side of PS II. However, neither Ca 2+ nor Mn 2+ could completely prevent inhibition by high concen- trations of Zn 2+ (>1 mM). We propose that elevated levels of Zn 2+ strongly perturb the conformation of the PS II core complex and might also affect the acceptor side of the photosystem.

Abbreviations: PMSF- phenylmethanesulfonyl fluoride; MES - 2-(N-morpholino)ethane sulphonic acid; Chl - chlorophyll; PS II - Photosystem II; DCIP - 2,6-dichlorophenolindophenol; D P C - sym-diphenyl- cabazide; DCBQ - 2,5-dichlorobenzoquinone

Introduction

Zinc is an essential element for many enzymatic reactions. It is a component of superoxide dis- mutase, carboxypeptidase, carbonic anhydrase and a range of dehydrogenase enzymes (Wool- house 1983). In plant systems, Zn 2+ deficiency hampers electron transport and photophosphory- lation activities (Schrotri et al. 1981). However, in the environment, Zn 2+ can accumulate to toxic levels in soil and water. There are many different sources of such accumulation such as mine wastes, slags from smelters, run-off from galvanized surfaces, and application of Zn 2+- containing sludges (Buchauer 1973, Woolhouse 1983). Toxic levels of Zn 2+ have been reported

to inhibit plant growth and metabolism (Ag- rawala 1977) and even relatively low levels of Zn 2+ have been reported to inhibit CO 2 fixation in marine phytoplankton (Davies and Sleep 1979).

The inhibitory effect of Zn 2+ on photosyn- thetic electron transport has not yet been ex- plored in great detail and there are contradictory reports in the literature concerning its mode of action. Several studies indicated that the active site of Zn 2+ is located on the donor side of PS II (Tripathy and Mohanty 1980, Baker et al. 1982, Miller and Cox 1983, Van Assche and Clijsters 1986). On the other hand, Mohanty et al. (1989) have recently suggested from their flash-induced thermoluminescence studies that Zn 2+ directly

124

modifies the QB (secondary plastoquinone elec- tron acceptor) site, leading to the loss of PS II activity. However, the above studies have been made on the basis of experiments using isolated chloroplasts. In these preparations, the donor side of the PS II complex is located within the lumenal surface of the thylakoid vesicles and is not readily accessible to exogenously added com- pounds like artificial electron donors (Witt 1975).

To avoid the above difficulties, we have studied the inhibitory action of Zn 2+ in (i) Triton X-100-derived PSII submembrane fractions, where both the donor and acceptor sides are exposed toward the external environment (Dunahay et al. 1984), and (ii) CaCl2-treated (protein-depleted) PS II submembrane prepara- tions, where three extrinsic polypeptides (16, 23 and 33 kDa) are removed (Ono and Inoue 1983), the inorganic ions associated with oxygen evolu- tion (CI-, Ca 2+ and Mn 2+) are disorganized and the water oxidation capacity is inhibited (Ono and Inoue 1983, Rashid and Carpentier 1989). It is shown for the first time that C a 2+ c a n reverse Zn 2+ inhibition. Our data provide strong evi- dence that Z n 2+ possesses an active inhibitory site on the oxidation side of PS II. The probable competition of Zn 2+ with Ca 2+ and/or Mn 2+ binding domain(s) in the vicinity of the PS II complex is discussed.

Materials and methods

Preparation of PS H submembrane fragments

Photosystem II submembrane fractions capable of oxygen evolution were prepared from Spinacia oleracea L. as in (Berthold et al. 1981) with modifications. Leaves were homogenized in a medium containing 50mM Tricine-NaOH (pH 7.6), 0.4M sorbitol, 10 mM NaC1, 5 mM MgCI2, 1 mM PMSF and 6 mM ascorbate. The homogenate was filtered through twelve layers of cheesecloth and the filtrate was centrifuged for 5 min at 2000 x g. The pellet was suspended in the same buffer but without sorbitol and PMSF and recentrifuged under the same conditions. The resulting pellet was resuspended in a buffer containing 40 mM MES-NaOH (pH 6.5), 0.3 M

sorbitol, 10 mM NaC1, 10mM MgC12 and 4% Triton X-100 to obtain a Chl concentration of 1 mg ml ~. After a 20 min incubation in the dark, on ice and with continuous magnetic stirring, the mixture was centrifuged for 10 min at 3600 x g. The PS II submembrane fractions having a Chl a/b ratio of 1.8-2.1, were harvested from the supernatant by centrifugation for 30min at 36 000 x g. They were washed in the same buffer (without Triton) and resuspended at a Chl con- centration of 2mgml J. Chlorophyll was de- termined according to the method of Arnon (1949).

Treatments of PS H particles with CaCl2

CaCI2-treatment was carried out according to Ono and Inoue (1983). After the treatments, the particles were washed with 20 mM MES-NaOH (pH 6.5) and suspended in the same buffer.

DCIP photoreduction

Photoreduction of DCIP was measured spec- trophotometrically at 600nm with a UV/VIS- spectrophotometer (Perkin-Elmer, model 553). The reaction medium in a 3 ml cuvette (20 mM MES-NaOH (pH 6.5), 30 I-~M DCIP and 15 txg Chl) was illuminated with the maximum intensity of a 150 W quartz halogen projector lamp. The light beam was initially filtered through a 3 cm water filter and additionally by Schott RG 665 and Ealing 35-6857 cut-off filters. The phototube was protected by a red cut-off filter (Ealing 35-5396 RTB).

Oxygen evolution

Oxygen evolution was monitored with a Clark- type electrode under continuous illumination, as described previously (Carpentier et al. 1984). The reaction medium contained 20mM MES- NaOH pH 6.5, 400/zM DCBQ, PS II fractions at a Chl concentration of 15/~gml -~ and indi- cated concentrations of ZnC12.

Determination of Mn content

All the glassware was washed with 5% HNO3, and then rinsed repeatedly with double distilled

125

de-ionized water. PS II samples (40ml, 15 p~g Chlml i) were treated with ZnC12 at the specified concentrations for 5 min in the dark at 20°C. Hydroxylamine treatment (NH2OH, 3 mM) was similar except that the sample was illuminated for 15 s during the treatment. All samples were centrifuged at 28000x g for 30 min, washed in MES-NaOH 20 mM (pH 6.5) and resuspended in the same buffer. Chlorophyll assay was performed on each sample and aliquots containing 300 p~g Chl were diluted to 5 ml with double distilled de-ionized water. 10 ~1 samples of this solution were then measured at A = 279.5 nm with an atomic absorption spec- trometer (Varian Model AA6) equipped with a graphite furnace for Mn determination.

Results

Effect of Ca :+ on Zn :+ inhibition of DCIP photoreduction in PS H submembrane fractions

Electron transport from H20 to DCIP and from H20 to DCBQ was monitored at various concen- trations of ZnC12 (Fig. 1). A similar pattern of Zn 2+ inhibition was obtained whether DCIP photoreduction of 02 evolution were measured

lO0- g

6 0 -

2o- g

g 6o

0 no oddition o o_ o= 20

0 IO -4 I0 - 3 I0 "2 I0 - I

Z n C l 2 ( M )

Fig. 1. (A) Effect of ZnCI 2 on oxygen evolution in PS II submembrane fractions. Control as 100% =302 p~mol 0 2 (mg Chlh) 1 (B) Effect of ZnC12 on DCIP photoreduction (control as 100%) in PS II submembrane preparations: (@), no addition, (©) 5 mM CaCI 2 added. The control rates are given in Table 2. Other conditions are described in Materials and methods.

and DCIP measurements were used throughout this study. Z n 2+ inhibited electron transport at relatively low concentrations. In fact, about 50 percent inhibition was already obtained at 1 mM Zn 2+, while complete inhibition was only pro- duced at approximately 40mM ZnCI 2. It was also observed that Z n ( N O 3 ) 2 could be substi- tuted for ZnC12 (data not shown) thus confirming that Z n 2+ is the inhibitory agent. On the other hand, concentrations of ZnCI 2 up to about 1 mM did not inhibit the activity of DCIP photoreduc- tion at all in the presence of 5 mM CaCI 2 (Fig. 1B). Nonetheless, at concentrations of ZnC12 above 1 mM, the activity did decline even in the presence of C a C I 2 but the overall inhibitory ac- tion was much weaker with CaC12 present. A biphasic nature of Z n 2+ inhibition is demon- strated in Fig. 2 in the absence of CaC12. CaCI 2 seems to eliminate the first part of the reaction since a monophasic behavior is obtained in its presence. The effect of Z n 2+ o n the Mn content of the preparations was also studied (Table 1). Zn 2÷ produced the release of Mn in proportion to its concentration. However, 10mM CaC12 strongly prevented this effect.

The concentration dependence of CaCI 2 on the photoreduction of DCIP in either the absence or presence of ZnC12 is shown in Fig. 3. In the absence of inhibitor, the control rates of photo- reduction were unaffected by CaC12 unless its concentration was raised above 20 mM (Fig. 3). Addition of 20mM ZnC12 to the preparation caused about 95% inhibition in the absence of CaCI 2 (Fig. 1). However, this effect was gradual- ly relieved as a result of increasing concen-

A o<

Z _o I--

" ' 0.05-

0 . 0 4 - o - r a. O.03- Q.

0.02-

ZnClz ( m M )

Fig. 2. Semi-reciprocal plot of the data presented in Fig. lB. The numbers in parentheses represent the apparent binding constants of Zn 2+ in mM.

126

Table 1. Content of Mn in PS II submembrane fractions. All values are normalized to the control value with the native PSII preparations (100% corresponds to 41-+2ppb per

sample)

Additives Mn content (%)

Native CaC12-treated

Control 100 69 2 mM NH2OH 15 - 3 mM H20 2 - 4 0.1 mM ZnCI 2 93 43 1.0 mM ZnCI, 62 44 1.0 mM ZnCI, 52 44 0.0 mM ZnCI 2 + 10 mM CaCI 2 100 - 0.1 mM ZnCI~ + 10 mM CaCI 2 96 - 1.0 mM ZnC1, + 10 mM CaCI2 103 - 10 mM ZnC12 + 10 mM CaC12 73 -

trations of CaC12. The maximum reduction of inhibition (around 50%) was obtained in the range of 5-10 mM CaCI:. Beyond that level, the activity decreased progressively. This decrease was probably due to the combined effects of the ZnCI 2 and the high concentrations of CaCI 2 be-

Z 0

::~ , 0 ~

o 45

o.~ 15

O ~

no odd i l l o n

- r~ -c eJ n t~ C 0 C ~ v ~ - - .o . . . . _ . o

2 0 r a M Z n C l 2

" ~ ' f ~ I I I I I I I I I I I I I I I I I I I I f f f l l l l

10 -4 10 -3 10 -2 i 0 - I

C o C l 2 (M)

Fig. 3. DCIP photoreduction in PS II preparations as a function of CaCl~ concentration: (©), no addition; (O), 20mM ZnCI, added. Conditions are described in the text.

cause activity observed in the absence of ZnCI 2 also declined above 20 mM CaC12 (Fig. 3). These results indicate that CaCI 2 significantly curtailed the inhibitory action of Zn 2÷ in PS II.

In order to evaluate the specificity of Ca 2÷ against the effect of Zn 2+, the inhibitory action was tested in the presence of various additives (Table 2). It was observed that the inhibition in

Table 2. Effects of various additives on Zn 2÷ inhibition in PS II submembrane preparations. Salts, including ZnCl 2, were used at 10 mM. H 2 0 2 w a s used at 3 mM, and DPC at 0.5 mM. The assay medium contained 20 mM M E S - N a O H (pH 6.5), 30/~M DCIP, and 5/xg Chl m1-1. Variations in the rates of DCIP photoreduction were within 5%

Additives DCIP photoreduction Inhibition (p, molmg ~ Chlh) (%)

Without With ZnCI 2 ZnCl 2

Native PS II preparations

CaCl,-treated PS II

None 74 14 81 MgCI 2 57 6 90 MnCI 2 63 14 78 CaC12 77 55 29 NaCI 74 17 77 KC1 74 17 77

H20 2 74 6 92 MnC12 + H20 2 80 17 79 DPC 87 40 54

None 0 0 -

H20 2 31 0 100 MgCl 2 0 0 - MgCl 2 + H202 17 0 100 MnCI 2 0 0 - MnCI 2 + H202 67 40 40 CaCI 2 0 0 - CaCl 2 + H202 11 0 100 NaCI 0 0 - NaCl 2 + H202 31 0 100 KCI 0 0 - KCI + H202 31 0 100 DPC 86 34 60

native PS II preparations was not attenuated at all by divalent (Mg 2+ or Mn 2+) or monovalent (Na ÷ or K +) cations other than Ca 2+. However, CI did show a very weak effect as can be seen in the case of NaCI or KCI (Table 2). On the other hand, a nearly 50% decrease of inhibition was observed in the presence of DPC, a specific electron donor to PS II (Table 2).

Effect of Mn :+ o n Z n 2+ inhibition of DCIP photoreduction in CaCl,-treated PS II submembrane fractions

In order to evaluate the role of three extrinsic polypeptides (16, 23 and 33 kDa) and their asso- ciated ions (Ca 2+, CI- and Mn 2+) in the acces- sibility of Zn 2+ into its inhibitory site within the PS II complex, CaCIz-treated PS II preparations were used. Electron transport was measured from U202 to DCIP. H20 2 acts as an electron donor in this type of preparation (Inoue and Nishimura 1971, Pan and Izawa 1979, Inoue et al. 1987, Schr6der and .~kerlund 1986). Alterna- tively, H20 2 was supplemented with MnCI 2 which catalyses H 2 0 oxidation (Rashid and Car- pentier 1989). The control activity in the pres- ence of U 2 0 2 (3 mM) alone was about 50% of the activity obtained with U 2 0 2 plus MnC12 (10 raM), see Table 2. MnC12 alone does not act on as an electron donor in this preparation (Table 2).

Figure 4 and Table 2 show that Zn 2+ severely inhibited the rate of DCIP photoreduction if H20 2 alone acts as an electron donor. The com-

Z 0 I-- I O 0 - u Q w ac 6 0 - 0 I- 0 1- ~L

O.

g 2 0 -

0 I0 -4

~ MnCIz

-J'/' , , ~ ~ , , l l l l ~ l iO -3 i0 -z

ZnCI 2 (M)

Fig. 4. Effect of ZnCL on DCIP photoreduction (control as 100%) in CaCL-treated PSII preparations: (©), 3mM H202 + 0.5 mM MnCI 2 added; (O), 3 mM H202 added; (11), 0.5 rnM DPC added. The control activity after each addition is shown in Table 2.

127

plete inhibition took place at about 2 mM ZnCl~ (Fig. 4), a concentration 20 times lower than for native PS II (see Fig. 1). However, the inhibitory effect was dramatically reduced if MnCI 2 was added together with H202. These results clearly indicate that exogenous Mn 2+, added in the form of MnC12 (which acts the same as Mn(NO3)2), extremely restricted the accessibility of Zn 2+ to its site of inhibition within the PS II complex. As seen in Table 2, CaCI 2 could not be used to substitute for MnCI 2 because it inhibits H202 electron donation in these protein-depleted prep- arations (Rashid and Carpentier 1989). When DPC was used as electron donor, the inhibition increased with the concentration of ZnCI 2 but it was always much weaker than with H20 as donor (Fig. 4 and Table 2). In such cases, inhibi- tion in the range of concentrations of ZnCI~ studied never exceeded 60%. In these prepara- tions, H202 produced almost complete release of Mn. Zn 2+ alone caused only the partial depletion of Mn from the submembrane fractions (Table 1).

The concentration dependence of MnCL to- wards the attenuation of inhibition by Zn 2+ in CaCl2-treated PS II preparations was evaluated (Fig. 5). In Fig. 4A, it is shown that in the presence of 3mM H202, the percentage of DCIP photoreduction strongly increases with

z 0 I--

~ " 70

o--6 o .E

~oo

Z 0 ~_ 6o 113

I z 20

3 mM H202

_ [ ] t I 1 I

B

- - f f l [ I I I I I I H I I I I I I 1 [ I I I I [11111 O [ 0 "6 ] 0 - 5 10-4 10"3

MnCI 2 (M)

Fig. 5. (A) DCIP photoreduction in CaCl2-treated PS II preparations as a function of MnCI 2 concentration; ( I ) , 3 mM H202 added; (©), 3 mM H202 + 1 mM ZnCL added. (B) Gradual reduction of ZnCI~ inhibition by increasing concentrations of MnCL in the above PS II preparations: (O), 3 mM H202 + 1 mM ZnCI_, present.

128

relatively low concentrations of MnC! 2 that are sufficient to catalyse H 2 0 2 oxidation and reaches the optimal level at around 6/zM MnC12. Beyond this concentration, the activity remained stable up to 1 mM M n C I 2. In the absence of M n C I 2, the addition of 1 mM Z n C I 2 with 3 mM H 2 0 2 brought down significantly the activity of DCIP photoreduction. The rates of photoreduc- tion could be gradually increased with the addi- tion of MnCI, at concentrations above 10 5 M. At 1 mM M n C I 2 (the highest concentration used in this case) the inhibition was attenuated by about 50% (Fig. 5B).

In the CaCl,-treated PSII, H20 2 probably acts as an electron donor to re-reduce the photo- oxidized M n 2+ (Inoue et al. 1987, Inoue and Wada 1987, Schr6der and Akerlund 1986, Vel- thuys 1983). Therefore, its contribution towards the attenuation of the inhibitory effects of Z n 2+

was tested. Figure 6A shows that in the presence of 1 mM M n C I 2, the protein-depleted prepara- tion only exhibits a weak DCIP photoreduction activity. The activity increased sharply as a func- tion of increasing H,O~ concentrations and reached the optimal level at around 5 mM U 2 0 2 .

Beyond this concentration, the activity remained stable up to at least 20 mM H 2 0 2 (the highest concentration used). Addition of 1 mM ZnCI, with 1 mM MnCI, reduced the activity monitored without H 2 0 2 by about 50%. Again, under these

Z ! 7o A , . . . . 2

o~E 42

.- / ~ --~ ~5 ~-

iO0___~/f I I I e

Z 0 6 0 - - ImM MnCI z ,m. ImM ZnCl 2

2o-

z _ - f f l I I I I I l l # l I I 1111[11 I I I I l l 0 10 -¢ I0 -3 10-2

H 2 0 2 ( M )

Fig. 6. (A) DCIP photoreduction in CaCl,-treated PS II preparations as a function of H20 _, concentration; (O), 1 mM MnC12 added; (©), 1 mM MnCl2 + 1 mM ZnCI 2 added. (B) reduction of ZnCl 2 inhibition by H_,O2; (11), i mM MnCI 2 + 1 mM ZnCI 2 were present.

Z 0

~ o. 12

W •

8g, 0.08 ~E

~ 0.o4

- 5 - 2

mM MnCl 2 ÷ l m M Z n C l 2 ~ -

I I I I I

2 6 I0

I / r a M H 2 0 2

Fig. 7. Double reciprocal plots showing the non-competitive interaction of ZnCI, with H20_~; (O), 1 mM MnCI 2 added; (©), 1 mM MnCL + 1 mM ZnCL added. Data obtained from Fig. 5A.

conditions, the activity increased as a function of increasing concentrations of H202 and reached its maximal level above 5 mM H202 (Fig. 6A). In Fig. 5b, it is shown that the inhibitory action decreased from 50 to 30% when the concen- tration of H20 2 was raised up to 10 mM. The apparent attenuation of Zn 2÷ inhibition by H202 is probably due to a stimulatory effect of H20 2 on Mn 2÷ activity, rather than its own antagonis- tic effect on Zn 2* inhibition (see also Fig. 4). The above interpretation is supported by Fig. 7, where it is shown that the interaction of Zn 2÷ with H202 is clearly non-competitive. The figure shows the same Km value (-0.2 mM) for H,_O 2 in both the absence or presence of ZnC12.

Discussion

At least three different inorganic cofactors, namely CI-, C a 2+ a n d Mn 2+, are established to be actively associated with the mechanism of photosynthetic water oxidation in green plants and algae. The proper function of this mecha- nism also necessitates the presence of three ex- trinsic polypeptides of 33, 23 and 16 kDa, respec- tively (Homann 1988, Nakatani 1984, for a re- view see Homann 1987)• Furthermore, Ca 2+ is supposed to be ligated to the residual portion of the D1 and/or D2 polypeptides at the water oxidation side of PS II (see a recent model in Dismukes 1988)• Thus, in the present investiga- tion, the significant prevention of Zn 2+ inhibition by Ca 2+ in the native form of PS II preparations

129

(Figs. 1, 3 and Table 2) clearly demonstrates that Zn 2+ possesses an active inhibitory site on the oxidizing side of PS II. This idea is further sup- ported by the release of Mn from the prepara- tions in the presence of Zn 2+ and by the higher accessibility of zinc to its active site(s) within the PS II complex found when the three extrinsic polypeptides are removed (Fig. 4 and Table 1).

In the polypeptide-depleted preparations, M n 2+ with H 2 0 2 also relieved the inhibition (Figs. 5 and 6). These data can be explained as follows. In CaCI2-treated PS II, two of the four Mn atoms are labile. Though the addition of H 2 0 2 renders the remaining Mn labile (Table 1 and Ghanotakis et al. 1984), residual Mn was present in the CaC12-treated PS II preparations. Thus, the observed rates of H202-supported DCIP photoreduction was probably a measure of Mn2+-supported DCIP photoreduction with U202 acting to re-reduce the photooxidized M n 2+ as proposed by Velthuys (1983) and San- dusky and Yocum (1988). Exogenously added M n 2+ enhanced the ability of H 2 0 2 to donate electrons to PS II (Rashid and Carpentier 1989). It is possible that the Mn released from the oxygen evolving complex by H 2 0 2 could also assume this function. It is reasonable to assume

2 + that in CaCl2-treated PS II, Zn , in combination with H 2 0 2 , might release all the residual Mn atoms from the oxygen evolving complex thus causing a severe inhibition of PS II activity. This is sustained by earlier reports (Miller and Cox 1983, Van Assche and Clijsters 1986), indicating a deleterious action of Z n 2+ o n the Mn-complex. In the protein-depleted preparations, it is ob- served that Z n 2+ affected PS II to a much lower extent when U 2 0 2 served as electron donor in the presence of exogenously added M n 2+ (Fig. 3 and Table 2). Addition of M n 2+ might reconsti- tute the native Mn concentration in the PS II core complex thus restricting the inhibitory ac-

~ + tion of Zn - . On the other hand, the binding behavior of exogenous M n 2+ is different between native and protein-depleted PS II preparations. Hence, we do not observe the protecting effect of exogenous M n 2+ a g a i n s t Z n 2+ inhibition in the native PS II preparations (Table 2).

Lastly, one of the most important observations was that neither C a 2+ n o r M n 2+ could efficiently protect against the inhibitory effects caused by

relatively high concentrations of Z n 2+ (Figs. 1 and 4). It is also observed that DPC could relieve Z n 2+ inhibition only partially (Table 2 and Fig. 4). Two possible interpretations can be put forward: i) there may exist a second Z n 2+ inhibitory site

in PS II beyond the DPC electron donation site, and/or

ii) at high concentration, Z n 2. treatment might impair QB-binding through a transmembran- ous conformational change of the D1 poly- peptide following its action on the donor side of PS II.

The above is in line with a recent report indicat- ing the interference of Zn 2+ with the Q~-site (Mohanty et al. 1989) and may be related to the biphasic nature of Z n 2+ inhibition demonstrated in Fig. 2.

Acknowledgments

Supported in part by a grant to R.C. (no. OGP000229) from the Natural Sciences and Engineering Research Council of Canada (NSERC). Authors would like to thank Mr Syl- vain Lepage for his technical assistance. M.B. was a recipient of a postgraduate fellowship from NSERC.

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