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Copper, iron, and zinc interactions with chlorophyll in extracts of

photosynthetic pigments studied by VIS spectroscopy

Article  in  Russian Journal of Physical Chemistry · September 2009

DOI: 10.1134/S0036024409090222

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ЖУРНАЛ ФИЗИЧЕСКОЙ ХИМИИ, 2009, том 83, № 9, с. 1718–1722

STRUCTURE OF MATTER
AND QUANTUM CHEMISTRY
УДК 543.42:541.64

COPPER, IRON AND ZINC INTERACTIONS WITH CHLOROPHYLL

IN EXTRACTS OF PHOTOSYNTHETIC PIGMENTS STUDIED
BY VIS SPECTROSCOPY
© 2009 J. Zvezdanovi c ***
 and D. Markovi c *
*University of Niš, Faculty of Technology, 16000 Leskovac
**Scholar of Ministry of Science and Technological Development of the Republic of Serbia, Belgrade
Email: jelite74@yahoo.com, dejan_markovic57@yahoo.com.

Abstract – Interactions of copper, iron and zinc with chlorophyll, the major photosynthesis pigment, were
studied by VIS spectrophotometry in extracts of photosynthetic pigments (in vitro). Copper predominantly
forms Cu–Chl complexes in all studied systems (Cu
, Cu/Fe
, Cu/Zn
, and Cu/Fe/Zn
incubated pigment
solutions). It is not clear whether iron forms Fe–Chl complexes or produces pheophytin. It’s effect is domi

nant over zinc (Fe/Zn
system) but highly minor compared to Cu
effect when all three metals are present
(Cu/Fe/Zn–Chl system) in the same concentrations.

INTRODUCTION formation of Chl–HMS, even in minor proportions

relative to the total Chl content, may inhibit photo

Chlorophyll (Chl), a major photosynthesis pig
synthesis completely [8]. The detailed consequences
ment, in chemical terms is a porphyrin derivative (a of Chl–HMS for higher plants and green algae have
cyclic tetrapyrrole with an isocyclic cyclopentanone been discussed by Kupper [8, 13, 14]. The general re

ring fused to a C
pyrrole ring between the C13 and actions of Chl with heavy metals in vitro [15] and in vi

C15 positions), where the central magnesium (Mg) at
vo [16, 17] have been already reported.
om coordinates four symmetric pyrrole rings (Fig.1).
Heavy metals can replace the labile bonded central Chl–HMS complexes show different spectroscop

Mg
atom of chlorophyll to form substitutional or ic behavior from Chl itself [1, 2, 8], qualitatively (shifts
“central” Chl
HMS complex (heavy metal complex), of the characteristic bands maximums, AQymax), as well
or they can play a coordinating role between two O
at
as quantitavely (different intensities of the corre

oms (C131 and C133, Fig. 1) at periphery of Chl
mol
sponding bands). This work deals with interaction and
ecule to form a cyclic 6
membered peripheral “che
possible formation of Chl–HMS complexes between
late” complex [1, 2].
Plants easily absorb many toxic heavy metals. Once
absorbed, they penetrate the plant tissues (including 32
31
the leaves) and, in higher concentrations, they may in
*
hibit photosynthesis [3–6]. In lower concentrations 2
3
5 *7
copper, iron and zinc themselves are essential micron
A 4 6
1 21 B 8
utrients for higher plants and algae, and “constitu
N 22
N 9 *
20
ents” of photosynthetic apparatus – photosystems I Mg 10
and II (Cu
protein – plastocyanin, Fe
containing 24
electron
carriers in cytochrom complexes) [7]. Zinc
H * 1819D N 23
N 11
17 16
porphyrins are formed during chlorophyll biosynthe
15 14C 12
* E
13
sis, but they are also found in Chl
degradation prod
171 2
ucts [8]. However, high external metal
concentrations H * 13 131
172 H
(copper, iron, zinc) – like the ones used in this work – O
may produce a lot of damaging effects. Zinc may be in
O C COOCH3
cluded in degradation of chloroplasts stromal proteins 173 133 134
O P2 3 P16
[9]. Copper itself may affect all kind of photosynthetic P 1
P P7 P11 P15
activities, like electron
transport and ATP production
[10, 11], or oxygen evolution [12]. In vivo experiments P17 CH3 H CH3 H P20
showed that the substitution of central magnesium at
P 18
P 19
om in chlorophyll by heavy metals (observed in vitro)
also takes place in living plants leading to permanent Fig. 1. Structure of chlorophyll a, with numerated C
posi

damaging effects on photosynthesis function [8]. The tions.

1718
 COPPER, IRON AND ZINC INTERACTIONS WITH CHLOROPHYLL
D
0.2
0.1
600 650
550 600
Fig. 2. Absorbance spectra of Cu/Fe
and Cu (left inserted spectra)
incubated pigment solutions for different time periods (tinc),
following beginning of the incubation (tinc = 0) in 550–700 nm spectral range (Qy
band).
chlorophyll and three chosen heavy metals in extracts of
photosynthetic pigments (i.e. pigment solutions) incu

bated with different combinations of the three metals

copper (Cu) & iron (Fe), Cu & zinc (Zn), Fe & Zn and,
Cu & Fe & Zn (in vitro). The interactions were then
examined by VIS spectroscopy.
EXPERIMENTAL
Photosynthetic pigments were extracted from spin

ach leaves (Spinacia oleracea L) by using Swec meth

od [18]. Chlorophylls content has been determined by
using spectrophotometric equations for chlorophylls in
acetone as reported [19]. The pigment extracts (con

taining two major Chl
forms, Chla and Chlb, and the
accessory pigments, carotenoids) were evaporated and
diluted in ethanol [8, 20]. Then, aqueous solutions of
CuSO4, ZnSO4, FeSO4 (combinations of metals in ratio
1:1) were added. The Chl concentration in the reaction
mixtures (cChl) has been adjusted to 8.0 µmol/dm3 and
the ratio of total metal ion concentration to chloro

phyll concentration ( c tot.,M2+ /cChl) was in the range of
1000:1. The time periods following the beginning of
the Chl incubation (tinc) ranged from 1 to 5 days. The
interaction was stopped by dissolving the reaction
mixture in cyclohexane and the visible spectra were re

corded after different incubation times tinc (on Varian
Cary
100 Spectrophotometer). The same experiments
were done simultaneously with individual metals to
observe the combination effects (in the presence of all
three metals) on chlorophylls. All experiments were
ЖУРНАЛ ФИЗИЧЕСКОЙ ХИМИИ том 83
1719
tinc = 0
1 day
3 days
4 days
5 days
700
650 700
λ, nm
performed in dark conditions to prevent chlorophyll
photodegradation.
RESULTS AND DISCUSSION
Chlorophyll has two major absorption regions in
visible range (400–800 nm), producing “red” (Qy)
band and “blue” (Soret or B) band [21]. Although the
changes were detected in incubated systems both with
B and Qy
bands, the interaction of heavy metals with
chlorophyll was followed in the pigments mixtures (in
vitro) by using Qy
band as sensible indicator, since it
belongs to chlorophyll only, and not to accessory pig

ments, carotenoids [22]. Before any metal ions addition
to pigment solutions, a control experiment has been
done in order to assign all possible absorption changes
to any possible other factors except the Me–Chl inter

action. The control was done in a period of 5 days with
pigments solution only, by controlling it’s absorption
spectra in the indicated period. A detectable changes
have not been detected (not shown), meaning that
changes in VIS spectra of chlorophyll in vitro in an in

cubation period of 5 days (with Cu, Zn, Fe and their
combinations) – and shown in Figs. 2–5 – should pri

marily be assigned to the Me–Chl interactions.
A hypochromic effect related to absorption maxi

mum of Chl Qy
band (AQymax) is observed in all cases,
proportional to the length of the incubation period
(Figs. 2–5). The changes in VIS spectra of Cu/Fe
,
Cu/Zn
, Fe/Zn
, and Cu/Fe/Zn
incubated pigment
solutions (with the same metals concentrations) were
clearly observed in the period of 5 days (Figs. 2–5).
№9 2009
1720 ZVEZDANOVI C , MARKOVI C

tinc = 0
1 day
3 days
0.15
4 days
5 days

0.10

600 700
650
0.05

550 600 650 700

λ, nm

Fig. 3. Absorbance spectra of Cu/Zn
compared to Cu (left inserted plots)
incubated pigment solutions for different tinc, follow

ing beginning of the incubation in 550–700 nm spectral range (Qy
band).

D tinc = 0
1 day
0.15 3 days
4 days
5 days

0.10

600 650 700

0.05

550 600 650 700

λ, nm

Fig. 4. Absorbance spectra of Fe/Zn
and Fe (left inserted plots)
incubated pigment solutions, for different tinc, following begin

ning of the incubation in 550–700 nm spectral range.

Clear hypsochromic shifts have been observed for Qy

band of Cu/Fe
, Cu/Zn
, and Cu/Fe/Zn
incubated
pigment solutions (Figs. 2, 3, 5), while batochromic
shift was detected for Fe/Zn
incubated pigment solu

tions only (Fig. 4) relative to tinc. Table shows the shifts
values for the Qy
band (∆λQymax) in VIS spectra of
heavy metals
incubated pigment solutions after 5 days
of incubation.
Comparison of Cu
effect only [23] with those ob

served in Cu/Fe
, Cu/Zn
, and Cu/Fe/Zn
incubated

ЖУРНАЛ ФИЗИЧЕСКОЙ ХИМИИ том 83 №9 2009

 COPPER, IRON AND ZINC INTERACTIONS WITH CHLOROPHYLL 1721

D tinc = 0
1 day
0.18 3 days
4 days
5 days
0.14

0.10
650 700

0.06

0.02
550 600 650 700
λ, nm

Fig. 5. Absorbance spectra of Cu/Fe/Zn
and Fe/Zn (left inserted picture)
incubated pigment solutions for different tinc, follow

ing beginning of the incubation in 550–700 nm spectral range.

pigment solutions led to two major conclusions, (a) in
all cases the interaction was finished after 1 day of in

cubation, and (b) in the same time a strong hypsoch

romic effect of Qy
band is observed in the correspond

ing VIS spectra (Figs. 2, 3, 5, table). The hypsochro

mic effect for “central” Cu–Chl complex (related to
Qy band) was already confirmed in cyclohexane (AQymax
at 649 and 654 nm for Cu–Chl and Cu
meth

ylpheophorbide, respectively) [1, 8]. In our earlier ex

periments with Cu
incubated chlorophylls in chloro

phyll fractions (a separated fraction of the pigment
extract, containing mainly Chla with small contribu

tion of Chlb) the same type of changes was detected,
and the formation of “central” Cu–Chl complex was
clearly confirmed in vitro by using additional experi

ments with FTIR and fluorescence spectrophotome

try [20, 23]. Our results strongly confirm that Cu–Chl
complex formation is not disturbed when one or the
two metals are present; the effect of Cu only is shown
in the form of the inserted plots (the left plots at Figs.
2, 3, concerning the Cu/Fe and Cu/Zn systems). The
spectral behavior shown at Fig. 5 is obviously very sim

ilar to the one produced by copper only: the inserted
plot related to Fe/Zn system shows the opposite shift
direction.
With the Fe/Zn
incubated pigment solutions the
observed interaction finished in 1 day only after incu

bation (Fig. 4). The same, batochromic effect, due to
a supposed Fe–Chl interaction, has been seen for the
Fe
incubated pigment solution only (Fig. 4 – the left
inserted plot). However, this batochromic or “red”
shifted absorption (in a range of few nanometers, like
these presented in this work) was already seen in pheo

phytins (Phe), a central Mg
depleted derivatives of
chlorophyll [24]. The possibility of pheophytins for

mation can not be neglected in this case.
In Zn
incubated pigment solutions 5 days after in

cubation, just a very small hypsochromic effect of Qy

band in VIS spectra was observed (of 1 nm, table).
Zinc interaction with chlorophyll in similar experi

mental conditions were examined earlier, suggesting
that Zn rather forms “chelate” complexes with chlo

rophyll (proved by FTIR spectra, [23]), than “central”
complexes (hardly detectable changes of Qy
band Amax
in VIS spectra), providing a shifted equilibrium be

tween “central” and “chelate” Zn–Chl complexes to

ward the latter ones [20, 23, 25].
Finally, when all three metals are present in the
same concentrations the overall effect is clearly very
similar to Cu
effect itself (Fig. 5). Metal interaction
with chlorophyll in solution can be explained by theo

retical analysis of Falk’s “stability factor” (includes
The observed shifts of Qyband absorbance maximums (∆λ)
in VIS spectra of heavy metal incubated chlorophylls in pig

ment solutions
M–Chl ∆λ, nm M–Chl ∆λ, nm
Cu–Chl –10.5 Cu/Fe–Chl –10.5
Fe–Chl +6 Cu/Zn–Chl –10.5
Zn–Chl –1 Fe/Zn–Chl +6
Cu/Fe/Zn–Chl –10.5

ЖУРНАЛ ФИЗИЧЕСКОЙ ХИМИИ том 83 №9 2009

1722 ZVEZDANOVI C , MARKOVI C
charge number of the metal ion, effective radius of the
metal ion in Å and Pauling’s electronegativity) [26].
According to Falk’s equation, the stability of “central”
Chl–HMS complexes is ordered as: Mg–Chl ~ Zn

Chl < Fe–Chl < Cu–Chl. Similar order for tendency
of metal ions to be bound in the centre of the chloro

phyll molecule was found [8]. Studies of interactions
of metal combinations (Cu/Zn, Cu/Fe, Fe/Zn,
Cu/Fe/Zn) used in this work in the same concentra

tions with chlorophyll in pigment solutions confirm
the established order.
CONCLUSIONS
From all three chosen heavy metals only copper
shows direct and dominant interaction with chloro

phyll (in vitro), independently of the other metals
presence. The Fe
effect dominates in combination
with zinc, though it is not clear whether Fe–Chl com

plex, or pheophytin is formed. Zinc has the smallest
affinity to build “central” Zn–Chl complex, and the
effect is completely negligible in the presence of either
iron or copper, or in combination with both of them.
ACKNOWLEDGMENTS
This work was supported under the project number
TR
19048, by the Ministry of Science and Technolog

ical Development of the Republic of Serbia.
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том 83 №9 2009