to photosystem I. Apart from the stoichiometric amount ligated to plastocyanine, endogenous copper was
also found in thylakoid membrane preparations and in isolated photosystem II submembrane fractions
[3,4] but it was shown to be associated with proteins or nuclear contaminants that can be removed together
with starch from Triton X-100 preparations using centrifugation at 10,000 g[5].
Addition of exogenous copper at concentrations greater than 1 M can cause a toxic response in most
photosynthetic organisms. Hence, it has been used extensively as an algaecide and herbicide. Prolonged
exposure of whole plants to relatively high copper concentrations can lead to complete disintegration of
the chloroplast lamellar system [6], whereas low concentrations have relatively minor effects on the pho-
tosynthetic apparatus [7]. In isolated chloroplasts or thylakoid membranes, copper is a relatively strong
inhibitor of photosynthesis as a micromolar range of its chloride or sulfate salt was shown to inhibit elec-
tron transport from water to artificial electron acceptors of photosystem II or photosystem I.
Copper was found to inhibit both photosystem I and II. A direct interaction with ferredoxin was in-
ferred to cause the inhibition on the acceptor side of photosystem I [8]. However, its action is much
stronger on photosystem II [9,10]. Energy storage measured by photoacoustic spectroscopy and P700
turnover measured under red light from absorbance changes at 820 nm in intact leaves were less affected
by copper than oxygen evolution [11–13]. This observation was attributed to the remaining energy stor-
age activity during cyclic electron transport in photosystem I, which is less inhibited than photosystem II.
The several proposed sites of copper inhibition in photosystem II are reviewed by Barón et al. [14].
It was readily found that variable chlorophyll fluorescence declines in the presence of copper. Fluores-
cence parameters such as Fv/Fm, representing the photochemical quantum yield of photosystem II, and
qP, which denotes the portion of absorbed energy that is trapped by open photosystem II centers, were
shown to decline in the presence of copper [15–19]. Further, diphenylcarbazide (DPC), an artificial elec-
tron donor to photosystem II, could not restore the inhibition in the green algae Ankistrodesmus falcatus,
indicating that the inhibitory site was located at the donor side of the photosystem near the DPC electron
donation site [20]. Similar conclusions were deduced from fluorescence measurements in microalgae
[21]. However other lines of evidence suggested an inhibition on the acceptor side beyond QB, the sec-
ondary quinone acceptor of photosystem II, and the inhibitory site of diuron in the QBpocket [22]. In fur-
ther studies by Hsu and Lee [23] using fluorescence induction experiments in pea thylakoids, the reduced
variable chlorophyll fluorescence in the presence of copper was interpreted as an inhibitory action at the
reaction center level affecting primary charge separation. This interpretation was challenged by Mohanty
et al. [24], whose thermoluminescence and delayed luminescence studies supported the idea of an in-
hibitory action directly at the QBsite or at the nonheme iron located between QA, the primary quinone ac-
ceptor of photosystem II, and QB. This site was also supported by thermodynamic and kinetic studies of
the electron transfer between QAand QBthat indicated a reduced affinity for atrazine binding in the QB
pocket [25]. Yet other studies involving variable fluorescence measurements also indicated a heteroge-
neous inhibition of photosystem II populations inhibiting the primary photochemistry in the QBreducing
photosystem II centers but not in the non-QBreducing centers [26–29].
Precise kinetic studies of electron transfer from QAto QB, from the manganese-containing water ox-
idizing complex to the redox-active TyrZOX, and from TyrZOXto P680, together with an analysis of the ex-
tent of charge separation between P680and QAusing flash-induced absorption and fluorescence
changes [25,30], provided evidence that copper does not affect charge separation but rather modifies
TyrZ. In copper-binding proteins, copper is coordinated with the imidazole nitrogen atoms of histidines.
It has been proposed that copper could bind His190 of the reaction center protein D1 [31]. This histidine
is located near TyrZand has also been proposed to take part in proton transfer from TyrZduring oxygen
evolution. However, thermoluminescence measurements have shown that recombination of the [HisQA]
couple was not affected by copper and only TyrZseems to be inactivated [32].
The preceding discussion indicates inhibition on both donor and acceptor sides of photosystem II.
Confirmation of this proposal came from electron paramagnetic resonance (EPR) experiments showing
that EPR signal II that reflects oxidation of TyrZcould not be induced in photosystem II preparations
treated with copper and further the EPR signal from the [QA-Fe^2 ] couple was also lost, indicating that
the nonheme iron located between QAand QBmay be displaced by copper [31]. It can be proposed that
Cu^2 interferes with the histidine ligands to which the nonheme iron is bound. To this effect, it is inter-
esting to note that added Fe^2 can partly prevent copper inhibition in photosystem II [22]. Alternatively,
Yruela et al. [33] proposed from picosecond time-resolved fluorescence experiments measuring charge
separation in photosystem II preparations that copper may be involved in a close attractive interaction
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