Marina A. Kozuleva and Boris N. Ivanov*
Photosynthetic Electron Transport lab., Institute of Basic Biological Problems, Pushchino, 142290, Russia
*Corrresponding author: E-mail, [email protected]; Fax, +7-496-7330532.
(Received November 29, 2015; Accepted February 10, 2016)
The review is dedicated to ascertainment of the roles of the
electron transfer cofactors of the pigment–protein complex
of PSI, ferredoxin (Fd) and ferredoxin-NADP reductase in
oxygen reduction in the photosynthetic electron transport
chain (PETC) in the light. The data regarding oxygen reduction in other segments of the PETC are briefly analyzed, and
it is concluded that their participation in the overall process
in the PETC under unstressful conditions should be insignificant. Data concerning the contribution of Fd to the oxygen
reduction in the PETC are examined. A set of collateral evidence as well as results of direct measurements of the involvement of Fd in this process in the presence of isolated
thylakoids led to the inference that this contribution in vivo
is negligible. The increase in oxygen reduction rate in the
isolated thylakoids in the presence of either Fd or Fd plus
NADP+ under increasing light intensity was attributed to
the increase in oxygen reduction executed by the membrane-bound oxygen reductants. Data are presented which
imply that a main reductant of the O2 molecule in the terminal reducing segment of the PETC is the electron transfer
cofactor of PSI, phylloquinone. The physiological significance
of characteristic properties of oxygen reductants in this segment of the PETC is discussed.
Keywords: Chloroplast Electron transport Ferredoxin Oxygen reduction Photosystem I Phylloquinone.
Abbreviations: A0, primary electron acceptor in PSI; A1, secondary electron acceptor in PSI, phylloquinone; DMF,
dimethylformamide; Em, midpoint redox potential; FA and
FB, terminal electron acceptors in PSI, FeS clusters; Fd, ferredoxin; FNR, ferredoxin-NADP+ reductase; FX, secondary electron acceptor in PSI, FeS cluster; PETC, photosynthetic
electron transport chain; PQ, plastoquinone; PQ
and
PQH , plastosemiquinone; PQH2, plastohydroquinone, plastoquinol; PhQ, phylloquinone; PhQ , phyllosemiquinone;
QA and QB, secondary electron acceptors in PSII; ROS, reactive oxygen species; SOD, superoxide dismutase.
Introduction
The elaboration of the problem of oxygen reduction in chloroplasts began in 1951 when Alan Mehler observed the appearance
of hydrogen peroxide (H2O2) under illumination of isolated
chloroplast fragments (Mehler 1951). The term ‘Mehler reaction’
is now used to designate the process of direct oxygen reduction
by components of the photosynthetic electron transport chain
(PETC). Kozi Asada was one of the first scientists who showed
that the superoxide anion radical, O2 , is the primary product
of O2 reduction in the PETC (Asada and Kiso 1973, Asada et al.
1974). In Asada’s studies, the characteristics of the Mehler reaction such as Km(O2) and the second-order rate constant (Asada
and Nakano 1978, Takahashi and Asada 1982) were first presented, convincing evidence for involvement of PSI cofactors in
the process of oxygen reduction (Takahashi and Asada 1988) was
obtained and the role of superoxide dismutase (SOD) located
near the acceptor side of PSI in a fast conversion of O2 produced there into H2O2 (Ogawa et al. 1995) was stressed. The
superoxide anion radical and H2O2 are reactive oxygen species
(ROS) capable of negatively affecting the photosynthetic reactions in chloroplasts, and therefore the concentrations of these
species are under control there. The above studies and the discovery of thylakoid-bound ascorbate peroxidase in chloroplasts
(Miyake and Asada 1992) led to the concept of the ‘water–water
cycle’ (Asada 1999, Asada 2000), the term originating from the
comprehension that electrons for both oxygen reduction and
scavenging of ROS in chloroplasts are derived from water, and
the final product of the reactions is water. The physiological
functions of the water–water cycle besides ROS scavenging
that provides protection from photooxidative damage (Rizhsky
et al. 2003) are defense against photoinhibition (Ort and Baker
2002) and participation in ATP production (formerly termed
pseudocyclic phosphorylation), additional to that coupled with
electron transport to NADP+ (Makino et al. 2002).
Data on the magnitude of the Mehler reaction and its contribution to total linear electron transport are presented in
numerous research papers, which are analyzed in several reviews (Badger 1985, Robinson 1988, Badger et al. 2000, Ivanov
et al. 2012, Ivanov et al. 2014), to which we refer readers. Briefly,
the Mehler reaction can account for from 5% to 50–70% of
total electron transport in different plant species under various
conditions. It may be concluded that the electron outflow from
the PETC components to oxygen is conditioned by the structure of the PETC as well as by environmental conditions, and
the genotype defines both this structure and its variability
under short and prolonged changes of environmental
Plant Cell Physiol. 57(7): 1397–1404 (2016) doi:10.1093/pcp/pcw035, Advance Access publication on 24 March 2016,
available online at www.pcp.oxfordjournals.org
! The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Special Focus Issue – Mini Review
The Mechanisms of Oxygen Reduction in the Terminal
Reducing Segment of the Chloroplast Photosynthetic Electron
Transport Chain
M. A. Kozuleva and B. N. Ivanov | Mechanisms of oxygen reduction in chloroplasts
conditions such as illumination, temperature, gas composition
of the atmosphere, mineral nutrients, the presence of pollutants, and so forth (Ivanov et al. 2014).
The Mehler reaction is the limiting crucial point of the
water–water cycle (Asada 2000); however, its mechanism, i.e.
from which components of the PETC the electrons are principally transferred to O2 molecules, is still under debate. In this
review, we will consider in particular the contribution of various
components of the terminal reducing segment of the PETC to
O2 reduction.
Conditions for O2 Reduction in Different
Media, and Possible Sites of This Reaction in
the PETC
The first step of oxygen reduction in the PETC is the transfer of
one electron to the O2 molecule, resulting in the generation of
the superoxide anion radical, O2 . The midpoint redox potential (Em) of the O2/O2 pair in aqueous media is 160 mV
at the normal partial pressure of O2 in ambient air (Asada and
Takahashi 1987) while, in the lipid bilayer of the membrane, the
Em (O2/O2 ) is close to that in dimethylformamide (DMF;
550 to 600 mV vs. normal hydrogen electrode) (Afanas’ev
1989). The thermodynamic basis is relevant for consideration of
sites of reduction of O2 molecules in the PETC because its
components are situated in the aqueous media of the lumen
and stroma, at membrane boundaries and within the interior of
hydrophobic membranes. The components of the PETC, which
may be considered as O2 reductants, should possess negative
redox potentials thermodynamically sufficient to reduce O2
molecules in the corresponding medium. The possibility of
O2
generation by intramembranous components of the
PETC was first proposed in the work of Takahashi and Asada
(1988), based on the data which showed the delayed appearance of O2 outside thylakoid membranes in response to a
10 ms flash. Recently, using reagents of different lipophilicity,
cyclic hydroxylamines producing EPR (electron parmagnetic
resonance)-detectable nitroxide radicals after oxidation by
O2 , the generation of O2 outside as well as within thylakoid membranes has been directly shown (Kozuleva et al. 2011,
Kozuleva et al. 2015). This implied an involvement of several
PETC components in the Mehler reaction.
Several segments of the PETC are considered as sites of O2
reduction. The data implying the possibility of this process at
the acceptor side of PSII were comprehensively reviewed by
Pospı́sil (2012). The redox potential of pheophytin, 610 mV,
shows that this PSII cofactor could be a reductant of O2 in
hydrophobic environments; however, the fast transfer of electrons from reduced pheophytin (200–500 ps) can prevent such
a reaction. The redox potential of QA/QA was recently found
to be 162 mV (Shibamoto et al. 2010), and this is the most
negative value for QA presented in the literature. The Em of QB/
QB was stated to be 45 mV (Crofts et al. 1984). Both these
values are not sufficiently negative for plastosemiquinone
(PQ ) at these sites to reduce O2 efficiently. Yadav et al.
(2014) suggested a role in O2 reduction of PQ , which
1398
might be formed in the recently revealed plastoquinone
(PQ)-binding QC site in PSII (Kruk and Strzalka 2001,
Hasegawa and Noguchi 2014) after one-electron oxidation of
plastoquinol (PQH2) by Cyt b559. It should be stressed, however,
that the experimental data demonstrating oxygen reduction in
PSII were obtained with PSII-enriched fragments of thylakoid
membranes with disturbed native membrane structure and,
possibly, with increased water content. In such fragments, the
presence of the photochemical generator producing the
reduced forms of cofactors with negative redox potentials
would inevitably lead to reduction of O2 as the sole available
acceptor. So, we must agree with Asada’s statement (Asada
1994) that oxygen reduction at the acceptor side of PSII in
intact thylakoids barely occurs.
Oxygen reduction in the PQ pool of undamaged thylakoids,
in which the oxidation of PQH2 by the Cyt b6f complex was
inhibited by the dinitrophenylether of 2-iodo-4-nitrothymol
(DNP-INT), was shown (Khorobrykh and Ivanov 2002). An analysis of data obtained in that and subsequent studies
(Khorobrykh et al. 2004, Mubarakshina et al. 2006) implied
that this reduction is executed by free PQ of the pool (Em
= 170 mV) at the membrane–water interface (Mubarakshina
and Ivanov 2010). The concentration of free PQ in the membrane as determined by the comproportionation reaction of PQ
with PQH2 is low; however, a very high second-order rate constant of the reaction PQ with O2 (108 M 1 s 1) ensured
that the calculated oxygen reduction rates in the PQ pool were
commensurable with those observed in isolated thylakoids in
the absence of the inhibitor (Khorobrykh and Ivanov 2002).
It was shown that semiquinone produced via oxidation of
quinol at the donor side of the Cyt bc1 complex can be oxidized
by O2, generating O2 (Muller et al. 2002). Baniulis et al. (2013)
found that the rate of O2 production in the purified Cyt b6f
complex was significantly higher than that in the isolated yeast
bc1 complex, and they presumed that there was a higher residence time of PQ /PQH2 in the niche near the Rieske protein
iron–sulfur cluster in the former. The rate of O2 generation
by PQ at that niche would depend on the competition between electron transfer to oxygen and the oxidized low-potential heme of Cyt b6; the latter process is highly favored under
conditions of normal PETC operation in vivo. Forquer et al.
(2006) proposed that semiquinone would leave the donor
niche of the b6f complex in order to react with oxygen. In
this case, however, this PQ
is incorporated into the PQ
pool and can dismutate with another PQ . It is difficult to
estimate to what extent oxygen reduction by free PQ of this
pool contributes to the total oxygen reduction in vivo during
operation of the intact PETC. It could be suggested that stressful conditions can promote the above-mentioned exit of PQ
produced at the donor side of the Cyt b6/f complex into the PQ
pool, increasing oxygen reduction in this PETC segment.
Convincing evidence in favor of PSI as the major site of oxygen
reduction in chloroplasts was already obtained at the beginning
of elaboration of this problem. In mutants with either a deficiency or absence of PSI, there was negligible electron leakage
from the PETC to oxygen (Fork and Heber 1968, Radmer and
Ollinger 1980). Discussion of other similar studies conducted
Plant Cell Physiol. 57(7): 1397–1404 (2016) doi:10.1093/pcp/pcw035
with undamaged leaves may be found in the reviews by Badger
(1985) and Robinson (1988). The common view now is that
oxygen reduction in the PETC occurs mainly, if not exclusively,
at its terminal reducing segment comprising the PSI complex,
ferredoxin (Fd) and ferredoxin-NADP-reductase (FNR).
Is it Possible That FNR is Involved in the
Reduction of Oxygen In Vivo?
In chloroplasts, FNR mediates electron flow from reduced Fd to
NADP+. The reduction of NADP+ requires two electrons,
while Fd is a one-electron carrier. Thus, FNR provides sequential
binding of two molecules of reduced Fd, which donate electrons
to the FNR prosthetic group, FAD. In the semiquinone form,
FAD in FNR is a good reductant of O2 molecules, with the
second-order rate constant of the reaction close to 108 M–1s–1
(Massey 1994).
The question of the involvement of FNR in oxygen reduction
in chloroplasts in vivo has been repeatedly raised in the literature (Robinson 1988). Isolated FNR added to a thylakoid suspension in either the presence or the absence of Fd increased
the rate of oxygen uptake in the light (Goetze and Carpentier
1994). Both the photochemical quenching of Chl fluorescence
and the rate of O2 generation were increased as a result of
FNR addition to a thylakoid suspension in the absence of Fd
(Miyake et al. 1998). In that work, the same effects were
observed in response to addition of isolated monodehydroascorbate reductase (MDAR), also an FAD-containing enzyme.
The authors assumed that MDAR can play a significant role
in oxygen reduction in vivo. The experimental technique in the
above studies implies that FB, the terminal cofactor of PSI, can
be the immediate electron donor to FNR, providing the oneelectron reduction of FAD to the semiquinone form.
However, the reduction of FNR by FB in vivo seems unlikely
since FB is located on the PsaC subunit of PSI, which, together
with PsaE and PsaD proteins, forms a docking site that was
evolutionarily optimized for efficient binding and redox
transformation of Fd. Also, in many species, isoforms of FNR
are firmly attached to thylakoid membranes, and their withdrawal to the stroma is under physiological control (Hanke and
Mulo 2013). This hampers the immediate interaction of FNR
with PSI.
Another question is whether the membrane-bound FNR can
be an effective electron donor to O2 molecules. It is believed
that during the catalytic cycle of NADP+ reduction by FNR,
NADP+ binding to FNR precedes the binding of reduced Fd
(Batie and Kamin 1984). In the absence of NADP+, the electron
transfer from reduced Fd to FNR is very slow, while pre-binding
of NADP+ greatly accelerates the rate of electron transfer
(k = 40–80 s 1 vs. 445 s 1; see Carrillo and Ceccarelli 2003).
Moreover, NADP+ is the top-priority electron acceptor from
FNR. Thus, oxygen reduction by FNR concurrently with NADP+
reduction can barely proceed. However, under conditions causing deficiency of oxidized pyridine dinucleotide in chloroplasts,
the electron transfer from Fd to FNR and then to O2 cannot be
excluded.
The Role of Ferredoxin in Oxygen Reduction
A detailed analysis of available literature data showed that the
rate of oxygen uptake in isolated thylakoids with stromal proteins removed is smaller than the rate of the process observed
in leaves (Asada 2000). This and some other facts led investigators to propose an involvement of stromal components in
oxygen reduction, first of all of Fd. Fd, an electron carrier from
PSI to FNR in linear electron flow, is a small, 12 kDa, watersoluble protein, containing an iron–sulfur cluster. In higher
plants, the cluster type is [2Fe–2S]. Fd possesses Em equal to
420 mV (Tagawa and Arnon 1968), which is considerably
lower than Em (O2/O2 ) in water solutions ( 160 mV). The
reduced Fd was shown to be oxidized by oxygen with formation
of O2
(Misra and Fridovich 1971). Nevertheless, Fd was
found to be a weak oxygen reductant. The rate constant of
the reaction of O2 with reduced Fd was found to be as low as
103 M 1 s 1 (Hosein and Palmer 1983, Golbeck and Radmer
1984, Kozuleva et al. 2007), while O2 reduction, e.g. by semiquinones possessing Em (Q/Q ) close to Em (Fd/Fd ), has a
second-order rate constant of approximately 109 M 1 s 1
(Wardman 1990).
In the work of Asada et al. (1974), the rate of O2 generation, measured in thylakoid suspension as the rate of Cyt c
reduction inhibited by SOD, was found to be almost unaffected
by Fd addition to thylakoids. The authors concluded that components of PSI rather than Fd are involved in oxygen reduction,
and the contribution of Fd to this process is negligible.
However, to date, Fd is sometimes considered as the main
oxygen reducing agent in chloroplasts in vivo. Indeed, addition
of purified Fd to a thylakoid suspension stimulated oxygen reduction when oxygen was the only final acceptor of electrons
from PETC components (Allen 1975, Ivanov et al. 1980, Furbank
and Badger 1983). The effect was explained to be the result of
Fd acting as a mediator between PSI and O2 molecules. In these
studies, the rate of oxygen reduction increased with increasing
Fd concentration. To achieve rates of oxygen reduction comparable with the maximum rates of electron transport through
the PETC, the Fd/Chl ratio in the suspension of isolated thylakoids should be close to 10 mol/mol (Ivanov et al. 1980, Golbeck
and Radmer 1984), i.e. approximately three orders of magnitude
higher than this ratio in chloroplasts in vivo. The above high Fd
concentrations in the thylakoid suspension were obviously necessary to reach a steady concentration of reduced Fd in the
light sufficient to provide the transfer of all electrons supplied
by the PETC to oxygen. The need for that concentration can be
seen by comparing the rate constant of the reaction of reduced
Fd with O2 molecules with the rate constant of Fd reduction in
the PETC, 106–107 M–1 s–1 (Golbeck and Radmer 1984). It is
important that in order to achieve the maximal rates of NADP+
reduction, which are close to the total capacity of PETC, in
experiments with isolated thylakoids the Fd/Chl ratio has to
be 0.5–1.0 mol/mol (Allen 1975, Red’ko at al. 1982, Furbank and
Badger 1983), indicating additionally that Fd reduction by PSI is
not a rate-limiting process in oxygen reduction. The above data
argued that the transfer of electrons from the PETC through Fd
to O2 molecules in thylakoids is an inefficient process.
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M. A. Kozuleva and B. N. Ivanov | Mechanisms of oxygen reduction in chloroplasts
In the studies of Kozuleva et al. (2007) and Kozuleva and
Ivanov (2010), the rates of oxygen reduction by reduced Fd and
by the membrane-bound PETC components were experimentally separated for the first time. It was directly shown that, in
the absence and in the presence of NADP+, the electrons for
oxygen reduction were supplied by both Fd and the membranebound components of the PETC. The relationship of these electron fluxes depended on the Fd concentration and the light
intensity. In the presence of only Fd, the fraction of oxygen
reduction mediated by Fd varied from 20% to 70%, depending
on the Fd concentration. At low Fd concentrations, the stimulation of oxygen reduction by Fd was negligible and this fraction
was low (Kozuleva et al. 2007). With increasing total Fd concentration, the steady-state level of the reduced Fd in the light
increased, leading to an increase in electron transport from
Fd to oxygen as well as its contribution to the overall oxygen
reduction (Kozuleva and Ivanov 2010). Gradually increasing
the light intensity resulted in the continuing increase in the
total rate of oxygen reduction, while the Fd-dependent
rate of oxygen reduction exhibited saturation at moderate
light intensities. This showed that when transfer of electrons from the reduced Fd to oxygen became limited, the increase in the total rate of oxygen reduction was determined
by electron flow to oxygen from the membrane-bound
components.
The most important question is the contribution of Fd to
oxygen reduction occurring simultaneously with NADP+ reduction. The simultaneous occurrence of these processes was discovered by Allen (1975), and then was repeatedly confirmed
(Ivanov et al. 1980, Robinson and Gibbs 1982, Furbank and
Badger 1983). Addition of NADP+ to a thylakoid suspension
containing Fd increased the entire photosynthetic electron
transport, i.e. to both NADP+ and O2, first of all as the result
of a high rate of NADP+ reduction. The considerable rate of
oxygen reduction found under conditions optimal for NADP+
reduction was initially ascribed to the involvement of Fd.
However, under such conditions, the concentration of reduced
Fd is rather low (Hosler and Yocum 1985, Kozuleva et al. 2007).
The Fd contribution to oxygen reduction at Fd concentrations
optimal for NADP+ reduction was found to be practically negligible, <7 %, and oxygen reduction is mainly performed by the
membrane-bound components of the PETC (Kozuleva and
Ivanov 2010). An increase in the light intensity in the presence
of NADP+ led to a noticeable increase in the rate of oxygen
reduction (Robinson and Gibbs 1982, Furbank and Badger 1983,
Kozuleva and Ivanov 2010). The rate of Fd-dependent oxygen
reduction was found to be saturated at very low light intensities
and remained negligible up to high light intensities (Kozuleva
and Ivanov 2010). This revealed that the increase in electron
flow to oxygen with increasing light intensity occurred at the
expense of the increased oxygen reduction by the membranebound components of the PETC.
Thus, direct measurements together with the set of indirect
evidence discussed above unequivocally prove that the transfer
of electrons from reduced Fd is an unlikely pathway of oxygen
reduction in vivo.
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Oxygen Reduction in the PSI Pigment–Protein
Complex
PSI, plastocyanin:ferredoxin oxidoreductase, contains the electron transport chain consisting of electron transfer cofactors
P700, A0, A1, FX, FA, FB (Fig. 1). Cofactors between P700 and FX are
arranged in two branches, named the A- and B-branches,
located on the PsaA/PsaB protein heterodimer along a
pseudo-symmetry axis. P700 is a dimer of Chl a molecules. A0
in both branches are also Chl a molecules, which were proposed
to be a dimer (Ptushenko et al. 2008) or a monomer (Müller
et al. 2010). The lifetime of A0 is around 30 ps, and this makes
the electron transfer to oxygen quite improbable despite low
redox potential, 1,250 mV (here and below, the Em values are
taken from Ptushenko et al. 2008). Phylloquinones (PhQs)
occupy two quinone-binding sites A1. These two PhQs differ
in Em values, 671 and 844 mV at the A1A and A1B sites,
respectively, and their reduced forms differ in their lifetimes:
electron transfer from PhQA
to FX possessing an
Em = 585 mV occurs within approximately 250 ns, while that
from PhQB
occurs in approximately 20 ns (Santabarbara
et al. 2015). FX, FA and FB are [4Fe–4S] clusters. FX is co-ordinated by four cysteine residues, two each from PsaA and PsaB,
and thus, besides its function as an electron transfer cofactor,
plays a crucial role in PsaA/PsaB heterodimer assembly
(Jagannathan et al. 2012). FX transfers electrons to the terminal
membrane-bound electron transfer cofactors at the acceptor
side of PSI, FA and FB, for 50 ns (Semenov et al. 2006). FA and FB
are situated at the PSI subunit PsaC, which protrudes into the
stroma. Oxidation of the terminal cofactors by Fd occurs within
500 ns to 1 ms in a pre-formed complex of Fd with PSI and in up
to 100 ms in the case of diffusion-limited electron transfer between Fd and PSI in solution (Setif 2006). The Em values of FA/
FA and FB/FB pairs are 479 and 539 mV, respectively
(Ptushenko et al. 2008), and they are sufficient for reduction
of O2 molecule in water. However, the values of the dielectric
constant in the immediate vicinity of these centers are close to
5 (Semenov et al. 2003), which could disable efficient O2 reduction by these cofactors due to positive values of G0 for this
reaction (one should take into account the Em value of O2/
O2
in DMF; see above). This estimation is confirmed by
low rates of oxygen reduction by both thylakoid membranes
(Asada et al. 1974, Khorobrykh et al. 2004) and isolated PSI
complexes (Kozuleva et al. 2014), when electron transfer from
the terminal PSI cofactors to O2 is the rate-limiting step of the
whole electron transport. All other PSI electron transfer cofactors are located in the hydrophobic region of the PsaA/PsaB
heterodimer with quite a low permittivity.
PSI was proposed as the main site of oxygen reduction in
numerous works. However, only a few attempts to establish
which PSI cofactor directly reduces O2 molecules were made.
Takahashi and Asada (1988) showed that superoxide-dependent
protein iodination in isolated thylakoids during the first seconds of
illumination occurred exclusively in the PSI proteins; FX was speculated to be the oxygen reductant. Involvement of PhQ in O2 reduction was proposed by Kruk et al. (2003), based on stimulation
of oxygen uptake by PhQ addition to the thylakoid membranes
Plant Cell Physiol. 57(7): 1397–1404 (2016) doi:10.1093/pcp/pcw035
Fig. 1 Schematic diagram of forward electron transfer in PSI with lifetimes and Em values of the cofactors. Em values of (O2/O2 ) in water and
dimethylformamide are shown. Pc, plastocyanin; P700, a dimer of Chl a molecules, the primary electron donor; A0, the primary electron acceptor;
PhQ, phylloquinone, a secondary electron acceptor; FX, 4Fe–4S cluster, a secondary electron acceptor; FA and FB, 4Fe–4S clusters, the terminal
electron acceptors.
containing no quinones after treatment with hexane. The dependence of the rate of oxygen reduction by membrane-bound components on the efficiency of electron withdrawal from PSI
permitted Kozuleva et al. (2007) to consider both FX and PhQ
as oxygen reductants. It should be noted that they both can
reduce O2 in media with a low permittivity.
The role of these membrane-bound components of PSI as
the main O2 reductants was further explored in Kozuleva and
Ivanov (2010). In that study, the unexpected dependence of
the O2 reduction rate on the presence of NADP+ was
observed in pea isolated thylakoids; addition of NADP+ at
high light intensities did not decrease and even increased the
oxygen reduction rate relative to the rate observed in the
presence of only Fd. This result was ascribed to peculiarities
of electron transfer reactions in PSI. Obviously, the reduction
of O2 by PhQ and/or FX should increase with an increase in
the quasi-steady-state concentration of these cofactors in the
reduced state. This concentration depends on the relationship between electron inflow from the donor side of PSI, the
electron outflow to downstream cofactors and the charge
recombination of their reduced forms with P700+. The latter
process is highly stimulated due to electrostatic repulsion
when the terminal cofactors, FA/B, become reduced, e.g. the
recombination time of P700+A1 is decreased by three orders
of magnitude, from 250 ms to 250 ns (Polm and Brettel 1998).
When electron withdrawal from FA/B is retarded, the higher
stationary negative charge holds there, and the electrostatic
effect on PhQ and FX does increase. At high light intensities, in the absence of NADP+, the retardation of electron
withdrawal from FA/B could be a result of delay in supply of
oxidized Fd due to a low rate of oxidation of reduced Fd by
oxygen, while the presence of NADP+ could maintain the
lower level of reduced Fd and consequently of FA/B . The
average retention of the electrons at FA/B in the presence
of only Fd was longer by an order of magnitude than with
Fd + NADP+ (Kozuleva and Ivanov 2010), so the electrostatic
repulsion in a larger population of PSI could be higher in the
absence of NADP+. It is assumed that the sum of rates of
forward electron transfer and the recombination from PhQ
(or Fx ) in the presence of both Fd and NADP+ is less than
in the presence of only Fd; and thus the higher quasi-steadystate concentration of PhQ (or Fx ) and hence the higher
rate of O2 reduction by these species should be in the presence of Fd and NADP+. The described increase in oxygen
reduction by isolated thylakoids owing to NADP+ addition
(Kozuleva and Ivanov 2010) can explain the higher rate of
oxygen reduction in intact chloroplasts as compared with
‘naked’ thylakoids, but whether it is inherent only for pea
may be ascertained after conducting corresponding studies
with other plants.
Experiments designed to identify the O2 reductant in PSI
were performed with PSI complexes isolated from cyanobacterium Synechocystis sp. PCC 6803 (Kozuleva et al. 2014).
Mutants with completely blocked biosynthesis of PhQ are
known for this organism (men mutants). These mutants are
still able to grow photoautotrophically in low light. It was
shown that, in the mutants, PQ occupies the A1 sites of PSI,
performing electron transfer from A0 to FX. However, the Em
value of PQ/PQ at the A1 sites is about 100 mV more positive
than that of PhQ/PhQ (Semenov et al. 2000). This decreases
the G0 value for O2 reduction at these sites. It was found that
in PSI isolated from the mutant menB, the dependence of
oxygen uptake reflecting O2 production on light intensity
exhibited saturation behavior already at moderate intensities,
while the uptake in PSI from the wild type continued to
increase up to notably higher light intensities (Kozuleva et al.
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M. A. Kozuleva and B. N. Ivanov | Mechanisms of oxygen reduction in chloroplasts
2014). Oxygen reduction by intermediate electron transfer cofactors in PSI occurred concurrently with such a process at the
terminal cofactors, FA/B, but the latter had the same rate in the
mutant as in the wild type, since the rate of electron supply to
FA/B was shown to be unaffected by the mutation (Kozuleva
et al. 2014). Comparison of oxygen reduction in PSI complexes
from the wild type and the mutant clearly showed that PhQ is
involved in oxygen reduction in PSI, and that its contribution to
this process increased with an increase in light intensity. It may
be relevant that in the work (Takahashi and Asada 1982), a
change in the affinity of oxygen for its reduction by the spinach
thylakoids with the change in the actinic light intensity was
observed.
Data demonstrating that PhQ is a basic reductant of oxygen
in the PETC are in good agreement with the evidence of O2
production within the thylakoid membrane (see above). They
support the proposition that H2O2 is formed within the membrane in the reaction of PQH2 with intramembrane O2
(Mubarakshina et al. 2006, Mubarakshina and Ivanov 2010).
Such a pathway of H2O2 formation, taking into account its
signaling function, may be important to explain the repeatedly
shown role of the PQ pool redox state as a trigger of acclimation
reactions in leaves (Borisova-Mubarakshina et al. 2015).
Conclusion
Mother Nature arranged the terminal reducing segment of the
PETC wisely, providing the presence there of the chain of low
potential components performing NADP+ reduction with minimal leakage of electrons to oxygen. The presented data show
that Fd cannot significantly contribute to oxygen reduction
in vivo. The slow oxidation of reduced Fd by oxygen has
evident physiological significance since the reduced Fd is the
central chloroplast component donating electrons to numerous metabolic reactions. It is believed that [2Fe–2S] Fds
with the clusters shielded by the protein are more tolerant to
oxygen than typical bacterial [4Fe–4S] Fds, whose clusters are
partially solvent exposed (Jagannathan and Golbeck 2009).
Therefore, recruitment of [2Fe–2S] Fds as mobile electron transfer proteins in organisms with oxygenic photosynthesis
was proposed to be an evolutionary adaptation for protection against interaction of PSI with oxygen (Jagannathan et al.
2012).
In isolated thylakoids, in the absence of natural or artificial
electron acceptors, the terminal cofactors of PETC, FA/B, transfer electrons to oxygen as the sole acceptor available. The FA
and FB centers, according to their redox potentials and surroundings, do not efficiently reduce O2 molecules, and the
rate of such a reduction is low. The low efficiency of oxidation
of FA/B by oxygen also has physiological reasons since it enables
FA/B to reduce Fd successfully. The evolution of PsaC carrying FA
and FB clusters from the bacterial dicluster Fd was proposed to
be initiated in order to protect FA and/or FX against oxidative
damage by shielding these cofactors by protein (Jagannathan
et al. 2012). The linkage of bacterial Fd to the ancestral reaction
center also could be designed to prevent electron leakage from
1402
its cofactors to oxygen. Moreover, the switch of Em (FA/FA ) to
more positive values providing uphill electron transfer from FA
to FB and preferential (90%) residence of electrons on FA was
proposed to be an evolutionary adaptation to minimize oxygen
reduction by FB in the absence of oxidized Fd (Shinkarev et al.
2000). It was shown that with partial damage of the thylakoid
membrane structure, the rate of oxygen reduction greatly increases (Asada and Nakano 1978, Takahashi and Asada 1982,
Navari-Izzo et al. 1996). Under stressful conditions in vivo, some
impairment of the membrane structure can occur, causing the
leakage of electrons from PSI cofactors to oxygen with production of O2 and H2O2 in the stroma, where their increased
amount can be the alarm signal.
It is widely accepted that the appearance of oxygen in
the atmosphere was accompanied by transformation of the
type I homodimeric reaction center to a heterodimeric one
(Jagannathan and Golbeck 2009, Rutherford et al. 2012). The
most conspicuous asymmetry in the electron transfer organization in PSI is the difference in Em values of PhQ at the A1 sites
and hence the rates of electron transfer from PhQ to FX in Aand B-branches. Under conditions of retarded electron transfer
from the terminal PSI cofactors, the charge recombination
occurs in this complex. This could lead to formation of the
triplet P700, which in the reaction with O2 molecules in the
ground state could produce 1O2, whose reactivity exceeds
that of O2
and H2O2. Rutherford et al. (2012) proposed
that the smaller energy gap between PhQA and FX than between PhQB and FX promotes charge recombination in the
A-branch; and the charge recombination occurring in this
branch with the more positive quinone leads to less probable
formation of the triplet P700 than charge recombination in the
B-branch. We propose that electron flow from PhQ to oxygen
can help maintain the forward electron transfer in PSI, thus
decreasing charge recombination and 1O2 production
(Kozuleva et al. 2014). This is especially relevant under conditions when charge separation takes place in both branches,
leading to formation of PhQ
at both A1 sites
(Santabarbara et al. 2015) and making two PhQ
compete
for one FX. In the case of retarded electron outflow from PSI, e.g.
at high light intensities and saturated NADP+ reduction,
oxygen reduction by PhQ may represent an essential mechanism of plants protection against photoinhibition.
Funding
This study was supported by the Russian Science Foundation
[research project # 14-14-00535].
Acknowledgements
The authors express their gratitude to Drs. V.V. Klimov and I.I.
Proskuryakov for helpful discussion.
Disclosures
The authors have no conflicts of interest to declare.
Plant Cell Physiol. 57(7): 1397–1404 (2016) doi:10.1093/pcp/pcw035
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