Akimasa Nakamura, Tomoyuki Suzawa, Yuki Kato* and Tadashi Watanabe
Institute of Industrial Science, the University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505 Japan
*Corresponding author: E-mail, [email protected]; Fax, +81-3-5452-6331
(Received February 12, 2011; Accepted March 15, 2011)
The redox potential of the primary electron donor P700,
Em(P700/P700+), of Photosystem I (PSI) has been determined for 10 oxygenic photosynthesis organisms, ranging
from cyanobacteria, red algae, green algae to higher plants,
by spectroelectrochemistry with an optically transparent
thin-layer electrode (OTTLE) cell to elucidate the scattering
by as much as 150 mV in reported values of Em(P700/P700+).
The Em(P700/P700+) values determined within error ranges
of ±1–4 mV exhibited a significant species dependence, with
a span >70 mV, from +398 to +470 mV vs. the standard
hydrogen electrode (SHE). The Em(P700/P700+) value appears to change systematically in going from cyanobacteria
and primitive eukaryotic red algae, then to green algae
and higher plants. From an evolutionary point of view, this
result suggests that the species believed to appear later
in evolution of photosynthetic organisms exhibit higher
values of Em(P700/P700+). Further, the species dependence
of Em(P700/P700+) seems to originate in the speciesdependent redox potentials of soluble metalloproteins, Cyt
c6 and plastocyanin, which re-reduce the oxidized P700 in
the electron transfer chain.
Keywords: Charge separation Electron transfer P700 PSI
Redox potential Spectroelectrochemistry.
Abbreviations: Fd, ferredoxin; LHC, light-harvesting complex;
OTTLE, optically transparent thin-layer electrode; Pc, plastocyanin; SHE, standard hydrogen electrode.
Introduction
PSI (Photosystem I), one of the two large pigment–protein
complexes with many cofactors involved in the linear electron
transfer chain of oxygenic photosynthesis, drives photoinduced transmembrane electron transfer from Cyt c6 and/or
plastocyanin (Pc) in the thylakoid lumen to ferredoxin (Fd) in
the stroma (for recent reviews, see Jensen et al. 2007, Sétif and
Leibl 2008). The redox potential span between the external
electron donor [Cyt c6 and/or Pc; Em & +360 mV vs. the standard
hydrogen electrode (SHE)] and the acceptor (Fd;
Em & 420 mV) is about 780 mV (Fig. 1), which acts energetically as an intermediary between water oxidation at PSII and
reduction of NADP+ to NADPH. This free energy gain is provided by light-induced charge separation from the primary electron donor P700 to the primary electron acceptor A0, this being
monomeric Chl a, and is characterized by the excitation energy
and redox potential of P700, Em(P700/P700+).
P700 is a heterodimer of Chl a and its C132-epimer, Chl a0 , as
verified by X-ray crystallography (Jordan et al. 2001) and pigment composition analysis (Nakamura et al. 2003). Since
Em(P700/P700+) is known to be roughly 400 mV lower than
the redox potential of monomeric Chl a in organic solvents
(approximately +800 mV; Watanabe and Kobayashi 1991), specific electrostatic interactions with the protein subunits and
electronic coupling of two p-electron systems of Chl a/a0
may control the value of Em(P700/P700+) in the PSI complex.
Recent studies employing site-directed mutations combined
with redox titration (Krabben et al. 2000, Witt et al. 2002,
Witt et al. 2003, Li et al. 2004, Sommer et al. 2004) revealed
how axial ligands and hydrogen bonding from the amino acid
residues neighboring P700 can affect Em(P700/P700+); however,
such site-directed mutagenesis studies were performed only on
a green alga Chlamydomonas reinhardtii, and it has not been
examined whether Em(P700/P700+) exhibits species dependence. Since the literature values of Em(P700/P700+) are still
ambiguous, we are unable to discuss this issue in detail. In
early literature, the values of Em(P700/P700+) were scattered
widely from +375 to +525 mV (reviewed in Golbeck 1987,
Ke 2001); though recent measurements, in addition to the
mutagenesis studies mentioned above, yielded more restricted
values, a scattering still prevails in a range of +420 to +480 mV
(reviewed in Sétif and Leibl 2008; see Discussion).
While surveying literature on the spectroscopic properties of
P700, we noticed that the light-induced difference spectra of P700
clearly differ among species (Hiyama and Ke 1972, Pålsson et al.
1998, Redding et al. 1998, Nakamura et al. 2003, Witt et al. 2003).
Regular Paper
Species Dependence of the Redox Potential of the Primary
Electron Donor P700 in Photosystem I of Oxygenic
Photosynthetic Organisms Revealed by
Spectroelectrochemistry
Plant Cell Physiol. 52(5): 815–823 (2011) doi:10.1093/pcp/pcr034, available online at www.pcp.oxfordjournals.org
! The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 52(5): 815–823 (2011) doi:10.1093/pcp/pcr034 ! The Author 2011.
815
A. Nakamura et al.
–1400
energetics on the electron transfer chain, from an evolutionary
point of view, and by invoking subtle interactions between the PSI
complex and Cyt c6 and/or Pc in the course of electron transfer.
P700*
–1200
A0 (Chl a)
Em /mV vs. SHE
–1000
A1 (PhQ)
–800
hn
Results
FB
FX
–600
FA
Fd
–400
–200
0
+200
+400
+600
+800
Cyt c6/Pc
P700
Photosystem I
Fig. 1 Energetic scheme of electron transfer through PSI based on the
redox potential Em of cofactors. Approximate potential values for
electron acceptors are from currently available data obtained by
redox titration or kinetic analysis (for details, see Moser and Dutton
2006, Sétif and Leibl 2008).
As stated by Witt et al. (2003), it is generally assumed that the
PSI core complexes, especially the cofactor arrangement in the
reaction center, are similar in all organisms despite diversification found in the peripheral and/or external architectures of
PSI. This situation is probably reflected by the highly conserved
primary sequences of the core subunits PsaA/B among species
(Fish et al. 1985, Mühlenhoff et al. 1993, Fromme et al. 2001),
and probably also holds true for Em(P700/P700+). Contrary
to this view, the aforementioned spectroscopic difference as
seen in the difference spectra suggests a difference in the geometrical arrangement of P700 among species (Witt et al. 2003).
This may, in turn, also lead to the species dependence of
Em(P700/P700+).
We showed, by spectroelectrochemistry, that the value of
Em(P700/P700+) for a cyanobacterium Thermosynechococcus
elongatus is more negative by about 50 mV than that of spinach
(Nakamura et al. 2005), suggesting a significant species dependence of Em(P700/P700+). This demonstrates that spectroelectrochemistry with an optically transparent thin-layer electrode
(OTTLE) cell, featuring rigorous potential control and rapid
redox equilibration within the cell, could shed light on the
species dependence of Em(P700/P700+) undiscovered in previous measurements, which in most cases employed chemical
titration with limited accuracy. In the present work, 10 different
oxygenic photosynthetic organisms, namely five cyanobacteria,
two red algae, one green alga and two higher plants, were submitted to spectroelectrochemical measurements of Em(P700/
P700+) to closely examine its species dependence. A significant
species dependence of Em(P700/P700+) was found under the
same conditions, and the result is discussed in terms of the
816
Fig. 2 shows the oxidized minus reduced difference absorption
spectra of P700 in PSI isolated from eight organisms measured
by spectroelectrochemistry, where the potential of the working
electrode was controlled at +650 and +50 mV for oxidation
and reduction, respectively. For the green algae and higher
plants in particular, the spectral change due to irreversible oxidation of bulk antenna Chl a molecules overlapped slightly with
the Qy absorption band (700 nm) of P700 (Nakamura et al.
2003, Nakamura et al. 2004). To eliminate any interference
from this phenomenon, the P700 difference spectra were obtained by starting the measurement of a spectrum for the fully
oxidized state (+650 mV), then changing the potential for the
measurement in the reduced state (+50 mV). The difference
spectra obtained spectroelectrochemically here are essentially
similar to the light-induced difference absorption spectra of
P700 reported elsewhere (Hiyama and Ke 1972, Pålsson et al.
1998, Redding et al. 1998, Nakamura et al. 2003, Witt et al.
2003), and close inspection clearly reveals subtle differences
in the spectral shape among organisms.
The maximum bleaching of the Qy band in the oxidized–
reduced difference spectra of P700 was slightly different among
organisms: 699 nm for Gloeobacter violaceus and Chlamydomonas
reinhardtii; 701 nm for T. elongatus, Cyanidium caldarium,
Synechococcus PCC 6301 and spinach; 702 nm for Spirulina
platensis; and 703 nm for Synechocystis PCC 6803. A smaller
bleaching band was observed for all species at 681–683 nm,
and a positive band between the two bleaching bands appeared
at 689–690 nm. The spectral shapes, reflecting the relative
ratios of these three band changes, were varied: the shapes
are similar in T. elongatus and spinach as observed also in the
light-induced difference spectra (Hiyama and Ke 1972, Pålsson
et al. 1998, Nakamura et al. 2003), and those in S. platensis and
Synechococcus PCC 6301 look similar, whereas G. violaceus and
Synechocystis PCC 6803 exhibit spectra quite different from
the others. The width of the most intense bleaching band
for the cyanobacteria was broader than those of C. reinhardtii
and spinach: half-width at half-maximum of the bands is
185–230 cm1 for cyanobacteria, 178 cm1 for C. reinhardtii
and 176 cm1 for spinach. The isosbestic points at the red-tail
of the band for the cyanobacteria shifted more to the red, of
which that of Synechocystis PCC 6803 shifted farthest to
738 nm, compared with those of C. reinhardtii and spinach.
The tendency observed in the differences of the peak position,
shape and isosbestic point is mostly consistent with that in
reported light-induced difference spectra (Nakamura et al.
2003, Witt et al. 2003).
Despite the difference in the spectral shape, the absorbance
change due to oxidation of P700 monitored at around
Plant Cell Physiol. 52(5): 815–823 (2011) doi:10.1093/pcp/pcr034 ! The Author 2011.
Species dependence of P700 redox potential
0
0
Gloeobacter violaceus
Synechococcus PCC 6301
DAbsorbance/ a.u.
0
0
Spirulina platensis
Synechocystis PCC 6803
0
0
Thermosynechococcus elongatus
Chlamydomonas reinharditii
0
0
Cyanidium caldarium
650
700
750
Wavelength/nm
800
Spinach
850
650
700
750
800
Wavelength/nm
850
Fig. 2 Oxidized minus reduced difference spectra of P700 in PSI complexes isolated from oxygenic photosynthetic organisms in the OTTLE cell, where
the potential of the working electrode was set at +650 V and +50 mV for oxidation and reduction, respectively. Each trace is an average of 2–8 scans.
800–820 nm, where a broad positive band appears by accumulation of P700+, was saturated at potentials higher than
+650 mV for all organisms. This means that P700 is fully oxidized at +650 mV (Nakamura et al. 2004). To determine the
redox potential of P700, the absorbance changes at 808 nm
(A808) for the species, with the exception of A700 for
G. violaceus, were measured during potential journeys as
shown in Fig. 3. By stepping the electrode potential to an
anodic value after thorough reduction at +50 mV, the A808
value increased rapidly and reached an equilibrium state within
80 s. Returning the potential to +50 mV induced a return
of A808 to the initial baseline, indicating that the redox reaction of P700 in these species is fully reversible, as previously
demonstrated for T. elongauts and spinach (Nakamura et al.
2004, Nakamura et al. 2005). This reversibility was also confirmed even after repetitive potential journeys for several hours.
The magnitude of A at the same anodic potential step
exhibited a remarkable difference among organisms: A was
significantly larger for the cyanobacteria and the red alga
C. caldarium than for C. reinhardtii and spinach (Fig. 3;
cf. Nakamura et al. 2005). This shows that P700s in the cyanobacteria and red alga are oxidized more readily.
The mole fraction of P700+, assessed from the A value
against the electrode potential, obeyed a theoretical oneelectron reversible redox process well, as verified by the
Nernstian curves in Fig. 4. The Em(P700/P700+) values determined by the Nernstian plots are summarized in Table 1. It is
obvious that the Em(P700/P700+) value covers a broad range
of >70 mV. Further, the Em(P700/P700+) value does not vary
randomly but rather somewhat systematically. Briefly, the
Em(P700/P700+) value appears to follow the order: cyanobacteria < red algae < green alga higher plants. It is also noted
that the Em(P700/P700+) values of cyanobacteria cover a range
of approximately 60 mV, in contrast to the eukaryotes; the
Em(P700/P700+) values of Synechococcus PCC 6301 and
Synechocystis PCC 6803 are higher than those of red algae.
Plant Cell Physiol. 52(5): 815–823 (2011) doi:10.1093/pcp/pcr034 ! The Author 2011.
817
A. Nakamura et al.
–6.0
–4.0
+425
–3.0
+400
–2.0
Synechococcus
PCC 6301
+650 mV
+550
+500
2.0
DA808/10–3
–5.0
DA700/10–3
G. violaceus
+650 mV
+500
+450
1.6
+475
1.2
0.8
+450
0.4
+425
+400
+375
–1.0
+350
0
0
0
50
0
100 150 200 250 300 350 400
50
100
1.2
0.8
0.4
S. platensis
+650 mV
+500
+475
DA808/10–3
DA808/10–3
1.6
150
200
250
Time/s
Time/s
+450
+425
+400
2.5
+650 mV
+550
2.0
+500
+475
1.5
+450
1.0
+425
0.5
+375
Synechocystis
PCC 6803
+400
0
0
0
50
100
150
200
250
0
50
100 150 200 250 300 350 400
Time/s
0.8
C. caldarium
+650 mV
+550
+500
+650 mV
+475
+500
1.0
+475
0.5
+425
+450
+425
+400
+400
0
0
0
C. reinharditii
+550
+450
0.4
1.5
DA808/10–3
DA808/10–3
1.2
Time/s
50
100
150
Time/s
200
0
250
50
100 150 200 250 300 350 400
Time/s
Fig. 3 Absorbance change A during potential journeys for P700 in PSI complexes from various organisms. The electrode potential was initially
held at +50 mV and then stepped to anodic potentials at the time indicated by a downward pointing arrow; after redox equilibration, the
potential was returned to +50 mV at the time indicated by an upward pointing arrow. A was monitored at 808 nm except for G. violaceus, of
which the A at 700 nm was followed and the vertical axis is inverted to facilitate comparison with those of other organisms. See Nakamura
et al. (2005) for the data for T. elongatus and spinach. Each trace is an average of two scans. The magnitudes of the redox-induced A were read
after smoothing the traces by simple moving average.
Hereafter, the cyanobacteria with Em(P700/P700+) values lower
and higher than those of the red algae are called group I and II,
respectively.
Discussion
The Em(P700/P700+) values covering a range of 72 mV between
the two extremes as summarized in Table 1 appear to follow
818
the order: cyanobacteria I < red algae < cyanobacteria II <
green alga higher plants. As reported for various PSI fractions
from T. elongatus and spinach, the potential shift induced by
different detergent treatments does not exceed 10 mV
(Nakamura et al. 2005). The present result hence demonstrates
that the difference in the observed Em(P700/P700+) values
reflects a species dependence. Though most of the early studies
on Em(P700/P700+) (see Golbeck 1987, Ke 2001) were
performed for higher plants, from recent Em(P700/P700+)
Plant Cell Physiol. 52(5): 815–823 (2011) doi:10.1093/pcp/pcr034 ! The Author 2011.
Species dependence of P700 redox potential
measurements, mostly via chemical titration with a lesser degree
of accuracy or reliability than the present spectroelectrochemical technique, such a species dependence has been implied
(reviewed in Sétif and Leibl 2008, and some reported values
are added here): +430 mV (Witt et al. 2003) for S. platensis;
+468 mV (Hamacher et al. 1996) for Synechococcus PCC 6803;
and +447 mV (Krabben et al. 2000), +450 mV (Li et al. 2004),
+473 mV (Sommer et al. 2004) and +478 mV (Witt et al. 2002)
for C. reinhardtii. However, to date, no report has dealt with the
species dependence of Em(P700/P700+).
Group I cyanobacteria possess the lowest Em(P700/P700+)
values among the organisms examined. These species
are thought to be the primitive forms of cyanobacteria.
Mole fraction of P700+
1.0
0.5
G. violaceus
S. platensis
T. elongatus
C. cardalium
P.purpureum
Synechococcus PCC 6301
Synechocystis PCC 6803
C.reinhardtii
Barley
Spinach
0
+300
+350
+400
+450
+500
+550
+600
E /mV vs. SHE
Fig. 4 Nernstian plots for the redox reaction of P700 in PSI complexes
from various organisms based on A values against electrode potentials. Each curve represents a theoretical curve for a one-electron
redox process, and the intersection with the broken line denotes
the Em(P700/P700+) value (see Table 1 for their values). The data
for T. elongatus and spinach are cited from Nakamura et al. (2005).
Gloeobacter violaceus is known to have branched off at a very
early stage in the phylogenetic tree of oxygenic photosynthetic organisms, based on the 16S rRNA sequence (Nelissen
et al. 1995). Thermosynechococcus elongatus also branched
off from the phylogenetic tree at a relatively early stage. In
contrast, group II cyanobacteria, Synechocystis PCC 6803 and
Synechococcus PCC 6301 branched off at a later stage (for example, see Mimuro et al. 2008). The chloroplasts of eukaryotic
algae and higher plants are considered to have evolved from
cyanobacteria by endosymbiosis of cyanobacteria and eukaryotes. Among eukaryotes, red algae branched off the phylogenetic tree at an earlier stage than green algae and higher plants
(Nelissen et al. 1995, Mimuro et al. 2008). In view of these
findings, the species dependence of the Em(P700/P700+) value
appears to reflect the evolutionary pathway of oxygenic photosynthetic organisms: the species which appeared later in
evolution exhibits the highest value of Em(P700/P700+).
In connection with evolution, it has been proposed that a
gradual transition from Cyt c6 to Pc employed as the electron
shuttle between Cyt b6f and PSI occurred as a consequence of
microbial adaptation to environmental conditions (De la Rosa
et al. 2006, Dı́az-Quintana et al. 2008). As the atmospheric
oxygen concentration rose due to the oxygenic photosynthetic
activity, the relative bioavailability of iron and copper went
down and up, respectively, and Cyt c6 was gradually replaced
by Pc. The biosynthetic ability to form the metalloproteins
depends primarily on the presence of the gene encoding
Cyt c6 and/or Pc in the genome of organisms (Moseley et al.
2000, Ho 2005). Most cyanobacteria and green algae employ
both Cyt c6 and Pc (De la Rosa et al. 2006, Dı́az-Quintana
et al. 2008), whose gene expression levels are regulated by
copper availability (Sandmann et al. 1983, Sandmann 1986,
Ho and Krogmann 1984). Cyanobacteria such as T. elongatus
(Ho 2005, Mulkidjanian et al. 2006) and S. platensis (Sandmann
1986), and red algae (Sandmann et al. 1983, Reith 1996) use Cyt
Table 1 Redox potentials of P700 determined spectroelectrochemically
Species
Classificationa
Em(P700/P700+)b
(mV) vs. SHE
Electron donorc
Gloeobacter violaceus
Cyanobacteria (I)
+398 ± 4
Cyt c6, Pc
Spirulina platensis
Cyanobacteria (I)
+416 ± 1
Cyt c6
d
Thermosynechococcus elongatus
Cyanobacteria (I)
+423 ± 1
Cyt c6
Cyanidium caldarium
Red algae
+431 ± 3
Cyt c6
Porphyridium purpureum
Red algae
+433
Cyt c6
Synechococcus sp. PCC 6301
Cyanobacteria (II)
+452 ± 1
Cyt c6, Pc
Synechocystis sp. PCC 6803
Cyanobacteria (II)
+454 ± 3
Cyt c6, Pc
Chlamydomonas reinhardtii
Green algae
+469 ± 3
Cyt c6, Pc
Barley (Hordeum vulgare)
Higher plants
+470 ± 3
Pc
Spinach (Spinacea oleracea)
Higher plants
+470 ± 2d
Pc
a
b
c
d
See text for the classification of cyanobacteria I and II.
Values are means ± SD of three or more independent measurements, except for P. purpureum for which a single measurement was taken.
See text for the type(s) of the metalloprotein employed in a species.
From Nakamura et al. (2005).
Plant Cell Physiol. 52(5): 815–823 (2011) doi:10.1093/pcp/pcr034 ! The Author 2011.
819
A. Nakamura et al.
c6 alone. In higher plants, Pc is the sole electron carrier (Ho
2005, De la Rosa et al. 2006, Dı́az-Quintana et al. 2008). In view
of the electron transfer from the metalloproteins to P700+
generated by light-induced charge separation, the species dependence of Em(P700/P700+) found here may be linked to the
redox properties of Cyt c6/Pc for the electron transfer
energetics.
The values of redox potentials of Cyt c6 and Pc have been
reported for evolutionarily distinct organisms, and exhibit a
scatter from approximately +320 to +390 mV (Fig. 5; see also
Supplementary Tables S1, S2 for details). Despite the scattering, a clear tendency can be found in the Em(Cyt c6/Cyt c+6 )
values, with the following order: cyanobacteria red algae <
green algae. This tendency is supported more strongly by
comparative results measured under the same conditions
(de Silva et al. 1988): +348 mV for a cyanobacterium and
+380 mV for a green alga. A similar tendency is also seen in
the Em(Pc/Pc+) values: cyanobacteria < green algae higher
plants (for comparative studies, see Meyer et al. 1987,
McLeod et al. 1996, Schlarb-Ridley et al. 2003). Further, in
such species as group II cyanobacteria having both metalloproteins, the values of Em(Pc/Pc+) are generally higher than those
of Em(Cyt c6/Cyt c+6 ). Comparative studies revealed more clearly
that Em(Pc/Pc+) is more positive by >30 mV than Em(Cyt c6/
Cyt c+6 ) under the same conditions for Synechocystis PCC 6803
(De la Cerda et al. 1997, De la Cerda et al. 1999), and a similar
tendency is also seen for other species (reviewed in DiazQuintana et al. 2003). Another comparative study showed
that the Em(Cyt c6/Cyt c+6 ) values in both cyanobacteria I and
II remain almost the same irrespective of its Pc availability
(Cho et al. 1999): +314 and +320 mV were reported for Cyt
c6 in Spirulina maxima and Synechocystis PCC 6803, respectively; the former is known to possess Cyt c6 alone (Cavet et al.
2003), as does S. platensis.
Cyt c6
300
Pc
P700
(This work)
Em /mV vs. SHE
325
350
375
400
425
450
475
500
Fig. 5 Comparisons of reported values of Em(Cyt c6/Cyt c+6 ) and
Em(Pc/Pc+) (see Supplementary Tables S1, S2 for details), and the
Em(P700/P700+) values determined in the present work among evolutionarily distinct organisms: light blue, cyanobacteria I; dark blue,
cyanobacteria II; magenta, red algae; light green, green algae; dark
green, higher plants.
820
A literature survey of the Cyt c6/Pc redox potential values
thus reveals a tendency for higher values to be found in the
organisms that appeared later in evolution for both Em(Cyt c6/
Cyt c+6 ) and Em(Pc/Pc+), and that Em(Pc/Pc+) is higher than
Em(Cyt c6/Cyt c+6 ). Such a tendency parallels our finding for
Em(P700/P700+) as shown in Fig. 5, and the energy difference
between P700 and metalloprotein(s) is apparently maintained
at a constant value of approximately 100 meV. In other words,
the redox potentials of P700 and of the metalloproteins
Cyt c6/Pc may have been optimized interdependently to
attain an efficient electron transfer between them in the
course of evolution.
Meanwhile, the relationship between the excited state
energy level of P700 (P700*), corresponding to Em(P700*/
P700+), and the reduction potentials of electron acceptors is
important for the energetics of light-induced charge separation
within PSI. As seen in the difference spectra of P700 of various
organisms (Fig. 2), the maximum absorbance dip is at around
700 nm, and shows little species dependence in spite of a significant difference in the spectral shape. The dip wavelength,
corresponding approximately to the minimum excitation
energy of P700, is in a narrow range from 703 to 699 nm, and
demonstrates a near-constancy of the excitation energy to
within 10 meV. Hence the variation in Em(P700*/P700+)
almost parallels that of Em(P700/P700+) for all the organisms
examined. Provided that the redox potential of the primary
electron acceptor A0 is common among the species, less favorable thermodynamics should exist in species that appeared
later due to a smaller Em(P700*/P700+) Em(A0/A0) difference resulting from a higher value of the former. It is possible,
however, that the Em(A0/A0) value is also tuned so as to keep
the Em(P700*/P700+) Em(A0/A0) difference nearly constant
for on efficient electron transfer from P700* to A0.
The Em(A0/A0) value has not been measured directly (for
reviews, see Brettel 1997, Moser et al. 2006). Thus, the free
energy change from P700* to A0 has only been estimated to
be 250 meV by Kleinherenbrink et al. (1994) by analysis of
delayed fluorescence emitted from membranes of a
PSII-depleted mutant of Synechocystis PCC 6803. A similar
value of 280 meV was estimated by an electroluminescence
measurement on spinach chloroplasts (Vos and van Gorkom
1988). Compared with the free energy difference between P700
and Cyt c6/Pc, the difference between P700* and A0 is larger
by >100 meV, and hence the species dependence of Em(P700/
P700+) or Em(P700*/P700+) may not be so crucial for
light-induced charge separation as the electron transfer at
the lumenal side. Further, subsequent electron transfer from
A0 to iron–sulfur clusters via phylloquinone A1 should prevent a reverse reaction, i.e. charge recombination from A0 to
P700+, and thus drive an efficient and rapid electron transfer from P700* to A0 (3 ps) and then to A1 (30 ps)
(Sétif and Leibl 2008). In any event, more comprehensive
studies on the energy level correlations among electron transfer cofactors within PSI are still needed to elucidate the
energetics.
Plant Cell Physiol. 52(5): 815–823 (2011) doi:10.1093/pcp/pcr034 ! The Author 2011.
Species dependence of P700 redox potential
Though electrostatic interaction and hydrogen bonding
provided by amino acid residues surrounding P700 are considered as factors tuning the Em(P700/P700+) value, it seems
to be difficult to pinpoint the possible factor(s) at the moment
because the amino acid residues are almost fully conserved.
Comparison of amino acid sequences allows us to confirm
that 32 of 39 amino acid residues within 10 Å distances from
the central Mg atoms of Chl a/a0 are conserved among the
organisms examined here. In particular, these amino acid residues are fully conserved among T. elongatus, Synechococcus
PCC6301, barley and spinach. The substitutions seem to
occur at random and no correlation can be found with species
dependence of the Em(P700/P700+) values. Therefore, the species dependence of Em(P700/P700+) is probably caused by the
difference in the three-dimensional structures of PsaA/B in the
species, which might confer small changes in arrangements of
both Chl a/a0 and surrounding amino acid residues. In any case,
high-resolution X-ray crystallographic data for other organisms,
though a higher plant X-ray structure is now available at 3.3 Å
resolution (Amunts et al. 2010), may allow us to explain how
the delicate changes in three-dimensional structure regulate
the Em(P700/P700+) value among organisms.
In conclusion, a significant species dependence of the
Em(P700/P700+) values was revealed for 10 species by spectroelectrochemistry in the present work. On the electron transfer
chain, the difference in the Em(P700/P700+) values seems to be
crucial for the electron transfer from the metalloproteins to PSI.
Much attention has so far been paid to such physiological
properties as charged states and the geometry of active sites
for the structurally different metalloproteins, relating the electron transfer to PSI (reviewed in De la Rosa et al. 2006, Hippler
and Drepper 2006, Diaz-Quintana et al. 2008), and kinetic
models have been proposed to account for the evolutionary
optimization of the reaction mechanism (De la Rosa et al. 2006,
Diaz-Quintana et al. 2008). The present finding, including the
literature survey of the redox potentials of the metalloproteins,
should contribute to a better understanding of the reaction
mechanism from thermodynamic points of view. For an exceptional species seen in Table 1, i.e. G. violaceus having the lowest
Em(P700/P700+) value and using both metalloproteins, determination of the values of Em(Cyt c6/Cyt c+6 ) and Em(Pc/Pc+)
values might be of much interest in future work.
Materials and Methods
Photosynthetic organisms
Gloeobacter violaceus PCC 7421 obtained from the SAG Culture
Collection (SAG 7.82), Synechococcus sp. PCC6301 from the
IAM Culture Collection (IAM M-6), T. elongatus strain BP-1
and Synechocystis sp. PCC6803 (kind gifts by Dr. Masahiko
Ikeuchi) were cultured in BG-11 medium (Allen 1968) for
5–10 d by aeration with air pumps under continuous light
from white fluorescent tubes. Spirulina platensis (IAM M-135)
was cultured in an enriched alkaline medium named AO
(Aiba and Ogawa 1977) by shaking under white illumination.
Cyanidium caldarium (IAM R-11) and Porphyridium purpureum
(IAM R-1) were cultured in Allen medium (Allen 1959) and in
an artificial seawater medium (Jones et al. 1963), respectively,
by aeration for 7 d. Chlamydomonas reinhardtii (IAM C-9) was
cultured in a Tris/acetate/phosphate medium (Gorman and
Levine 1965) at room temperature by shaking for 6 d under
continuous white illumination. Most organisms were cultured
at room temperature, except for T. elongatus (55 C), and
Synechocystis PCC 6803 and C. caldarium (37 C). Barley was
cultivated in a planter filled with vermiculite for 7 d, and spinach was obtained from local markets.
Isolation of PSI
PSI core complexes were isolated from cyanobacteria and
PSI-light-harvesting complex (LHC) I from a green alga and
higher plants as described previously (Nakamura et al. 2003).
PSI-LHC I from red algae was isolated according to a previous
report (Yoshida et al. 2003). Thylakoid membranes were isolated from cyanobacteria and algae after cell disruption with
glass beads, and from higher plants after homogenization.
Thylakoid membranes from cyanobacteria and algae were solubilized with dodecyl-b-D-maltoside. The detergent/Chl a weight
ratio was adjusted depending on the organisms: 10 for cyanobacteria, 12–15 for C. reinhardtii, 20 for C. caldarium and 37 for
P. purpureum. Thylakoid membranes from higher plants were
solubilized with Triton X-100 at a Triton X-100/Chl ratio of
14 : 1. PSI complexes were then purified from solubilized thylakoid membranes by overnight sucrose density gradient centrifugation. After recovering the PSI fraction, PSI complexes from
cyanobacteria and algae were further purified with
anion-exchange perfusion HPLC. For higher plants, the PSI fraction was employed without further purification. Finally, PSI
complexes were concentrated on a 100 kDa ultrafiltration
device, and were kept frozen until use.
Spectroelectrochemistry
Spectroelectrochemistry with an OTTLE cell was carried out as
described previously (Nakamura et al. 2004). The OTTLE cell
with an optical path length of about 180 mm is equipped with a
gold mesh (100 mesh per inch), a Pt black and an Ag–AgCl (in
saturated KCl) electrode as the working, counter and reference
electrode, respectively. The electrode potential in this work is
referred to a SHE (+199 mV vs. Ag–AgCl). PSI complexes were
suspended with 50 mM Tris–HCl (pH 8.0), 0.2 M KCl, 0.3%
dodecyl-b-D-maltoside at a concentration of 1–2 mM Chl a
corresponding to approximately 10 mM P700. The following
agents were employed as redox mediators; 10 mM N-methyl
phenazonium methosulfate (Em = +80 mV), 20 mM tetrachlorobenzoquinone (Em = +260 mV), 30 mM 1,10 -dimethyl ferrocene (Em = +337 mV), 30 mM ferrocene (Em = +418 mV) and
30 mM 1,10 -ferrocene dimethanol (Em = +476 mV).
Absorption spectra and absorbance change for samples
within the OTTLE cell were measured on a double beam
Plant Cell Physiol. 52(5): 815–823 (2011) doi:10.1093/pcp/pcr034 ! The Author 2011.
821
A. Nakamura et al.
spectrophotometer Model V560 (JASCO). The electrode potential was controlled with a potentio/galvanostat Model 2020
connected to a function generator Model FG-01 (Toho
Technical Research).
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Japan Society for the
Promotion of Science (JSPS) [Grant-in-Aid for Scientific
Research (C) (No. 19614003) and a Global Centers of
Excellence Program for ‘Chemistry Innovation through
Cooperation of Science and Engineering’].
Acknowledgments
We are grateful to the IAM Culture Collection (the University of
Tokyo; their cultures are now kept by the National Institute for
Environmental Studies) for donating cultures of cyanobacteria
and algae.
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