(Received for publication, August 22, 1995; and in revised form, October 24, 1995)
From the
We compare primary charge separation in a photosystem II
reaction center preparation isolated from a wild-type (WT) control
strain of the cyanobacterium Synechocystis sp. PCC 6803 and
from two site-directed mutants of Synechocystis in which
residue 130 of the D1 polypeptide has been changed from a glutamine to
either a glutamate (mutant D1-Gln130Glu), as in higher plant sequences,
or a leucine residue (mutant D1-Gln130Leu). The D1-130 residue is
thought to be close to the pheophytin electron acceptor. We show that,
when P680 is photoselectively excited, the primary radical pair state
P680Ph
is formed with a time
constant of 20-30 ps in the WT and both mutants; this time
constant is very similar to that observed in Pisum sativum (a
higher plant). We also show that a change in the residue at position
D1-130 causes a shift in the peak of the pheophytin
Q
-band. Nanosecond and picosecond transient
absorption measurements indicate that the quantum yield of radical pair
formation (
), associated with the 20-30-ps
component, is affected by the identity of the D1-130 residue. We
find that, for the isolated photosystem II reaction center particle,
>
>
>
. Furthermore, the spectroscopic and quantum
yield differences we observe between the WT Synechocystis and
higher plant photosystem II, seem to be reversed by mutating the
D1-130 ligand so that it is the same as in higher plants. This
result is consistent with the previously observed natural regulation of
quantum yield in Synechococcus PS II by particular changes in
the D1 polypeptide amino acid sequence (Clark, A. K., Hurry, V. M.,
Gustafsson, P. and Oquist, G.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11985-11989).
Photosystem II (PS II) ()is unique in that it is the
only complex of photosynthetic organisms that is able to catalyze the
oxidation of water. Upon light absorption, primary charge separation
results in the oxidation of P680, the primary electron donor of PS II,
and the reduction of pheophytin (Ph). It is the high oxidizing
potential of P680
that drives the secondary electron
donor-side reactions of tyrosine and manganese oxidation and that leads
ultimately to the splitting of water and release of molecular oxygen.
All oxygenic photosynthetic organisms contain PS II; these include
higher plants, algae, and cyanobacteria. The most commonly isolated
reaction center complex from PS II (the D1/D2 cytochrome b complex) binds six chlorophylls, two
pheophytins, two
-carotenes, and one cytochrome b
(1, 2) . Since this PS II
reaction center lacks the secondary acceptors, Q
and
Q
, and has little effective tyrosine Z
activity(3) , its photochemistry is limited to the formation of
the primary radical pair state P680
Ph
and charge recombination pathways from this state (for review,
see (4) ). The isolated reaction center is, however, an ideal
system for spectroscopic studies of PS II primary photochemistry since
the complications usually associated with energy transfer from antenna
complexes and secondary electron transfer processes are absent.
We
have determined that primary charge separation, leading to the radical
pair state P680Ph
, is largely
associated with a time constant of 21 ps (5, 6, 7) (
)and that it is
preceded by sub-ps equilibration of excitation energy between P680 and
some of the other reaction center chlorins(9) . We have also
observed slower energy transfer processes (of the order of tens of ps)
but have found that these can be avoided by photoselective excitation
of P680(10, 11) .
Other spectroscopic studies of the isolated higher plant PS II reaction center have characterized the charge recombination processes; these occur on a nanosecond timescale. Early transient absorption measurements indicated that the primary radical pair decays with a lifetime of the order of 36 ns(12, 13) . This lifetime was confirmed by time-resolved fluorescence measurements(14, 15, 16, 17) . More detailed analysis of time-resolved fluorescence data indicated that the kinetics of charge recombination of the primary radical pair are in fact multiexponential, exhibiting at least two lifetimes of 20 and 52 ns (18, 19) .
Site-directed mutagenesis has been found to be a useful tool in identifying residues of the protein matrix that affect primary and secondary electron transfer reactions in purple bacterial reaction centers. The majority of work involving site-directed mutagenesis of PS II has employed the cyanobacterium Synechocystis PCC 6803 (20) , although it is now also possible to make mutations in PS II of Chlamydomonas reinhardtii(21) .
Site-directed mutagenesis employing the cyanobacterium Synechocystis PCC 6803 has been applied to the study of secondary electron transfer reactions in PS II(20) . More recently this technique has also been applied to the identification of those residues important to the function and assembly of the oxygen-evolving complex (22, 23, 24) . However, to date, there have been no reports of the application of site-directed mutagenesis to the study of primary photochemistry in PS II. Although studies of primary photochemistry in PS II have been performed, these have been limited to reaction centers isolated from higher plants (see above) and, more recently, from the green alga C. reinhardtii ( (25) and see (26) for isolation procedure of PS II reaction centers from Chlamydomonas). Higher plants, however, are not readily amenable to genetic engineering. It is therefore highly desirable to study the primary photochemistry of PS II reaction centers isolated from organisms that are easy to manipulate genetically, in order to determine whether they are suitable model systems for the higher plant PS II.
Comparison of the primary structures of the Synechocystis D1 and D2 polypeptides with those of the higher
plant, Pisum sativum(27) indicates several
significant differences in primary structure between the two organisms.
Of particular relevance to this paper is the difference in the residue
at position 130 on the D1 polypeptide. In Synechocystis, the
D1-130 residue is a glutamine, while in P. sativum, and
all other higher plants studied, it is a glutamate; this residue is
thought to lie close to the putative binding site of the pheophytin
electron acceptor(28) . This particular residue is analogous to
residue 104 on the L subunit in Rhodobacter capsulatus. In a
study of these bacterial reaction centers, it was found that,
converting the L-104 residue from a glutamate to a glutamine or a
leucine resulted in a blue shift (of different amounts) in the
absorption maximum of the pheophytin Q-band and
also in slightly slower primary electron transfer than in the wild
type(29) .
In this paper we report the isolation of a PS II reaction center preparation from Synechocystis, using a different version of the isolation procedure described in (30) . Our main aim has been to produce a Synechocystis PS II particle that is active and stable and that contains sufficiently low levels of chlorophyll to allow photoselective excitation of P680. Photoselective excitation of P680 has been used in the transient absorption experiments to avoid those slow energy transfer steps that can confuse the interpretation of the kinetic data(10) . We present data comparing the primary photochemical properties of the PS II reaction center preparation isolated from a wild-type control strain of Synechocystis and from two different site-directed mutants of Synechocystis, one in which the D1-130 residue has been changed from a glutamine to a glutamate (mutant D1-Gln130Glu), so as to mimic the situation in higher plants, and another in which it has been changed to a leucine (mutant D1-Gln130Leu). This is the first report of such studies on PS II reaction center particles that have been subjected to protein engineering.
The D1-Gln130Glu mutant and the D1-Gln130Leu mutant were created using methods described in (23) . The mutations were verified by sequencing the psbA3 gene after amplification by the polymerase chain reaction(23) .
TC31 cells were grown (20-liter cultures) and
harvested as described in (30) . All subsequent steps were then
carried out in the dark. The concentrated TC31 cells were resuspended
in 20 mM Mes-NaOH (pH 6.35) and 25% (w/v) glycerol to give a
final volume of approximately 200 ml with 90-100 mg of
chlorophyll. The cells were then frozen in liquid N and
stored at -80 °C.
To isolate thylakoid membranes, the TC31
cells were initially washed, after thawing, with an equal volume of
``break'' buffer (20 mM Mes-NaOH (pH 6.35), 5 mM CaCl, 5 mM MgCl
, 25% (w/v)
glycerol, 1 mM
-amino caproic acid, and 1 mM benzamidine) and pelleted by centrifugation at 4 °C.
(
-amino caproic acid is a protease inhibitor). The pellet of
washed cells was resuspended in break buffer and then broken
immediately (without any incubation period) in a prechilled 360-ml
beadbeater chamber (Biospec Products), as described by Kirilovsky et al.(32) . After breaking the cells and removing the
cellular material and residual beads(32) , the thylakoid
membranes in the supernatant were pelleted by centrifugation at 45,000
rpm for 45 min, at 4 °C. The pellet was resuspended in a buffer
containing 50 mM Mes-NaOH (pH 6.5), 20 mM sodium
pyrophosphate, 1 mM
-amino caproic acid, and 1 mM benzamidine, and the membranes were pelleted again. This
additional wash step helped to remove the phycobiliproteins from the
membranes. The pelleted thylakoid membranes were resuspended in break
buffer to a final volume of approximately 90 ml, and homogenized with
approximately 5-8 passes at 4 °C. The membranes were frozen
in liquid N
and stored at -80 °C. A 20-liter
carboy typically yielded membranes containing 60-75 mg of
chlorophyll.
Washed thylakoid membranes, typically 60-75 mg of
chlorophyll, were subjected to three further washing steps, at very low
chlorophyll concentrations, to remove any residual phycobiliproteins.
For the first two washes, the membranes were diluted to a concentration
of approximately 0.1 mg/ml chlorophyll with 50 mM Mes-NaOH (pH
6.5), 1 mM -amino caproic acid, 1 mM benzamidine
(buffer A), containing 20 mM sodium pyrophosphate, and
centrifuged at 14,000 rpm for 40 min in a Beckman JA14 rotor
(
30,000
g) at 4 °C. The supernatant and any
soft pellet were discarded. For the third wash, the hard pellet, from
the second wash, was resuspended in buffer A only, to give a final
chlorophyll concentration of approximately 0.14 mg/ml. The diluted
membranes were centrifuged as for the previous washes, but for only 30
min, and the supernatant was again discarded. The hard pellet of washed
membranes was resuspended in as little buffer A as possible, and a
chlorophyll concentration determination was carried out according to
the method of Arnon(33) . The solution was then diluted with a
30% (w/v) stock of Triton X-100 and more buffer A, if necessary, to
give final concentrations of 0.45 mg/ml chlorophyll and 4.5% (w/v)
Triton X-100. This material was then homogenized, with 12 passes, at
room temperature and left to incubate in the dark, on ice and stirring
gently, for 2 h. It was then centrifuged at 45,000 rpm for 45 min in a
Kontron TFT 65.38 rotor (
145,000
g) at 4 °C.
The pellet of unsolubilized material was discarded, and the supernatant
containing the solubilized material was subjected to three anion
exchange chromatography columns for the separation of PS I and PS II
and the subsequent purification of PS II.
The supernatant of
solubilized material was applied to a column (16 200 mm) of
Fractogel TSK DEAE-650(S) (Merck-BDH) maintained at 4 °C and
equilibrated with buffer A containing 0.2% (w/v) Triton X-100 (Triton
buffer). The packed column length was approximately 150 mm. The sample
was loaded at 2.0-2.5 ml min
and then washed
overnight with 550-650 ml of Triton buffer containing 50 mM NaCl, at a flow rate of approximately 0.5 ml
min
. At this concentration of 50 mM NaCl,
some of the PS I contained within the sample was eluted from the
column. Following this overnight wash, the absorbance at 450 nm of the
eluant was reduced to a level where the absorbance was approximately
0.25-0.35 optical density units and no longer changed very much.
The column was then washed with 200-250 ml of Triton buffer
containing 75 mM NaCl, at a flow rate of approximately
2.5-2.8 ml min
. Again, the absorbance at 450
nm of the eluant was monitored, and washing was continued until the
absorbance was approximately 0.20-0.30 optical density units and
again no longer changed very much. A NaCl gradient in Triton buffer
(75-300 mM) was then applied at 2 mM NaCl
ml
and a flow rate of 0.5 ml min
.
Absorption spectra of most fractions were recorded, and those eluted at
about 110-120 mM NaCl were pooled, subject to the
following criteria: (i) that the position of the peak of the Q
absorption band be in the range from approximately 674 to 675.3
nm (this can be somewhat variable), but what is more critical is that
the position of the Q
absorption peak of the fractions
either side of the required fractions are redder; (ii) that there be an
observable absorption peak at 542 nm for the pheophytin Q
band; and (iii) that the ratio of the Soret peaks (435 nm/420 nm
(approximate peak positions)) be less than or equal to 1.1. Fractions
satisfying these criteria were pooled, the absorption spectrum of the
pooled material was recorded, and the chlorophyll a to
cytochrome b
ratio estimated. The pooled
fractions after the first column typically contained 16-20
chlorophyll a/cytochrome b
and a total
of 240-270 µg of chlorophyll. It should be noted that, when
determining the chlorophyll a to cytochrome b
ratio of the pooled fractions after this and the second columns,
care was taken not to overestimate the oxidized minus reduced
cytochrome b
change due to a contribution to the
signal by cytochrome c
(34) . This
contaminating cytochrome c
was usually removed
completely after the second column. Its loss could be followed clearly
by monitoring the absorption spectrum of the eluant; cytochrome c
has a large absorption in the Soret region
(data not shown).
All of the pooled material from the first column
was diluted 4-fold with Triton buffer and applied to a second column
(10 98 mm), packed, and equilibrated with Triton buffer, as
described for the first column. The packed column length was
approximately 5 cm. The sample was then washed with 60-80 ml of
Triton buffer containing 75 mM NaCl, at a flow rate of
approximately 1.4 ml min
, in repeated cycles of
washing with 20 ml and stopping the flow for 5 min. The purpose of this
wash was to remove chlorophyll and any residual cytochrome c
. The absorption spectrum of the eluant was,
therefore, monitored periodically to ensure that this was the case and
that reaction center particles were not also being washed off (the
latter would occur with prolonged washing of the column). The sample
was then exchanged from Triton X-100 to n-dodecyl
-D-maltoside (maltoside) by washing with approximately 15
ml of buffer A containing 2 mM maltoside (maltoside buffer)
and containing 75 mM NaCl. PS II particles with reduced
chlorophyll content were then eluted in a single salt step with
maltoside buffer containing 200 mM NaCl and at a flow rate of
approximately 0.6 ml min
. The eluted material after
the second column typically contained 9-10 chlorophyll a/cytochrome b
and a total of
90-120 µg of chlorophyll. This material was frozen in liquid
N
and stored at -80 °C overnight.
A third
column was prepared as for the second column. All of the PS II
particles from the second column were slowly thawed on ice and then
diluted 4-fold with Triton buffer. They were applied to a third column
and then washed, exchanged from Triton X-100 to maltoside, and eluted
as described above for column two. The eluted material after the third
column typically contained 7-8 chlorophyll a/cytochrome b and a total of 40-50 µg of
chlorophyll. This material was frozen in liquid N
and
stored at -80 °C. It was subsequently thawed slowly on ice
for determination of pigment and cofactor composition and spectroscopic
characterization.
Higher plant PS II reaction center particles were isolated from P. sativum as described previously(35) .
Quantitative analyses of the individual pigments were carried out by
high performance liquid chromatography (HPLC). The samples were
protected from light at all stages of the procedure. The pigments were
extracted from the proteins dissolved in buffer by addition of 9
volumes of 100% HPLC grade cold acetone (0.9 ml of acetone in 0.1-ml
sample). After stirring and centrifugation for 5 min at 10,000 rpm in a
bench centrifuge, at 4 °C, the green supernatant was filtered
through a 0.2-µm fluoropolymer filter (ACRO(TM) LC13). 20-µl
aliquots (equivalent to 200-300 ng of chlorophyll) were injected
in the chromatograph for analysis on a reverse phase HPLC column
(Spherisorb S5ODS1; 5 mm, 250 4.6 cm inner diameter). An
isocratic mobile phase of methanol/ethyl acetate (68:32, v:v) with a
flow rate of 1.0 ml min
was used for separation. The
peaks were detected with a UV-visible Kontron spectrophotometer at 450
nm for
-carotene and xanthophylls, at 663 nm for chlorophyll a and pheophytin a, at 645 nm for chlorophyll b,
and at 225 nm for plastoquinone-9. The extinction coefficients for
chlorophyll a, pheophytin a, and
-carotene were
calculated from the absorption spectra of the pigments in a
methanol/ethyl acetate (68:32 (v:v)) mixture, i.e. the
isocratic mobile phase used in the HPLC analysis, and by comparison
with the known extinction coefficients of these pigments in methanol or
80% acetone(36) . The extinction coefficients calculated were,
in units of mM
cm
, as
follows: 86.9 for chlorophyll a at 663 nm; 49.3 for pheophytin a at 663 nm; 135.0 for
-carotene at 450 nm. These values
are only slightly different to those in (36) . The pigment
stoichiometry given in Table 1, as determined by HPLC, also
assumes that there are 2.0 pheophytin a/PS II reaction center
particle.
Chlorophyll a to cytochrome b ratios were measured by determining the concentration of
cytochrome b
from the reduced (dithionite) minus
oxidized (ferricyanide) absorption difference spectrum obtained for the
sample resuspended in buffer A; this difference spectrum peaked at 559
nm for the Synechocystis PS II preparations. An extinction
coefficient of 23.4 mM
cm
(37) was used for the concentration of cytochrome b
at 559 nm. The chlorophyll a concentration of the sample was calculated from the reaction
center concentration, assuming a chlorophyll a to pheophytin a ratio of 7.9:2.0 (taken from the HPLC data, Table 1).
The reaction center concentration was calculated from the absorbance at
663 nm for the 80% acetone extracted reaction center particle and using
an extinction coefficient calculated by assuming 7.9 chlorophyll a and 2.0 pheophytin a/reaction center (from the HPLC data)
and applying Lichtenthaler's (36) extinction coefficients
for these pigments in 80% acetone. On this basis, the extinction
coefficient for an acetone-extracted reaction center particle
containing 7.9 chlorophyll a and 2.0 pheophytin a, is
780.4 mM
cm
. It should
be noted that, for these samples, the absorbance at the Q
absorption peak for the aqueous suspension was found to be the
same as the absorbance at 663 nm for the 80% acetone extracted sample.
Femtosecond transient
absorption measurements were carried out as described
previously(5, 10) . The time resolution of the
spectrometer was approximately 250 fs for the experiments reported
here. The excitation pulses were centered at 694 nm for selective
excitation of P680(10) . Transient absorption data were
collected using a multichannel detector at 150 time delays between 0
and 60 ps and were globally analyzed as described in (10) . The
experiments were repeated several times on different samples in order
to assess the reproducibility of the data and to establish the
precision of the results. Precision in the determination of the
measured lifetimes is quoted as ± one standard error. Data were
collected with the polarization of the probe beam rotated by 54.7°
relative to the pump. This ``magic angle'' configuration
avoids any contributions to the data from depolarization processes. The Synechocystis PS II reaction center particles used in these
experiments were those that were eluted directly from the third
chromatographic column, without further dilution apart from the
addition of the oxygen trap, 5 mM glucose, 0.1 mg
ml glucose oxidase, and 0.5 mg ml
catalase(16) , and contained typically 30-35 µg
of chlorophyll (in a volume of approximately 0.75 ml). The samples were
rotated in a circular cuvette at 60 Hz and maintained at 10 °C.
Nanosecond transient absorption measurements were carried out as
described previously(18) , with the following modifications.
Output pulses from a dye laser, pumped by a nitrogen laser, were used
as the excitation source; the pulses were 500 ps in duration, at a
repetition rate of 2 Hz, and they were passed down an optical fiber
with a diameter of 5 mm. The samples were excited either at 630 nm
(pulse energy 44 µJ/cm) or 680 nm (pulse energy 18
µJ/cm
). Under these excitation conditions, a 50%
reduction in excitation power resulted in a 50% reduction in the
transient signal, i.e. the excitation pulses were
nonsaturating. The probe wavelength was at 820 nm, and the optical
pathlength was 4 mm. All samples (WT and mutant Synechocystis PS II reaction center particles or higher plant PS II reaction
centers, used as control samples) were resuspended in buffer A,
containing 2 mM maltoside and 200 mM NaCl, which had
been made anaerobic by the addition of the oxygen trap (see above). The
samples contained approximately 2 µg of chlorophyll (in a volume of
0.25 ml), and they all had the same optical density at the excitation
wavelength being used. The samples were maintained at room temperature
during the nanosecond absorption experiments. The data analyzed were an
average of 100 flashes. The absorption decays were fit to three
exponentials. In these fits, one lifetime was fixed (at 4.7 µs) to
represent the electronically filtered decay of triplet P680 in the PS
II reaction centers (see (18) ), while all other parameters
were free running.
For the transient absorption experiments, the
position of the chlorophyll Q band absorption maximum was
used as a monitor of the stability of the sample; this peak shifted by
less than 1 nm during the course of the experiments, suggesting an
activity loss of less than 10%(18) .
Fig. 1shows the room temperature steady state
absorption spectrum for the control WT Synechocystis PS II
reaction center preparation compared with that for the higher plant, P. sativum, PS II reaction center. The room temperature steady
state absorption spectrum for the PS II preparation isolated from the
D1-Gln130Glu and D1-Gln130Leu mutants is similar to that for WT Synechocystis (data not shown). The Q band of the
WT Synechocystis PS II reaction center particle peaks at
675.0-675.3 nm, which is slightly to the blue of the Q
band maximum of the higher plant PS II reaction center, which
peaks at 675.5-676.0 nm. In the Soret region, the short
wavelength peak at approximately 417 nm predominates, as it does in
higher plant PS II reaction centers; however, differences can be seen
at approximately 434 nm. Another notable difference is the magnitude of
the absorbance in the carotenoid region at approximately 483-489
nm; it is nearly a factor of 2 lower in the WT Synechocystis preparation than in the higher plant one, indicative of reduced
carotenoid content (confirmed by HPLC data; see Table 1).
Figure 1: Steady state absorption spectrum of the WT Synechocystis PS II reaction center particle compared with that for the higher plant, P. sativum, PS II reaction center. Samples were resuspended in buffer A, containing 2 mM maltoside and 200 mM NaCl.
The
pigment and cofactor composition of the WT Synechocystis PS II
reaction center preparation was determined by combining the results
from three different types of analyses, the results of which are given
in Table 1. It contains approximately seven to eight chlorophyll a, two pheophytin a, one cytochrome b, and one
-carotene; no quinone was
detected. This pigment and cofactor composition is similar to that for
the higher plant PS II reaction center(2) , apart from the
presence of one to two extra chlorophyll and the carotenoid content,
which is reduced by half. The Synechocystis PS II preparation
contains no PS I, as confirmed by the lack of the 725 nm fluorescence
peak, originating from PS I, in steady state fluorescence measurements
at 77 K (data not shown). It does, however, contain some extra
polypeptides, over and above those observed for the higher plant PS II
reaction center (data not shown), and as also observed by Oren-Shamir et al.(38) for another isolated Synechocystis PS II reaction center preparation. These extra polypeptides may be
responsible for the extra one to two chlorophylls found in this
preparation compared with higher plant reaction centers. Despite this
contamination, it is still possible to photoselectively excite P680,
which is a crucial requirement for examining radical pair formation. We
have carried out femtosecond transient absorption experiments on higher
plant PS II reaction centers, which contain 5, 6, 7, or 8 chorophylls
and have shown that, with photoselective excitation of P680, the
kinetics for primary charge separation are not affected greatly by the
presence of these extra chlorophylls. (
)The pigment and
cofactor composition of the PS II preparations isolated from the
D1-Gln130Glu and D1-Gln130Leu mutants was analyzed by the reduced minus
oxidized cytochrome b
difference spectrum and
pheophytinization. The composition of these PS II Synechocystis mutant preparations was similar to that reported for the WT Synechocystis.
Fig. 2compares transient absorption
data collected in the region of the pheophytin Q absorption
band for the WT Synechocystis PS II reaction center particle
and for the higher plant, P. sativum, PS II reaction center.
The pheophytin Q
band was chosen as it can be most easily
assigned (10, 11) . Data were collected using pulses
centered at 694 nm that achieved photoselective excitation of
P680(12, 13) . Global analyses of these data resolved
two components, an exponential decay component and a component that did
not decay on the detection timescale (0-60 ps). The exponential
component had a lifetime of 22 ± 3 ps for the WT Synechocystis PS II reaction center particle and 21.5 ±
1 ps for the higher plant PS II reaction center. (The error margins
quoted here are one standard error and result from approximately 20
analyses of independent data sets; it can therefore be concluded that
the lifetimes of these two components are indistinguishable.) Spectra
of the amplitudes of these components are shown in Fig. 2.
Figure 2:
Spectra of the amplitudes of two kinetic
components resolved following excitation of PS II reaction center
preparation particles isolated from WT Synechocystis (a) and PS II reaction centers isolated from the higher
plant P. sativum (b). The solid lines are
spectra of a component that did not decay on the 0-60-ps
timescale used for the experiment; this component is assigned to the
radical pair state P680Ph
. The dotted lines are the spectra of a component with a lifetime of
22 ± 3 ps (WT Synechocystis reaction center
preparation) and 21.5 ± 1 ps (higher plant, P. sativum,
reaction centers); this component is assigned to the formation of the
radical pair state.
For the higher plant, P. sativum, the minimum at 544 nm in
the spectrum of the nondecaying component, which has a lifetime much
longer than 60 ps (Fig. 2b, solid line),
results from bleaching of the Q band of the photoactive
pheophytin associated with formation of the radical pair state
P680
Ph
(for a full discussion of the
assignment of these spectra see Refs. 5, 6, 9, 10, 25). As mentioned
earlier, the decay of this radical pair state occurs primarily on the
nanosecond time scale(18) . The maximum in the spectrum of the
higher plant 21.5 ps component (Fig. 2b, dotted
line), also at 544 nm, allows us to identify this component with
the production of reduced pheophytin. The amplitude of this feature
indicates that 40-50% of the final pheophytin Q
bleach grows in with the 21.5 ps component, indicating that a
lower limit of 40% of the total pheophytin reduction occurs with a
21.5-ps time constant.
The spectral features observed in the WT Synechocystis data (Fig. 2a) show similarities
with those observed for higher plant reaction centers of P.
sativum. As in the higher plant data, the spectrum of the
nondecaying component (which has a lifetime much longer than 60 ps) (Fig. 2a, solid line) shows a bleach of a
pheophytin Q absorption band. The spectrum of the 22 ps
component (Fig. 2a, dotted line) exhibits a
maximum coincident with this bleach, thereby allowing us to associate
this component with the formation of reduced pheophytin. The amplitude
of this feature indicates that 45-65% of the final pheophytin
Q
bleach grows in with the 22 ps component. Thus the 22 ps
component resolved in the WT Synechocystis data not only has
the same lifetime, within error, as the 21.5 ps component resolved in
the higher plant data, but it also produces a similar increase in the
bleach of the pheophytin Q
absorption band. We therefore
conclude that the 22 ps component resolved in the WT Synechocystis data should be assigned to formation of the radical pair state
P680
Ph
.
There are, however, some
clear differences between the spectra obtained for the higher plant, P. sativum, and WT Synechocystis PS II reaction
center particles (see Fig. 2). It is of particular interest to
note that the bleaching of the pheophytin Q absorption band
is blue-shifted by 2-3 nm in the WT Synechocystis data.
After taking account of background absorption changes, we estimate from
these data that this absorption band has a maximum at 541.5 ±
0.5 nm for WT Synechocystis, in contrast to the 544 ±
0.5 nm maximum observed for higher plants. A similar blue shift of the
WT Synechocystis pheophytin Q
band has also been
observed in steady state pheophytin anion photoaccummulation
experiments(39) . Other differences in the spectrum of the
nondecaying component spectrum for the WT Synechocystis PS II
preparation, when compared with that of higher plants, are discussed
below.
Fig. 3compares the absorption difference spectra of
the nondecaying component, following photoselective excitation of P680,
for higher plant, P. sativum, PS II reaction centers and for
WT and mutant Synechocystis PS II reaction center particles,
the Synechocystis mutants being D1-Gln130Glu and D1-Gln130Leu.
The most striking effect is that the spectrum obtained for the
D1-Gln130Glu mutant is very similar to that of higher plants. The
pheophytin Q absorption band has a maximum at 544 ±
0.5 nm for the D1-Gln130Glu mutant and at 540 ± 0.5 nm for the
D1-Gln130Leu mutant. Similar shifts in the maximum of the pheophytin
Q
absorption band, compared with higher plants, have also
been observed in steady state pheophytin photoaccumulation studies of
these particular mutants(39) . The spectrum of the nondecaying
component observed for the D1-Gln130Glu mutant is not positively
shifted as is observed for WT Synechocystis PS II, but is more
like that observed for higher plant PS II. The spectrum for the
D1-Gln130Leu mutant is, however, even more positively shifted than that
for WT Synechocystis PS II.
Figure 3:
Comparison of the spectrum of the
nondecaying component for PS II reaction centers isolated from higher
plant (P. sativum), WT Synechocystis, and the
D1-Gln130Glu and D1-Gln130Leu Synechocystis mutants, following
photoselective excitation of P680. The peak of the pheophytin
Q band is given for each spectrum. Radical pair
spectrum for wild-type Synechocystis, -;
D1-Gln130Glu mutant, - - - -; D1-Gln130Leu
mutant, -
-
-; P.
sativum,
.
Fig. 4compares the
decay kinetics of the WT and mutant Synechocystis PS II
reaction center preparations at the peak of the pheophytin Q absorption band, following photoselective excitation of P680. The
variability in signal to noise on the data is purely a function of the
apparatus signal to noise and the concentration of sample used for the
experiment. The kinetics in Fig. 4show that the lifetime most
strongly associated with primary charge separation is similar in the
higher plant, P. sativum, and in the WT Synechocystis and the D1-130 mutants of Synechocystis and is of
the order of 20-30 ps. Similar kinetics were also obtained from
data collected in the Q
spectral region. The final
absorption change associated with primary charge separation, in Fig. 4, is, however, variable and is a function of the residue
at position D1-130. When the residue at position D1-130 is
the same as in higher plants (i.e. the D1-Gln130Glu mutant),
the final absorption change at 60 ps is similar to that of higher
plants, i.e. it is negative.
Figure 4:
Comparison of the decay kinetics at the
peak of the pheophytin Q absorption band,
following photoselective excitation of P680 in PS II reaction centers
isolated from higher plants (P. sativum), WT Synechocystis, and the D1-Gln130Glu and D1-Gln130Leu Synechocystis mutants. The probe wavelength used and the
lifetime of the kinetic component associated with the formation of the
radical pair state P680
Ph
are given
for each of the decay kinetics. The lifetimes shown were determined
from global analyses of multiple data sets collected at 128 wavelengths
between 500 and 600 nm. For the D1-Gln130Leu mutant, an additional
faster (approximately 3 ps) component can also be
resolved.
Nanosecond transient
absorption measurements at 820 nm were carried out to obtain
information regarding the quantum yield of radical pair formation
() in the isolated WT and mutant Synechocystis PS II reaction center preparations, compared with the isolated
higher plant, P. sativum, PS II. These nanosecond kinetics are
shown in Fig. 5. Measurements at 820 nm monitor the presence of
P680
Ph
and triplet P680;
contributions from unbound chlorophyll singlet excited states are
relatively small (see ``Discussion''). Analysis of the decay
kinetics in Fig. 5resulted in the following lifetimes, with
absolute amplitudes (
10
), being given in
parentheses: P. sativum, 48 ns (3.26) and 4.8 ns (3.43); WT Synechocystis, 36 ns (1.07) and 4.8 ns (2.84); D1-Gln130Glu
mutant, 45 ns (1.70) and 4.9 ns (2.57); D1-Gln130Leu mutant, 44 ns
(0.55) and 6.4 ns (2.39). (The amplitude of the 4.7-µs component,
due to an electronic filter, was of the order of 0.23
10
for all samples). The 20 ns component resolved in (18) is not resolved in the data shown in Fig. 5, due
to poorer signal to noise, but its amplitude will contribute to both
the nanosecond components that are resolved in the data of Fig. 5. These nanosecond components represent charge
recombination of the primary radical pair(18) , although the
faster nanosecond component may also contain a contribution from
chlorin singlet excited states formed in active reaction centers. The
amplitudes of the nanosecond components can, therefore, be taken to be
a measure of the quantum yield of radical pair formation
(
) in the sample. Since the optical densities of the
samples at the excitation wavelength used were identical and the
excitation pulses were nonsaturating, it is valid to compare the
amplitude of the nanosecond components in the different samples
directly. Table 2estimates the
from the
nanosecond transient absorption data (i) by comparing the total
amplitude of the decay kinetics in Fig. 5and (ii) by comparing
the relative amplitudes of just the long-lived nanosecond component. In
both cases the amplitudes are normalized to 100% for
in isolated higher plant PS II reaction centers. As can be seen
in Table 2, the relative quantum yield of radical pairs, at 1 ns,
is in the following order: higher plant > D1-Gln130Glu mutant >
WT > D1-Gln130Leu mutant. This order is, in fact, apparent by
inspection of the nanosecond data presented in Fig. 5. Similar
results (i.e. lifetimes and amplitudes) were obtained for the
nanosecond data when either 630 nm or 680 nm excitation wavelengths
were used.
Figure 5: Comparison of the nanosecond decay kinetics, monitored at 820 nm and following excitation at 680 nm, in PS II reaction centers isolated from higher plants (P. sativum), WT Synechocystis, and the D1-Gln130Glu and D1-Gln130Leu Synechocystis mutants.
Time-resolved fluorescence studies of the WT Synechocystis PS II reaction center preparation were used to assay the level of uncoupled chlorophyll present in the samples. We obtained similar time-resolved fluorescence data for both WT Synechocystis and higher plant, P. sativum, PS II reaction centers (data not shown). As discussed in (17) and (18) (in which the higher plant data is interpreted), this indicates that in the WT Synechocystis PS II reaction center preparation, up to 94% of the chlorophyll is energetically coupled to active PS II reaction centers, assuming the equilibration kinetics between the radical pair state and the antenna are similar for Synechocystis and higher plants. Therefore, we can establish that the additional one to two chlorophylls present in the Synechocystis preparation are functionally connected to active reaction centers (also confirmed by the nanosecond data described above; see ``Discussion'').
In this work we have compared the primary photochemistry of a PS II reaction center preparation isolated from the WT cyanobacterium Synechocystis PCC 6803 and from two different site-directed mutants of the D1-130 residue in Synechocystis, one in which this residue has been changed from a glutamine to a glutamate (the D1-Gln130Glu mutant), as is the case for higher plants, and another in which it has been changed to a leucine (the D1-Gln130Leu mutant).
In order for the ultrafast transient absorption measurements to be interpreted in a straightforward manner, it is necessary to photoselectively excite P680. We have shown that this can be done with PS II reaction centers isolated from the higher plant, P. sativum(8, 9) and that if P680 is not photoselectively excited, then slow energy transfer processes interfere with the kinetics observed(10) . A criterion, therefore, that had to be satisfied in this study, was that the PS II preparation isolated from the transformable cyanobacterium Synechocystis had to have its chlorophyll content reduced sufficiently to allow photoselective excitation of P680, while retaining maximum stability and activity.
The procedure we have used to isolate a Synechocystis PS II reaction center preparation employed only a single detergent treatment for solubilization of the membranes rather than two, as published previously(30) . Further changes to the Gounaris et al.(30) preparation included an extra chromatographic column, to remove further chlorophylls, and exchange of the sample on the second and third columns from Triton X-100 to maltoside, for stabilization of the sample (40) .
The Synechocystis PS II reaction center preparation we have
isolated from WT and the D1-130 mutants has a pigment and
cofactor composition similar to that reported for higher plant PS II
reaction centers(2) , except for the presence of some extra
chlorophyll and the loss of one -carotene. The chlorophyll content
of this preparation has therefore been reduced sufficiently to allow
photoselective excitation of P680 and to allow the ultrafast absorption
data to be interpreted straightforwardly. The composition of our Synechocystis PS II preparation is similar to that reported
recently for another isolated Synechocystis PS II reaction
center preparation(38) . It is, however, different to the Synechocystis PS II particle of Gounaris et al.(30) ; this particular particle was reported to contain
only one pheophytin a/eight chlorophyll a, one
cytochrome b
, and 0.75
-carotene.
The stability of our Synechocystis PS II reaction center preparation, during the course of these experiments, was the same as the P. sativum PS II reaction center (see ``Materials and Methods'').
We have shown, in our ultrafast transient
absorption studies, that formation of the primary radical pair state
P680Ph
occurs mainly with a time
constant of 20-30 ps (see Fig. 4) in the WT and both
D1-130 mutants of Synechocystis; this time constant is
very similar to that observed in the higher plant, P. sativum.
The residue at D1-130, however, does affect the wavelength of
the active branch pheophytin Q transition (Fig. 3).
The peaks are at 541.5 ± 0.5 nm for WT Synechocystis,
at 544 ± 0.5 nm for the D1-Gln130Glu mutant, and at 540 ±
0.5 nm for the D1-Gln130Leu mutant. These peaks are to be compared with
the 544 ± 0.5 nm peak observed in the higher plant, P.
sativum. Hence, only when the residue at position D1-130 is
the same as in higher plants (i.e. the D1-Gln130Glu mutant) is
the peak of the functional pheophytin Q
absorption band
located at the same wavelength as that of higher plants. Shifts in the
peak of the pheophytin Q
absorption band, when comparing WT
and mutant Synechocystis and higher plants, have also been
observed in steady state pheophytin anion photoaccumulation
experiments(39) . Recent Fourier transform infrared data
indicate that the residue at position D1-130 probably lies close
to the binding site of the pheophytin electron acceptor, (
)as predicted from sequence similarities to the bacterial
reaction center(34) .
In studies of R. capsulatus mutants(29) , a change in the residue at position L-104
(equivalent to the higher plant position D1-130) was also
reported to cause a shift in the position of the pheophytin Q absorption peak (see the Introduction). Furthermore, it was
observed (29) that the primary electron transfer event in the
glutamine mutant was only slightly slower than in the wild type (4.6
± 0.7 ps compared with 3.4 ± 0.5 ps, respectively). These
observations of Bylina et al.(29) using purple
bacterial mutants, therefore, show some similarity to those reported
here.
Also observed in our ultrafast absorption data, are differences in the overall absorption change associated with the spectrum of the nondecaying component (Fig. 3). The difference in the spectrum of the nondecaying component observed for WT Synechocystis, when comparing it with that for the higher plant, P. sativum, can be reversed by changing the D1-130 residue (to a glutamate) so that it is the same as in higher plants.
This variability in overall absorption change
associated with the nondecaying component, for the isolated WT and
mutant Synechocystis PS II preparations, may be related to
their quantum yield for radical pair formation ().
This quantum yield has been estimated (a) from the nanosecond transient
absorption data in two different ways and (b) by simulation of the
spectrum of the nondecaying component resolved in the ultrafast data (Fig. 3). The results are presented in Table 2.
It is interesting to note that Oren-Shamir et
al.(38) observe that the quantum yield for the
photoaccumulation of Ph, in their WT Synechocystis PS II reaction center preparation, is about half
of that activity observed in PS II reaction centers of higher plants.
At first sight, it seems unlikely that the reduced quantum yield that
we have observed in wild-type Synechocystis PS II reaction
centers reflects the situation in vivo. It is widely believed
that a reaction center should be optimized for maximum quantum yield,
but this expectation is somewhat naive. The cyanobacterium Synechococcus sp. PCC 7942 has two forms of PS II that differ
by 25 amino acids in the D1 polypeptide(27) . One form is
expressed under low light and one under high light, and it has been
shown that the high light form is less easily photoinhibited than the
low light form(8) . Moreover, it was also demonstrated that the
high light form of PS II exhibits a 25% higher quantum yield than the
low light form(8) . What makes this observation particularly
relevant is that one of the amino acids that changes between the two
forms of Synechococcus PS II reaction center is in fact the
D1-130 residue; in the low light form it is a glutamine, while in
the high light form it is a glutamate. We have shown here that the
mutation D1-Gln130Glu increases the quantum yield in the isolated Synechocystis PS II reaction centers by approximately
20-30%. It is therefore tempting to conclude that the quantum
yield in Synchococcus is controlled largely by the
D1-130 residue and that the PS II reaction center isolated from
wild type Synechocystis probably does show a reduced quantum
yield even in vivo.
In conclusion, we have shown that the
time constant observed for the formation of the radical pair state
P680Ph
is similar for WT Synechocystis, for the D1-130 Synechocystis mutants, and for the higher plant, P. sativum. However,
while the rate of radical pair formation is relatively insensitive to
the residue at position D1-130, a mutation at this site can
result in a shift in the peak of the Q
band of the acceptor
pheophytin and in a change in the quantum yield for the formation of
P680
Ph
. It is remarkable that the
quantum yield of radical pair formation in isolated Synechocystis PS II reaction centers can be increased by mutating the
D1-130 residue from a glutamine to a glutamate, in a manner
similar to that found naturally in Synechococcus.