From the
In oxygenic photosynthesis, photosystem II is the
chlorophyll-containing reaction center that carries out the
light-induced transfer of electrons from water to plastoquinone.
Fourier transform infrared spectroscopy can be used to obtain
information about the structural changes that accompany electron
transfer in photosystem II. The vibrational difference spectrum
associated with the reduction of photosystem II acceptor quinones is of
interest. Previously, a high concentration of the photosystem II donor,
hydroxylamine, has been used to obtain a spectrum attributed to
Q
PSII
Although there is no crystal structure
of PSII, there is an atomic level structure of a related protein, the
reaction center from purple, non-sulfur bacteria
(3) .
Comparison of the acceptor side of PSII to the bacterial reaction
center reveals both sequence and functional homology
(3) .
However, the bacterial reaction center does not oxidize water and,
thus, does not contain redox-active tyrosines or a manganese cluster.
Other electron transfer reactions occur in PSII that are not
described above and do not occur in the bacterial reaction center. For
instance, redox-active tyrosine D can be oxidized by
P
Difference FTIR can be used to obtain dynamic
structural information about PSII. Such ``reaction-induced''
infrared spectroscopy allows the study of the structural changes in
cofactors and protein components that accompany function. In
application to PSII, light-minus-dark difference spectra provide
``dynamic'' structural information concerning light-induced
electron transfer events. For example, previous studies have identified
vibrational modes associated with the oxidation of the two tyrosine
radicals, D
The contributions of the acceptor side of PSII to the
light-minus-dark infrared spectrum are certainly of interest. A high
concentration of hydroxylamine is sometimes used as a donor in steady
state illumination experiments in order to accumulate
Q
High concentrations of hydroxylamine have been
used previously in difference (light-minus-dark) FTIR spectroscopy in
an attempt to identify structural changes on the acceptor side of PSII
(24) . PSII membranes were used in this work
(25) . These
membranes were treated with the inhibitor, DCMU, to block electron
transfer from Q
However, some FTIR studies of PSII are not consistent with these
previous assignments to Q
Therefore, we have reinvestigated
the redox state of PSII reaction centers in the presence of
hydroxylamine. We have used EPR spectroscopy as a control for our
difference infrared studies. Samples were prepared in the same way for
both EPR and FTIR experiments, that is, dehydrated on solid substrates.
Our work shows that steady state illumination in the presence of
hydroxylamine results in the reversible production of a chlorophyll
cation radical in approximately 5% of the centers. We present evidence
that this level of chlorophyll contribution to the difference infrared
spectrum is detectable. Further, we show that global labeling of
photosystem II with
The membranes were manganese-depleted
by the addition of hydroxylamine from a stock that contained 50 m
M hydroxylamine, 1 m
M EDTA, 50 m
M MES-NaOH, pH
6.0, 15 m
M NaCl, and 25% glycerol. This stock was made up
immediately before use. The final concentration of hydroxylamine in the
membrane sample was 10 m
M. After addition of hydroxylamine,
the membranes were agitated in darkness for 80 min at 0 °C. The
membranes were diluted 1:4 by the addition of a buffer containing 50
m
M MES-NaOH, pH 6.0, 15 m
M NaCl, and 25% glycerol.
After hydroxylamine treatment, the membranes were pelleted by
centrifugation for 30 min at 19,000 rpm in a SS-34 rotor. The pellets
were resuspended in a buffer that contained 5 m
M MES-NaOH, pH
6.0. The membranes were pelleted again as described above and
resuspended in 5 m
M MES-NaOH, pH 6.0. The final chlorophyll
concentration was 4.0 mg/ml.
PSII complexes were prepared,
manganese-depleted, and concentrated as in
(11, 31) . As
purified, the PSII complexes had a specific activity of 1100-1200
µmol of O
Cyanobacterial PSII
particles from Synechocystis sp. PCC 6803 were prepared as
previously described
(35) and had a specific activity of
2300-3000 µmol of O
Immediately before EPR and FTIR
spectroscopy, hydroxylamine was added to either the PSII membranes or
PSII complexes to a final concentration of 10 m
M. For the
cyanobacterial PSII complexes, the final hydroxylamine concentration
was 6 m
M. In some cases, potassium ferricyanide also was added
to a final concentration of 3 m
M.
Oxygen-evolving PSII
complexes used for cryogenic FTIR were prepared as previously described
(11) and were in 25% glycerol, 50 m
M MES (pH 6.0), 7.5
m
M CaCl
PSI was purified from the
cyanobacterium, Synechocystis sp. PCC 6803, by the procedure
described in Noren et al. (35) .
Spin quantitation was performed at
-160 °C on samples that had been illuminated at -9
°C and then frozen in darkness. The amount of the light-induced
radical was quantitated by double integration of the EPR signal using
the program, IGOR (Wavemetrics, Lake Oswego, OR). Fremy's salt
was used as a spin standard
(38, 39) .
The g value was determined by using the chlorophyll radical in purified
PSI as a standard; the PSI sample was dehydrated on a mylar substrate.
The EPR spectrum was obtained at -9 °C. The g value
of the chlorophyll radical in PSI is known to be 2.0025
(40) . FTIR Spectroscopy at -9 °C-Samples contained 50-70
µg of chlorophyll for spinach samples and 20-30 µg of
chlorophyll for Synechocystis PSII particles. All PSII
preparations were dried on a germanium-coated window as described by
MacDonald and Barry
(11) . Spectral conditions were as
previously described
(11) . The absorbance in the amide I band
at 1655 cm
EPR Spectra of PSII Membranes at -9 °C-In Fig.
1, we present EPR spectra recorded on PSII membranes in the presence of
hydroxylamine alone (Fig. 1 B) and in the presence of
hydroxylamine and potassium ferricyanide (Fig. 1 A). The
presence of hydroxylamine will lead to accumulation of
Q
A difference in the decay of this chlorophyll
signal is observed when samples containing hydroxylamine alone
(Fig. 1 B) are compared to samples containing
ferricyanide and hydroxylamine (Fig. 1 A). In the presence of
ferricyanide, the chlorophyll radical is reduced by approximately 15%
in 8 min (Fig. 1 A, compare dot-dashed and
solid line). Approximately 30% of the radical has decayed
after 90 min (Fig. 1 A, compare the dotted line and solid line). If ferricyanide is not present in the
PSII membranes, only <10% reduction is seen after 90 min of dark
adaptation (Fig. 1 B, dotted line), showing
that, in the presence of hydroxylamine alone, the formation of the
chlorophyll radical is relatively irreversible.
To summarize, our
EPR results show that, when both hydroxylamine and ferricyanide are
present, a chlorophyll radical is produced reversibly in approximately
5% of the centers. However, in the presence of hydroxylamine alone,
less than 1% of the centers reversibly produce a chlorophyll radical.
We would expect that Q
It should be noted that the EPR line shape of the
radical varies from sample to sample, as does the amount of radical
produced. Some samples exhibit a broader EPR signal that contains
contributions from the tyrosine radical, D
Our experiments provide evidence for a correlation
between the appearance of a reversibly oxidized chlorophyll radical
(EPR spectroscopy) and an increase in the intensity of the infrared
spectrum. To demonstrate that an approximately 5% (moles of chl
radicals/total moles of chl) contamination from a chlorophyll radical
would be observable in the difference infrared spectrum, we present
light-minus-dark difference infrared spectra (Fig. 3) on purified
photosystem I from the cyanobacterium, Synechocystis sp. PCC
6803. The antenna size
(35) of the PSI samples is approximately
90 chlorophyll per P
These results
show that the oxidation of chlorophyll makes an intense contribution to
the difference infrared spectra. Such a conclusion is consistent with
the results of previous infrared studies on bacterial reaction centers
(for example, see Ref. 32). These studies have shown that the
vibrational spectrum associated with the oxidation of the
bacteriochlorophyll donor dominates the difference spectrum. For
example, vibrational modes of the primary donor are an order of
magnitude more intense than those observed for the acceptor quinones
(32) .
We also point out that because of the large sample
sizes needed to obtain high signal-to-noise EPR data, the light source
is not saturating under the EPR conditions. For FTIR experiments, a
much smaller amount of sample is required. For this reason, we believe
that the EPR results probably underestimate the number of chlorophyll
radical-producing centers in the infrared experiment.
Our difference
spectra resemble data obtained previously on hydroxylamine-treated PSII
membranes
(24) . In order to demonstrate this, we have
reproduced the difference spectrum reported by Berthomieu et al. (24) (Fig. 4, solid line) and have compared
it directly to one of our spectra (Fig. 4, dotted line),
which was also recorded on hydroxylamine-treated PSII membranes. The
dark adaptation time was 90 min (Fig. 4, dotted line).
The superimposition shows that the spectra are similar. The difference
observed in the 1670-1650 cm
EPR spectra recorded on PSII
complexes in the presence of both hydroxylamine and ferricyanide are
shown in the inset of Fig. 1; these spectra were
recorded under illumination ( solid line), after 8 min of dark
adaptation ( dot-dashed line), and after 90 min of dark
adaptation ( dotted line). The EPR spectrum of a chlorophyll
radical is observed under illumination, and this radical is reduced in
the dark. Spin quantitation of the radical indicates approximately 10%
of the PSII complexes exhibit the light-induced radical when
hydroxylamine and ferricyanide are present. If only hydroxylamine is
present, the chlorophyll radical is produced under illumination, but
the radical does not decay appreciably in 90 min of dark adaptation
(data not shown). This is a similar result to the one described above
for PSII membranes. The broader line width and higher g value
of the signal shown in the inset are due to an increased
contribution from D
Fig. 5
shows FTIR difference spectra
recorded on manganese-depleted PSII complexes under the conditions used
to record the EPR data. The signal-to-noise is increased upon
comparison of Fig. 5 to Fig. 2, because PSII complexes have a
smaller antenna size. The reduced antenna size will allow vibrational
modes associated with structural changes in redox-active tyrosine, D,
and other amino acids to be more easily observed. Thus, we expect there
to be small differences in intensity and frequency between the two
samples, even if chlorophyll and quinone vibrational lines contribute
to both difference spectra. Spectra in Fig. 5, A and
B, were recorded in the presence of hydroxylamine and
ferricyanide with either an 8-min (Fig. 5 A) or a 90-min
(Fig. 5 B) dark adaptation between the
``light'' and ``dark'' spectra. Intense difference
spectra are observed (Fig. 5, A and B), and an
increase in intensity is observed that correlates with the length of
dark adaptation. Examination of Fig. 5, A and
B, reveals positive lines with frequencies of 1456, 1478,
1506, 1718, and 1737 cm
In light of these observations, we decided
to investigate the origin of the intense 1478 cm
In Fig. 6 B, we present
light-minus-dark difference spectra obtained on control ( dashed
line) and
On the basis of the
The assignment of the 1478 cm
Our FTIR
difference spectra show spectral similarities with other infrared data
recorded on bacteriochlorophyll and chlorophyll both in vitro and in vivo. For example, in all these cases, oxidation
of chlorophyll or bacteriochlorophyll leads to differential features in
the spectral region between 1750 and 1650 cm
In addition to these
characteristic lines in the 1750-1650 cm
It is of
interest to compare our data, obtained in the presence of
hydroxylamine, to the chl
In the
data reported here, Z is not oxidized, because hydroxylamine is present
in these samples
(23, 43) . Under conditions where
Z
We thank the University of Minnesota Agricultural
Experiment Station and T. Krick for assistance in obtaining the mass
spectra. We are also grateful to Mary Bernard for assistance in sample
preparation.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is found in plants, green algae,
and procaryotic cyanobacteria and is one of two chlorophyll-containing
reaction centers that cooperate to transfer electrons from water to
NADP
in oxygenic photosynthesis. PSII carries out the
light-induced transfer of electrons from water to plastoquinone. This
reaction center is composed of integral membrane proteins that bind the
prosthetic groups required for efficient charge separation. Extrinsic
polypeptides are also required for oxygen evolution under physiological
conditions. The prosthetic groups associated with PSII include the
primary chlorophyll donor, P
, accessory chlorophylls,
pheophytin, and two plastoquinone molecules, Q
and
Q
. Upon absorption of light, P
and Z
(11, 12) . These spectra may
contain contributions from the vibrational difference spectrum,
Q
to Q
. The resulting difference
spectrum was considered to be the vibrational spectrum of
Q
N causes a 2 cm
shift of the most intense spectral feature, the positive 1478
cm
line. A 2 cm
N shift is
inconsistent with the previous assignment of the 1478 cm
line to Q
Sample Preparation
PSII membranes were prepared
as in Berthold et al. (25) and resuspended in a buffer
containing 50 m
M MES-NaOH, pH 6.0, 15 m
M NaCl, and
25% glycerol. This preparation is not monodisperse, but is in the form
of membrane sheets that can be pelleted by centrifugation. The purified
PSII membranes evolved oxygen at rates of approximately 600 µmol of
O/mg of chl
h.
/mg of chl
h. The final resuspension
buffer was 5 m
M MES-NaOH, pH 6.0. Unlike PSII membranes, this
preparation is detergent solubilized and is monodisperse. The PSII
complexes do not contain functional Q
(11) . The
final chlorophyll concentration was 1.3 mg/ml.
/mg of chl
h. PSII
particles were hydroxylamine-treated as described elsewhere
(11, 31) , except the incubation time was only 40 min.
The manganese-depleted PSII particles were then concentrated as in
MacDonald and Barry
(11) . Global isotopic labeling with
N was performed as previously described
(31, 36) . Mass spectral analysis
(37) of
acetone extracted chlorophyll was used to verify isotopic
incorporation. Mass spectra were obtained on a Kratos MS25 mass
spectrometer. The sample was ionized by fast atom bombardment through
the use of an 8-KeV xenon beam. The matrix was 3-nitrobenzyl alcohol,
and positive ions were detected.
, 0.05% lauryl maltoside (Anatrace, Maumee,
OH), and 260 m
M NaCl.
EPR Spectroscopy
The manganese-depleted PSII
membranes and complexes were dehydrated on mylar substrates as in
MacDonald and Barry
(11) . Membrane samples contained 280 µg
of chlorophyll. The PSII complex samples contained 400 µg of
chlorophyll. Spectra were recorded at -9 ± 2 °C as
described by MacDonald and Barry
(11) . Spectra were obtained at
X band using the following conditions: modulation amplitude, 3.2 G;
microwave power, 0.67 mW; scan time, 4 min; time constant, 2 s; gain,
3.2 10
.
was always <0.9 absorbance units.
Spectra were recorded at -9 °C. The spectral resolution was 4
cm
, the mirror velocity was 1.57 cm/s, and 1000
scans were co-added for each interferogram. The difference spectrum was
constructed by directly ratioing the interferogram obtained under
illumination to the interferogram obtained in the dark and then
converting to absorbance units. Five to seven difference spectra were
averaged in order to obtain the final data.
Cryogenic FTIR Spectroscopy
The sample contained
30 µg of chlorophyll. Spectral conditions were as described above.
The temperature was controlled to ±0.1 K with a High-Tran liquid
nitrogen cryostat (R. G. Hansen & Associates, Santa Barbara, CA)
and a temperature control unit (Scientific Instruments model 9620, West
Palm Beach, FL). The absorbance of the amide I band was 0.7 absorbance
units. Spectra were recorded at 80 K, and 1000 scans were co-added to
construct each interferogram. Difference spectra were constructed by
the method described above. Two difference spectra were then averaged
in order to obtain the final spectrum.
Figure 1:
EPR spectra of manganese-depleted
spinach PSII membranes at -9 °C in the presence of
hydroxylamine and potassium ferricyanide ( A) or hydroxylamine
alone ( B). The inset shows low temperature (-9
°C) EPR spectra of manganese-depleted PSII complexes in the
presence of hydroxylamine and potassium ferricyanide. In all cases, the
solid lines are spectra recorded under continuous
illumination, while the dot-dashed and dotted lines are spectra recorded after 8 and 90 min of dark adaptation,
respectively. Spectral condi-tions are given under ``Experimental
Procedures.''
In the presence of hydroxylamine alone or in the presence
of hydroxylamine and ferricyanide, EPR spectroscopy (Fig. 1,
A and B, solid line) shows that a
chlorophyll radical is present under illumination. The line width (7.5
G) and g value (2.0025) of this radical are both consistent
with a chlorophyll origin
(40, 48) . Spin quantitation
of the spectra recorded under illumination (Fig. 1, A and B, solid line) indicates that the
chlorophyll radical is present in approximately 10-20% of the
centers. This is in contrast to earlier results, which reported no
chlorophyll radical present when hydroxylamine was added as a reductant
(26) . Note that the level of detection was not specified in
this earlier study.
, in addition to
chlorophyll contributions (data not shown). The presence of
hydroxylamine in high concentrations leads to this variability, which
is not observed in infrared studies of PSII in the absence of
hydroxylamine (for example, see Refs. 11 and 12). Therefore, high
concentrations of hydroxylamine are problematic in experiments
conducted under steady state illumination. FTIR Spectra of PSII Membranes at -9 °C-Light-minus-dark
difference FTIR spectra recorded on PSII membranes are shown in Fig. 2.
In Fig. 2, A and B, both ferricyanide and
hydroxylamine are present. In Fig. 2 A, there was a short
dark adaptation time of 8 min between the light and dark spectra. In
Fig. 2B, there was a long dark adaptation time of 90 min
between the light and dark spectra. Both sets of data were obtained on
the same sample, and both difference spectra exhibit strong vibrational
modes. These are the conditions where our EPR experiment predicts that
a chlorophyll radical will make the largest contribution to the
spectrum. An increase in intensity is observed when Fig. 2 B is compared to Fig. 2 A. This increase in intensity
roughly correlates with the results of our EPR experiment
(Fig. 1 A) that showed a more extensive decay of the
chlorophyll radical in 90 min than in 8 min. The use of hydroxylamine
leads to some variability in the relative intensities of the difference
infrared spectrum and in the amplitudes of the EPR signal, so an exact
correlation of EPR and FTIR spectral intensities is not expected.
Figure 2:
Light-minus-dark FTIR spectra recorded at
-9 °C of manganese-depleted spinach PSII membranes. Samples
were treated with either hydroxylamine and potassium ferricyanide
( A and B) or hydroxylamine alone ( C and
D). The dark adaptation was 8 min in A and C and 90 min in B and D. The tick marks on the y axis correspond to 4 10
absorbance units. Spectral conditions are given under
``Experimental Procedures.''
Fig. 2
, C and D, present difference spectra
recorded on PSII membranes in the presence of hydroxylamine alone. In
Fig. 2C, the dark adaptation was 8 min, and in Fig.
2 D, it was 90 min. Both sets of data were obtained on the same
sample. These difference spectra exhibit only weak features (see
Fig. 2
, C and D). These are conditions where
our EPR control experiment showed only a small change in the
concentration of chlorophyll radical even after prolonged (90 min) dark
adaptation.
. Illumination of photosystem I
photooxidizes the primary chlorophyll donor,
P
Figure 3:
FTIR difference spectra recorded at
-9 °C of purified PSI particles from the cyanobacterium,
Synechocystis sp. PCC 6803. Samples contained chlorophyll in
the amounts of 4.0 µg ( A), 2.0 µg ( B and
D), and 0.4 µg ( C) plus 1.5 m
M potassium
ferricyanide. A, B, and C are
light-minus-dark difference spectra in which the dark adaptation time
was 8 min. D is a dark-minus-dark difference spectrum obtained
after illumination. The tick marks on the y axis
correspond to 4 10
absorbance
units.
In Fig. 3 A,
the P
spectral region
may be due to an increased contribution of D
in Berthomieu et
al.'s experiments, which involved much longer dark
adaptations. Our previous work has assigned lines to redox active
tyrosine D through the use of isotopomers of tyrosine
(12) . In
addition, the 1670-1650 cm
spectral region may
include contributions from the amide I band of the peptide backbone
(31) . The spectra compared in Fig. 4 were obtained at a
different temperatures, and this temperature difference could result in
small differences in protein conformation and, thus, in amide I
frequencies and intensities.
Figure 4:
Light-minus-dark FTIR spectra of
hydroxylamine-treated spinach PSII membranes either from this work
( dotted line) or as reproduced from Berthomieu et al.
(24) ( solid line). The data reproduced from Berthomieu et
al. (24) were obtained in the presence of DCMU at 15
°C.
The spectrum of Berthomieu et
al., was attributed to Q
EPR and FTIR Spectra of PSII Complexes
The PSII
membranes used in the experiments described above may contain small
amounts of PSI. At room temperature, the primary chlorophyll donor of
PSI, P, gives rise to an EPR signal. To distinguish
whether the chlorophyll observed in Fig. 1is due to residual PSI
or originates from PSII, EPR data were obtained on a PSII complex
preparation that has no PSI contamination. PSI quantitation was
performed on the same samples according to the method described in
Noren et al. (35) ; PSI was not detectable in these
samples by optical techniques.
.
and negative modes at 1522,
1557, 1630, 1645, 1672, 1682, 1724, and 1743 cm
. All
of these frequencies are within 1-2 cm
of
corresponding lines in Fig. 2, A and B.
Figure 5:
Light-minus-dark FTIR spectra recorded at
-9 °C of manganese-depleted spinach PSII complexes. Samples
were treated with either hydroxylamine and potassium ferricyanide
( A and B) or hydroxylamine alone ( C and
D). The dark adaptation was 8 min in A and C and 90 min in B and D. The tick marks on the y axis correspond to 4 10
absorbance units. Spectral conditions are given under
``Experimental Procedures.''
In
contrast, in the presence of hydroxylamine, spectral features are weak,
regardless of the length of dark adaptation (Fig. 5, C and D). However, when hydroxylamine is added in strict
darkness and samples are then dark-adapted for 3 h or more, intense
light-minus-dark difference spectra are obtained (data not shown).
These spectra resemble those shown in Fig. 5, A and
B. This strong infrared spectrum is again correlated with the
production of the chlorophyll cation radical in an EPR control
experiment (data not shown). This radical does not appreciably decay.
In reference Berthomieu et al. (24) a very slow
spectral decay behavior was also observed.
The
work that we have presented thus far shows a correlation between the
reversible production of a chlorophyll cation radical and an intense
positive line at 1478 cmN-Labeling of Photosystem II
in the difference infrared
spectrum (Figs. 1, 2, and 5). This line is observed either in spinach
PSII membranes (Fig. 2) or in spinach PSII complexes (Fig. 5).
There is also a correlation with the appearance of differential
features in the 1750-1650 cm
region (Figs. 2
and 5). Although this chlorophyll cation radical is produced in only 5%
of the centers, we have presented evidence that this a detectable
amount of chlorophyll radical, as assessed by infrared spectroscopy
(Fig. 3). We have also provided evidence that the frequency of
the 1478 cm
line is not responsive to the presence
of DCMU (Fig. 4).
line that is observed in the presence of hydroxylamine.
Vibrational lines in complex biological spectra can be assigned through
isotopic labeling. As the first step in our attempts to label
chlorophyll and quinones specifically for infrared spectroscopy, we
carried out global
N-labeling of photosystem II. This can
be performed by growing the cyanobacterium, Synechocystis sp.
PCC 6803, in the presence of [
N]nitrate and
isolating photosystem II from the labeled cultures. This procedure
should have the effect of replacing every
N in the cell
with
N. To verify isotope incorporation, mass spectral
analysis was performed on extracted chlorophyll from the same samples
used to generate Fig. 6 B. Mass spectral analysis
(37) shows that the total amount of
N incorporation
into chlorophyll is 97% (data not shown). 90% of the chlorophyll is
labeled with
N at all four ring positions
(
N
), while the remaining 7% is either
N
,
N
, or
N
. This analysis is in agreement with previous
N-labeling experiments in cyanobacteria
(31, 36) .
N-labeled ( solid line) PSII
particles from Synechocystis. A 2 cm
shift
of the 1478 cm
line is observed upon global
N-labeling (Fig. 6 B, or see the expanded
spectrum in Fig. 6 A). It should be noted that overlay of two
control,
N, difference spectra gives a reproducible
frequency of 1478 cm
for this line (data not shown).
This
N shift was observed in PSII samples isolated from
three different
N-labeled cultures. Also, extensive
H
O exchange had no effect on the frequency of
this line. Since plastoquinone contains no nitrogen atoms, a 2
cm
N shift precludes assignment of the 1478
cm
line to Q
Figure 6:
Light-minus-dark FTIR difference spectra
recorded at -9 °C of control (N) ( dashed
line) or of
N-labeled ( solid line) PSII
particles from Synechocystis sp. PCC 6803. Panel A shows an expanded spectrum from 1500 to 1450
cm
, while Panel B shows the 1900 to 1300
cm
region. Samples contained hydroxylamine and
ferricyanide. The dark adaptation time was 8 min. The tick marks on the y axis correspond to 4
10
absorbance units. Spectral conditions are given under
``Experimental Procedures.''
A
difference infrared spectrum of N-labeled spinach
photosystem II preparations that were treated with hydroxylamine has
been described previously
(26) . In this work, it was reported
that the 1478 cm
line did not shift as a result of
isotopic labeling. The discrepancy with our result is probably the
result of incomplete
N-labeling of chlorophyll in the
earlier study. No mass spectral analysis of chlorophyll was described
in the previous study, and the extent of labeling was not reported.
N shift obtained, we favor the
assignment of the 1478 cm
line to a chlorophyll
cation radical. This chlorophyll may be the primary donor or may be
another chlorophyll oxidized by P
line to
chlorophyll implies that the spectrum observed in the presence of
hydroxylamine is actually dominated by the vibrational changes
associated with chlorophyll oxidation, not by acceptor side changes.
Assignment of this spectrum to a dominating chl
- chl contribution accounts for the fact that a similar
spectrum is observed in the absence of hydroxylamine in some PSII
membrane preparations
(27, 28) . Such a result could be
obtained if contributions from a PSII chlorophyll oxidized in inactive
centers dominated the spectrum
(27, 28) .
(51, 52) . These lines have been assigned to ester
and carbonyl vibrations (Fig. 7) that are perturbed by oxidation.
Note, however, that a recent study suggests that some lines in this
spectral region may be more properly assigned to nonfundamental
vibrations
(53) . In in vitro studies of chlorophyll
a oxidation, a differential feature at 1718/1693
cm
was assigned to the C
=O
vibration (Fig. 7), and a differential feature at 1751/1738
cm
was assigned primarily to the C
ester carbonyl vibration
(52) . By analogy, a strong
differential feature at 1717/1700 cm
, observed after
photoxoidation of P
in photosystem I, was assigned to the
C
keto group
(52) . However, unambiguous assignment
of the C
ester carbonyl vibration was not possible, since
two differential features, not one, were observed at higher frequency:
1753/1748 and 1741/1734 cm
. The authors favored the
assignment of both these spectral features to a C
ester
carbonyl vibration. They reasoned that the individual chlorophylls,
making up a presumed dimeric cation radical, contributed independently
to the difference spectrum. However, other possible explanations could
not be excluded
(52) .
Figure 7:
Structure of chlorophyll
a.
Based on these previous studies, we
tentatively assign the differential feature at 1718/1701
cmin our spectrum (Fig. 2, A and
B) to the C
keto C=O group of chlorophyll.
Our difference spectra also exhibit two differential features in the
1725-1750 cm
region. If the cation radical is
dimeric (see discussion below), these features could arise from the
C
C=O groups of the component monomers. These
frequencies are slightly shifted in PSII complexes, as compared with
PSII membranes. This may be an effect of the solubilization of the
preparation in detergent, since the frequencies of these lines would be
expected to be sensitive to changes in solvent. Increased contribution
from amino acids could also have the observed effect of shifting
observed vibrational lines of chlorophyll.
spectral region, the infrared difference spectrum associated with
the oxidation of the primary bacteriochlorophyll donor, P
- P, also exhibits an intense positive mode at 1474
cm
(54) . This line may be analogous to the
1478 cm
line observed in our spectra. However, the
primary chlorophyll donor of PSI does not exhibit a strong line in the
1470 cm
region (see Fig. 3, for example), and
this line is not observed either in bacteriochlorophyll
(51) or
in chlorophyll
(52) upon oxidation in vitro. This
spectral feature is also not very intense in Rb. capsulatus mutants that contain a bacteriochlorophyll-bacteriopheophytin
heterodimer as the primary donor
(55) . The significance of the
appearance of this spectral feature is not understood, but we speculate
that its appearance may be correlated with whether the
chlorophyll/bacteriochlorophyll cation radical is a dimer.
Q
Figure 8:
Light-minus-dark FTIR difference spectra
of spinach PSII complexes. In A, data were recorded at
-9 °C on manganese-depleted preparations in the presence of
hydroxylamine and potassium ferricyanide (same as Fig. 5 B). In
B, data were recorded at 80 K on manganese-containing
preparations. In both cases, cytochrome bis
known to be in low potential (15).
Although
superficially Fig. 8, A and B, are dissimilar,
these spectra do have common vibrational lines. Observed spectral
differences between Fig. 8, A and B, could have
several possible causes. First, changes could be due to an alteration
in the relative concentrations of the chlorophyll cation radical and
the semiquinone anion radical. Second, differences could be due to an
effect of hydroxylamine on the Q
can be accumulated under illumination, isotopic labeling has
shown that the tyrosine radical also makes a contribution to the 1478
cm
spectral region
(12) . This contribution
can be distinguished from that of chlorophyll, since mass spectral
analysis has shown that labeling of tyrosine with either
C
or
H does not change the isotopic composition of
chlorophyll
(12) . When Z
contributes to the spectrum,
i.e. in the absence of hydroxylamine, a small
N-induced change in line shape and intensity is observed
in this region
(31) . However, there is no significant
alteration of the frequency of the line. Comparison of this result with
Fig. 6
indicates that the 1478 cm
mode,
observed in the presence of hydroxylamine, contains fewer components.
This is consistent with our conclusion that difference infrared
spectrum, obtained in the presence of hydroxylamine, is dominated by
the oxidation of chlorophyll.
Summary
We conclude that infrared difference
spectra, recorded on hydroxylamine treated samples, reflect structural
changes associated with both the oxidation of chlorophyll and the
reduction of quinone. Therefore, this procedure cannot be used to
isolate the acceptor side contribution to the difference infrared
spectrum. Definitive identification of quinone contributions to the
vibrational spectrum awaits isotopic labeling of plastoquinone.
, primary chlorophyll donor of PSII; P
,
primary chlorophyll donor of PSI; MES, 4-morpholineethanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.