From the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota 55108-1022
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Photosystem II (PSII) is the photosynthetic
enzyme catalyzing the oxidation of water and reduction of plastoquinone
(Q). This reaction occurs at a catalytic site containing four manganese atoms and cycling among five oxidation states, the Sn states,
where n refers to the number of oxidizing equivalents stored. Biochemical and spectroscopic techniques have been used previously to conclude that aspartate 170 in the D1 subunit influences the structure and function of the PSII active site (Boerner, R. J., Nguyen, A. P., Barry, B. A., and Debus, R. J. (1992)
Biochemistry 31, 6660-6672). Substitution of glutamate for
aspartate 170 resulted in an assembled manganese cluster, which was
capable of enzymatic turnover, but at lower steady-state oxygen
evolution rates. Here, we obtained the difference (light-minus-dark)
Fourier transform IR spectrum associated with the
S2Q Photosystem II (PSII)1
is the photosynthetic reaction center that is responsible for the
light-driven oxidation of water and the reduction of quinone in plants,
green algae, and cyanobacteria. The catalytic site contains a
tetranuclear manganese cluster, and the catalytic cycle of water
oxidation has been proposed to consist of five oxidation states
(Sn) called the Joliot-Kok S states. The subscript n
refers to the number of oxidizing equivalents stored at the active
site. Absorption of four photons and four corresponding charge
separations are necessary to complete one catalytic cycle. Each cycle
results in the oxidation of two water molecules to form one molecule of
molecular oxygen and four protons. Calcium is required for oxygen
evolution and may be bound in proximity to the manganese cluster
(reviewed in Refs. 1 and 2).
Many studies of PSII have focused on the S2 state (for
review, see Ref. 3). Dark adaptation at room temperature sets the catalytic center to the S1 state because S0
converts to S1 by reduction of tyrosine radical, D·
(4, 5). Two different EPR signals, a g = 2 multiline signal (6)
and a g = 4.1 signal (7), are observed from the S2
state. Illumination of plant or cyanobacterial PSII at 200 K results in
the formation of the g = 2 multiline EPR signal (for examples, see
Refs. 6 and 8) and an EPR signal from
Fe2+QA A structural model for the manganese cluster in the S1
state has been suggested; this model is based on x-ray absorption
studies of the manganese cluster (reviewed in Ref. 10). According to this model, the manganese cluster has been proposed to contain four
manganese atoms arranged in two bis-µ-oxo-bridged dimers. These
dimers are linked by two carboxylato bridges and one bis-µ-oxo bridge
to form a C-shaped tetranuclear metal center. Because each manganese
atom must be five- or six-coordinate (15), amino acids must provide
ligation to the metal atoms. Site-directed mutagenesis suggests that
some of these ligands are provided by aspartate and glutamate residues
in the lumeral regions of the D1 and D2 polypeptides (reviewed in Ref.
16). Chemical modification experiments support this conclusion (17).
Ligation by carboxylate groups would have the effect of stabilizing
high oxidation states of the manganese ions (15).
Mutations have been made in carboxylate residues in the D1 polypeptide,
with multiple substitutions generated at each site (for reviews, see
Refs. 16, 18, and 19). The effect of mutations on oxygen evolution
activity, photoautotrophic growth, and the variable fluorescence yield
was assessed. From this work, likely metal ligands have been identified
(reviewed in Refs. 16, 18, and 19).
The most thoroughly characterized mutations in this group were those
introduced at aspartate 170 of the D1 polypeptide (20, 21). Eleven
different amino acids were substituted for aspartate 170. All
mutations, except glutamate and histidine, abolished photoautotrophic
growth (21). Most mutations resulted in dramatic decreases in oxygen
evolution activity (21), although residual low levels of oxygen
evolution were detected in the DC170D1, DW170D1, DR170D1, DY170D1,
and DM170D1 mutants (21). PSII particles containing the DN170D1
mutation had no detectable activity and showed electron transfer
kinetics that were consistent with the absence of a functional manganese cluster (20). The manganese content of the DN170D1 mutant was
reduced dramatically compared with manganese content in wild-type PSII
(20).
On the other hand, an aspartate-to-glutamate mutation did not abolish
oxygen evolution, but the mutation resulted in a 2-3-fold decrease in
the steady-state rate of oxygen evolution activity (20, 21).
Spectroscopic characterization showed that a functional manganese
cluster was assembled in this mutant (20, 21). The manganese content of
the DE170D1 mutant was similar to the manganese content of wild-type
PSII (20). Characterization of DE170D1 cells showed that oxygen
evolution exhibited a period 4 oscillation with flash number; this
oscillation was similar to the pattern observed in wild-type cells
(21). The lifetime and oxidation kinetics of the S2 state
were perturbed (20, 21), giving a slightly more stable S2
state in the mutant.
From the studies described above (20, 21), it was concluded that
aspartate 170 influences the assembly and/or activity of the active
site (see also Ref. 17). To investigate the mechanism by which
aspartate 170 exerts its influence on the PSII catalytic site, we have
employed vibrational spectroscopy. Use of oxygen-evolving PSII
particles from the DE170D1 mutant provides an opportunity to obtain
information about the catalytic site in a perturbed but active form. We
report here the difference FT-IR spectrum associated with the
S1-to-S2 transition in wild-type PSII and in
PSII isolated from the DE170D1 mutant. Our data provide evidence for
shifts in carboxylate coordination of manganese or calcium when
wild-type and DE170D1 PSII are compared. These spectra provide new
information about the catalytic site of this enzyme.
Chlorophyll and oxygen evolution assays were performed (22).
Spinach PSII complexes were isolated (23). The steady oxygen evolution
rates (22) of this preparation were Infrared spectra were recorded on a Nicolet 60-SXR spectrometer
equipped with a liquid nitrogen-cooled MCT-B detector (12, 13). The
sample temperature was controlled to ±0.3 K with a High-Tran liquid
nitrogen cryostat (R. G. Hansen & Associates, Santa Barbara, CA)
and a temperature control unit (Model 9620, Scientific Instruments,
West Palm Beach, FL). The temperature was continuously monitored during
data acquisition. The liquid nitrogen cryostat was equipped with TnSe
(see Fig. 1) or CaF2 windows (see Figs. 2, 3, and 5).
Illumination was provided with a Dolan-Jenner fiber optic annular
illuminator equipped with a red filter and a heat filter. Spectral
resolution was 8 cm To construct a difference FT-IR spectrum, an interferogram recorded
under illumination was ratioed directly to interferograms recorded in
the dark before illumination. The resulting difference spectra were
then averaged. The length of the scans was 5 min in Fig. 1 and 7.5 min
in Figs. 2, 3, and 5. The absorbance of the amide I band at 1655 cm EPR spectra were obtained on a Bruker EMX spectrometer equipped with a
temperature-controlled Wilmad flow-through Dewar inserts. A stream of
cold nitrogen was used to maintain the EPR sample temperature at 80 K;
the sample temperature was continuously monitored (22). PSII samples
containing 1 eq of potassium ferricyanide were frozen in liquid
nitrogen, and EPR spectra, corresponding to a dark scan, were recorded.
PSII samples were then illuminated at 200 K with red- and heat-filtered
light for 7.5 min and re-equilibrated in liquid nitrogen. EPR spectra,
corresponding to the light scan, were then recorded. Spectra were
obtained with the following instrument settings: microwave frequency,
9.383 GHz; microwave power, 1 milliwatt; modulation amplitude, 3.2 G;
and time constant, 1.3 s. Twelve scans of 83 s were recorded
and averaged. EPR spectra were integrated and analyzed through the use
of the program IGOR (Wavemetrics, Lake Oswego, OR).
Difference FT-IR Studies of the
S1QA-to-S2QA
Because illumination of PSII complexes at this temperature is
associated with the production of multiline and
Fe2+QA
Fig. 2 also presents the result of a second illumination of PSII at 200 K. The sample was preilluminated, generating an
S2QA
Our previous experiments, in which spectra were obtained as a function
of illumination temperature, have led to the conclusion that the
S2QA Difference FT-IR Studies of Oxygen-evolving Wild-type and DE170D1
PSII--
In Fig. 3, we present the
results of 200 K illumination of oxygen-evolving PSII preparations
isolated from the cyanobacterium Synechocystis sp. PCC 6803. The cyanobacterial
S2QA
In Fig. 3B (solid line), we present a difference
FT-IR spectrum obtained by illumination at 200 K of oxygen-evolving
PSII particles of the DE170D1 mutant. The spectrum exhibits an overall resemblance to the wild-type cyanobacterial
S2QA
Although there is an overall resemblance between
S2QA EPR Studies of Oxygen-evolving Wild-type and DE170D1
PSII--
When PSII preparations are illuminated at 200 K in
glycerol-containing buffers, there are three possible donors to
P680+: the S1 state, tyrosine D,
and Chl (12-14). Cytochrome b559 is low
potential in these preparations and already oxidized (20, 31).
Redox-active tyrosine D is preoxidized in both wild-type (12-14) and
mutant PSII and does not significantly contribute to the
light-minus-dark difference spectrum (Fig.
4, A and B). In centers that lack a manganese cluster or are inhibited in oxygen evolution, 200 K illumination will oxidize Chl, and not the
S1 state of the manganese cluster (12-14, 28, 29).
To obtain an indirect measure of the amount of S2 formation
in the DE170D1 mutant, an EPR control experiment was performed to
record the amount of Chl cation radical generated by 200 K illumination. These data are shown in Fig. 4. In wild-type
cyanobacterial PSII (Fig. 4, A and a), EPR spin
quantitation showed that the amount of Chl cation radical generated is
0.2 ± 0.1 spin/tyrosyl radical D· (or per reaction
center). This result is similar to the amount of Chl+
generated in spinach PSII complexes at 200 K, which has been reported
as 0.33 ± 0.09 spin/D· (13). In DE170D1 PSII (Fig. 4,
B and b), EPR spin quantitation showed that the
amount of Chl cation radical generated at 200 K is ~2-fold the amount
generated in wild-type PSII, giving again a result similar to the
Chl+ content determined in spinach PSII (13). Because this
experiment was performed on DE170D1 samples with a somewhat lower
oxygen evolution rate (300 µmol of O2 (mg of
Chl/h)
This conclusion is consistent with previous characterization of these
preparations that showed that the amount of variable fluorescence,
produced after a single saturating flash to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea containing, dark-adapted samples in the S1
state, was indistinguishable in wild-type and DE170D1 PSII preparations (20). Because variable fluorescence is an indirect measure of the
amount of QA Difference FT-IR Studies of Hydroxylamine-treated DE170D1
PSII--
Based on the reasoning described above, the light-minus-dark
difference spectrum obtained by illumination of DE170D1 PSII at 200 K
(Fig. 3B) reflects oxidation of the S1 state and
not just the oxidation of Chl. To further test this idea, the
difference FT-IR spectrum associated with illumination at 200 K was
obtained from hydroxylamine-treated DE170D1 PSII (Fig.
5). If the spectrum shown in Fig.
3B reflects the production of the S2 state in
the DE170D1 mutant, then manganese removal is expected to change the spectrum (14). The contribution of Chl oxidation to the difference FT-IR spectrum will increase upon hydroxylamine treatment of
DE170D1 PSII.
In Fig. 5, we present a comparison of difference FT-IR data obtained
from oxygen-evolving DE170D1 PSII (Fig. 5A) and from hydroxylamine-treated, manganese-depleted DE170D1 PSII (Fig.
5B). Alterations in frequency and intensity are observed in
the 1770 to 1700 cm
Because the difference spectra in Fig. 5 (A and
B) are normalized on a protein and path length basis (27),
the full amplitude of QA Assignment of the Double-difference Spectrum
DE170D1-minus-Wild-type--
Our aim is the interpretation of the
spectral alterations observed in the DE170D1
S2QA
As shown in Fig. 3C (striped areas), the
double-difference spectrum DE170D1-minus-wild-type exhibits positive
lines at 1719 and 1683 cm
In the double-difference spectrum constructed from wild-type and
DE170D1 data (Fig. 3C), positive features are due to the wild-type S1 state or the mutant S2 state,
whereas negative features are due to the wild-type S2 state
or the mutant S1 state. Amino acid residues that are
perturbed by oxidation of manganese and by the DE170D1 mutation will
contribute to this double-difference spectrum. Thus, contributions from
both aspartate/aspartic acid 170 (wild-type) and glutamate/glutamic
acid 170 (mutant) would be expected in Fig. 3C, if these
residues are ligating or close to the manganese cluster. Also,
contributions from other amino acid residues close to or ligating the
metal cluster and perturbed by the DE170D1 substitution are expected.
Vibrational lines may originate from amino acid residues in the DE170D1
mutant or in wild-type PSII.
These alternatives can be evaluated by comparison with the spectrum
shown in Fig. 5C, which compared data obtained from
oxygen-evolving and manganese-depleted DE170D1 PSII. Lines assignable
to the S1-to-S2 transition in the mutant will
be in common and have the same sign when these
two double differences (Figs. 3C and 5C) are
compared. Another possible contributor to Fig. 3C is
vibrational modes of Chl+-minus-Chl. In such a case, ester
and keto contributions to the spectrum, arising from Chl, should be
observed in the 1750 to 1650 cm Assignment of the 1750 to 1670 cm Interpretation of the Lines in the 1750 to 1670 cm
There are three types of structural changes that would result in a
carboxylic acid contribution to the spectrum. The first is a frequency
shift of the C=O stretching vibration, perhaps due to a perturbation in
the pKa or hydrogen bonding of these groups (12).
This first type of structural change would give rise to
derivative-shaped spectral features in the 1750 to 1720 cm Frequencies--
The frequencies of carboxylic acid C=O stretches
reflect changes in double-bond character in the C=O bond as well as
changes in the basicity of the oxygen (35). A typical range of
frequencies is from 1750 to 1720 cm
However, the other frequencies, 1696 cm
On the basis of these considerations, spectral features at 1696, 1683, and 1669 cm
The direction of the shift provides additional information
about the origin of vibrational lines. The four lines discussed above
are components of two derivative-shaped lines, 1719/1696 and 1683/1669
cm Assignment of the 1600 to 1400 cm Interpretation of the Lines in the 1600 to 1400 cm
The energy gap between the asymmetric and symmetric
stretching vibrations of carboxylates is influenced by metal binding
(38, 40) and provides information about the origin of spectral
features. The
Consideration of the spectral features assigned to wild-type PSII (Fig.
3C, dotted areas) gives
The direction of the shift provides additional information
about the origin of vibrational lines. These broad lines are assigned to wild-type PSII; therefore, the double-difference spectrum is an
S1-minus-S2 spectrum. When
manganese is oxidized, both asymmetric and symmetric vibrations of
bridging carboxylate ligands downshift (39). For example, the
asymmetric stretching vibration of bridging carboxylates downshifts
from 1636 to 1540 cm Upon illumination of PSII at 200 K, the
S1-to-S2 transition can be studied
independently of other S state transitions (12-14, 42), and the
S2QA Carboxylate rearrangements involving conversion between bridging and
unidentate coordination are known to occur on metal oxidation in
methane monooxygenase (43, 44) and ribonucleotide reductase (45). Our
work has now documented carboxylate shifts at the catalytic site for
photosynthetic water oxidation. Therefore, the large line width of the
carboxylate ligand vibrations in wild-type PSII (Fig. 3C)
may arise from homogeneous broadening due to carboxylate rearrangements
on the time scale of the measurement (13, 14). Alternatively, the line
width may arise from inhomogeneous broadening due to a contribution to
the spectrum from multiple carboxylate groups in slightly different
protein environments (13).
A change in inner-sphere coordination of a metal is also observed when
an aspartate-to-glutamate mutation is introduced at a Ca2+
ligand in staphylococcal nuclease (46). In this case, the substitution of glutamate for aspartate 21 results in a change from unidentate ligation by aspartate 21 to bidentate ligation by glutamate 21. Other
substantial changes in coordination of calcium occur, including a
change in the number of coordinating water molecules and new ligation
by glutamate 43, which is not coordinating in the wild-type protein. In
addition, more long-range alterations in protein structure are also
observed (46). This work provides a precedent for the interpretation of
our vibrational spectra.
The question of whether aspartate 170 and/or glutamate 170 is directly
ligating manganese is of importance. The simplest interpretation of our
results is that aspartate 170 is a bridging/bidentate ligand in the
S1 and S2 states and that glutamate 170 is a
unidentate ligand in the S1 and, possibly, S2
states (Fig. 6). If correct, other changes in coordination must also
occur to explain our data. For example, in the S1 state,
another bridging/bidentate ligand must be replaced with a unidentate
ligand (Fig. 6). These additional changes in coordination may be
rearrangements of bound water, which may not contribute to the spectral
region investigated here (38), or additional carboxylate
rearrangements, contributing to the spectra presented here. Although
there is precedent for this interpretation (see discussion of
staphylococcal nuclease above (46)), the possibility of long-range
structural changes induced by the mutation must be considered. In this
scenario, substitution of glutamate for aspartate 170 causes a
structural change that results in the substitution of unidentate for
bridging carboxylate ligands.
The catalytic site of the DE170D1 mutant is functional, but perturbed.
This is evident in the decreased steady-state rate of oxygen evolution,
in the longer lifetime of the S2 state, and in observed
changes in the rate of oxidation of the catalytic site (20, 21). Also,
we have not yet observed a g = 2 multiline signal from this mutant
(data not shown), indicating that alterations in magnetic interactions
have occurred. We attribute these functional alterations to the change
in ligation described above (Fig. 6). For example, ab initio
calculations have associated a 10 kcal/mol stabilization of energy with
the exchange of a bidentate ligand in the first solvation shell of
Mg2+ with a unidentate ligand (30). A decrease in energy
upon substitution of unidentate for bidentate coordination rationalizes
our results with previous studies showing a stabilization of the
DE170D1 S2 state (20, 21).
Additional indirect evidence for a perturbation of manganese binding is
observed in data obtained after hydroxylamine treatment of DE170D1. In
spinach and wild-type cyanobacterial PSII, treatment with hydroxylamine
and EDTA is required to remove manganese and to generate a
Chl+QA We have shown that substitution of glutamate for aspartate 170 gives
rise to a change in coordination of manganese or calcium in PSII. The
spectral changes observed are consistent with an alteration from
bridging/bidentate carboxylate ligands to unidentate carboxylate
ligation in the mutant. The cluster, which exhibits vibrational spectra
characteristic of unidentate ligation, is still active in water
oxidation (20, 21). Our work is the first characterization of a
mutation that alters the structure and function of the photosynthetic
water-oxidizing complex through the use of vibrational spectroscopy.
These studies have established that carboxylate shifts are possible at
the metal center, which carries out photosynthetic water oxidation.
This result indicates that PSII is a member of the same class of
enzymes as ribonucleotide reductase and methane monooxygenase,
which undergo carboxylate shifts at their metal centers (43-45).
-minus-S1Q transition by
illumination of oxygen-evolving wild-type and DE170D1 PSII preparations
at 200 K. These spectra are known to be dominated by contributions from
carboxylic acid and carboxylate residues that are close to or ligating
the manganese cluster. Substitution of glutamate for aspartate 170 results in alterations in the
S2Q
-minus-S1Q spectrum; the
alterations are consistent with a change in carboxylate coordination to
manganese or calcium. In particular, the spectra are consistent with a
shift from bridging/bidentate carboxylates in wild-type PSII to
unidentate carboxylate ligation in DE170D1 PSII.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(9). The
S1-to-S2 transition is associated with
oxidation of manganese, probably from Mn3+ to
Mn4+ (reviewed in Ref. 10). Recently, we have obtained the
difference FT-IR spectrum associated with the
S1-to-S2 transition and have shown that this
spectrum is dominated by contributions from carboxylate and carboxylic
acid side chains that are close to or ligating the metal cluster
(12-14).2
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1000 µmol of O2 (mg
of chl-h)
1. Construction of a wild-type
kanamycin-resistant strain of Synechocystis sp. PCC 6803 and
the DE170D1 mutant has been described (20). Cyanobacterial PSII
particles were purified (20, 22, 24). Wild-type cells had average
oxygen rates of 480 µmol of O2 (mg of
Chl-h)
1; DE170D1 mutant cells had average oxygen rates of
270 µmol of O2 (mg of Chl-h)
1. Wild-type
PSII particles, employed for FT-IR experiments, exhibited rates of
oxygen evolution of ~2000 µmol of O2 (mg of
Chl-h)
1, and DE170D1 PSII particles, employed for FT-IR
studies, exhibited rates of ~500 µmol of O2 (mg of
Chl-h)
1. These steady-state oxygen evolution rates are in
the range of values previously obtained (20). We have also shown that
the chlorophyll antenna sizes of the wild-type and DE170D1 PSII
preparations are similar (20). Hydroxylamine treatment of spinach and
cyanobacterial PSII was performed (25). For infrared and EPR
spectroscopy, each sample contained 1 molar eq of potassium
ferricyanide. The chlorophyll antenna sizes of spinach and
cyanobacterial PSII, used to calculate the molarity from the measured
chlorophyll concentration, have been reported (26).
1 in Fig. 1 and 4 cm
1 in
Figs. 2, 3, and 5. A Happ-Genzel apodization function was used; two
(Fig. 1) or three levels (Figs. 2, 3, and 5) of zero filling were
employed; double-sided interferograms were collected; and the mirror
velocity was 1.57 cm/s.
1 was <0.9 absorbance units. Spectra were normalized
to an amide II absorbance of 0.5 absorbance units. Such a normalization
is equivalent to a correction for protein concentration and path length
(12, 13, 27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Transition--
Our previous work has shown that the difference FT-IR
spectrum,
S2QA
-minus-S1QA,
can be acquired by 7.5 min of continuous illumination at 200 K
(12-14). In Fig. 1, we present such
S2QA
-minus-S1QA
FT-IR spectra, and accompanying negative controls, obtained from
spinach PSII complexes. To show that illumination for 7.5 min with red-
and heat-filtered illumination has no deleterious effects on the
sample, Fig. 1 presents an experiment conducted with shorter, 5-min
illumination times. Fig. 1A presents the dark-minus-dark negative control experiment, which, as expected, gives a flat base
line. The first light-minus-dark difference spectrum (Fig. 1B) was acquired with 5 min of illumination. These data are
similar to difference spectra obtained with 7.5 min of illumination
from spinach PSII (Fig. 2A).
Illumination for an additional 5 min (Fig. 1C) generates a
difference spectrum indistinguishable from data obtained during the
first 5 min of illumination (Fig. 1B). The light-minus-light
spectrum (Fig. 1D), which gives a flat base line, confirms
that there are no accumulating, deleterious effects due to a 10-min
illumination of the sample. The base lines shown are typical of base
lines obtained for both spinach and cyanobacterial PSII samples.
View larger version (23K):
[in a new window]
Fig. 1.
The light-minus-dark difference FT-IR spectra
obtained at 200 K from spinach PSII complexes are shown in B
and C. Data were obtained with 5 min of illumination, and
the data acquisition scheme was as follows: dark (1), dark (2), light
(1), light (2). Here, (1) and (2) refer to the
order in which spectra were acquired. The dark (1)-minus-dark (2)
(A) and light (1)-minus-light (2) (D) controls
are also shown. The tick marks on the y
axis represent A = 1 × 10
3 absorbance unit. Other spectral conditions are given
under "Experimental Procedures."
View larger version (24K):
[in a new window]
Fig. 2.
Light-minus-dark difference FT-IR spectra
obtained from spinach PSII complexes. In A, the PSII
preparation was oxygen-evolving; the temperature was 200 K; and the
spectrum corresponds to
S2QA -minus-S1QA.
In B, the PSII preparation was depleted of manganese with
hydroxylamine and EDTA; the temperature was 200 K; and the spectrum
corresponds to
Chl+QA
-minus-ChlQA.
In C, the PSII preparation was oxygen-evolving, but the
temperature was 80 K, so the spectrum corresponds to
Chl+QA
-minus-ChlQA.
Finally, an oxygen-evolving PSII preparation was illuminated at 200 K,
yielding an
S2QA
-minus-S1QA
spectrum as shown in A, and then this illuminated sample was
incubated at 200 K for 1 h in the dark. The results of
reillumination of this sample are shown in D. The tick
marks on the y axis represent
A = 1 × 10
3 absorbance unit.
Other spectral conditions are described under "Experimental
Procedures."
EPR signals (12, 13), the
spectra shown in Fig. 1B and Fig. 2A were
assigned to
S2QA
-minus-S1QA.
Positive features in the difference spectrum are due to
S2QA
, and negative features are
due to S1QA. This conclusion is verified by the
200 K difference spectrum shown in Fig. 2B, which was
obtained from non-oxygen-evolving spinach PSII. In generating this
spectrum, PSII samples treated with hydroxylamine and the metal
chelator EDTA were employed. Hydroxylamine treatment of PSII, under
these conditions, inactivates water oxidation and causes the release of
bound manganese from the enzyme (14). EDTA treatment chelates remaining
bound manganese (14). In the absence of manganese, Chl is the electron
donor to P680+ (12-14, 28, 29). When the data
in Fig. 2B are compared with spectra obtained from
oxygen-evolving PSII (Fig. 2A), intensity and amplitude
changes are observed in the 1770 to 1700 cm
1 and 1500 to
1200 cm
1 regions. This result is consistent with the
assignment of Fig. 2A to an
S2QA
-minus-S1QA
spectrum. Furthermore, Fig. 2B resembles the spectrum obtained through 80 K illumination of oxygen-evolving spinach PSII
(Fig. 2C). At this temperature, oxidation of S1
is blocked, and Chl is oxidized instead of the manganese cluster
(12-14, 28).
-minus-S1QA
spectrum (as shown in Fig. 2A), and then the sample was
incubated in the dark for 1 h at 200 K. Another round of data acquisition (i.e. dark scan, dark scan, light scan, light
scan) generated the light-minus-dark difference spectrum shown in Fig. 2D. This spectrum, shown on the same scale as Fig. 2
(A-C), has no well defined vibrational features. This
result is consistent with the conclusions that a second, full charge
separation cannot occur in this sample at 200 K and that there are no
deleterious effects due to the use of continuous illumination.
-minus-S1QA
spectra are dominated by contributions from the donor side (12-14).
This conclusion was reached on the basis of alterations in the electron
donor, changes in the temperature of illumination, and manganese
removal (see Fig. 2, for example). We have recently identified
QA
and QA contributions to the
spectrum through the use of isotopic labeling and confirmed that these
vibrational lines are small in amplitude compared with the overall
intensity of the
spectrum.3
-minus-S1QA
spectrum (Fig. 3A) is similar to data obtained from spinach
PSII particles (Figs. 1B and 2A). Previous global
15N labeling experiments have led to the conclusion that
the cyanobacterial spectrum is dominated by contributions from
glutamic/aspartic acid and glutamate/aspartate residues that are
ligating or close to the manganese cluster (12-14).2
View larger version (36K):
[in a new window]
Fig. 3.
The light-minus-dark difference FT-IR spectra
obtained at 200 K from wild-type and DE170D1 mutant PSII are shown in
A and B, respectively. The spectrum shown in
A has been reproduced in B as the dashed
line to aid comparison. The double-difference spectrum
mutant-minus-wild-type (B-minus-A) is shown in
C. A control double-difference spectrum, constructed by
averaging wild-type-minus-wild-type and mutant-minus-mutant, is shown
in D. The tick marks on the y
axis represent A = 1 × 10
3 absorbance unit. Striped areas are
assigned to the S2-minus-S1 spectrum in DE170D1
PSII. Dotted areas are assigned to the
S1-minus-S2 spectrum in wild-type PSII. Other
spectral conditions are described under "Experimental
Procedures."
-minus-S1QA
spectrum (Fig. 3A). Although we have not observed a g = 2 multiline signal from this mutant, our previous characterizations have shown that this mutant contains a functional manganese cluster and
is capable of advancement from the S1-to-S2
state (20). Also, we have observed an
Fe2+QA
EPR signal upon 200 K
illumination of DE170D1 PSII (data not shown); this result indicates
that charge separation has occurred upon illumination.
-minus-S1QA
spectra obtained from wild-type and DE170D1 PSII, a detailed comparison
of spectra obtained from mutant (Fig. 3B, solid
line) and wild-type (dashed line) PSII revealed
spectral changes in the carbonyl stretching region (1720 to 1670 cm
1) and broad spectral changes between 1600 and 1400 cm
1. These spectral alterations will be discussed in
detail below.
View larger version (24K):
[in a new window]
Fig. 4.
The EPR spectra obtained from wild-type
(WT) and DE170D1 PSII are shown in A and
B, respectively. In each panel, spectra shown by the
dashed lines were obtained before illumination, and spectra
shown by the solid lines were obtained after 200 K
illumination. In a, the wild-type light-minus-dark
difference EPR spectrum corresponding to Chl+ is shown. In
b, the DE170D1 light-minus-dark difference EPR spectrum
corresponding to Chl+ is shown. For A and
a and B and b, the chlorophyll
concentrations of the samples were 0.1 mg of Chl/ml, and the gain was
6.3 × 102. Other spectral conditions are described
under "Experimental Procedures."
1) compared with samples employed for FT-IR
spectroscopy, this result provides an upper limit for the amount of
Chl+ that could be generated in an FT-IR experiment.
Wild-type cyanobacterial and plant PSII give rise to an intense
S2 multiline signal upon illumination at 200 K (12, 13).
Taken together, these results imply that PSII reaction centers in the
DE170D1 sample can advance from the S1 to the
S2 state.
produced in the absence of
fluorescence quenchers (20), this experiment suggests that the amount
of charge separation is indistinguishable in oxygen-evolving wild-type
and DE170D1 PSII. This deduction can explain the intensities of the
difference FT-IR spectra obtained, which exhibit an overall similarity
when wild-type and mutant PSII are compared (Fig. 3, A and
B).
View larger version (28K):
[in a new window]
Fig. 5.
Shown are the light-minus-dark difference FT-IR
spectra obtained at 200 K from oxygen-evolving (A) and
hydroxylamine-treated (B) PSII, which were isolated from the
DE170D1 mutant of PSII. The spectrum shown in A has been
reproduced from Fig. 1B. The double-difference spectrum
(A-minus-B) is shown in C. The
tick marks on the y axis represent
A = 1 × 10
3 absorbance unit.
Striped areas are assigned to the
S2-minus-S1 spectrum in oxygen-evolving
DE170D1. Other spectral conditions are described under "Experimental
Procedures."
1 and 1500 to 1200 cm
1
regions when these spectra are compared. Note the apparent increase in
intensity of a 1477 cm
1 spectral feature, which, under
these conditions, can been assigned to Chl+ (32, 33).
Spectra obtained from manganese-depleted DE170D1 PSII (Fig.
5B) are similar to
Chl+QA
-minus-ChlQA
data obtained from plant PSII (see Fig. 2 (B and C) and also Refs. 12-14, 32, and 33).
is expected under
both conditions, and the amplitude of these quinone and semiquinone
contributions is small (12-14), direct one-to-one subtraction (Fig.
5C) yields a DE170D1 double-difference spectrum,
(S2/S1)-minus-(Chl+/Chl). This
double-difference spectrum reflects a contribution from amino acid
residues that are close to or ligating the manganese cluster in the
DE170D1 mutant, as well as from the oxidation of Chl. This
double-difference spectrum (Fig. 5C) exhibits sharp spectral
features in the 1734 to 1659 cm
1 region as well as broad
spectral features between 1560 and 1339 cm
1. The
frequencies of these lines are 1719 cm
1 (positive), 1699 cm
1 (negative), 1684 cm
1 (positive), 1668 cm
1 (negative), 1560 cm
1 (negative), 1462 cm
1 (negative), and 1339 cm
1 (negative).
Because the intensities of these lines decrease in amplitude
upon removal of the manganese cluster, we assign these lines or a
component of these lines to amino acid residues that are close to or
ligating manganese or calcium in the DE170D1 mutant. These data will
aid in the assignment and interpretation of the S2QA
-minus-S1QA
spectrum obtained from oxygen-evolving DE170D1 PSII (Fig.
3B).
-minus-S1QA
spectrum (Fig. 3B). This interpretation will yield structural information about the catalytic site of DE170D1. The S2QA
-minus-S1QA
spectra obtained from wild-type (Fig. 3A) and DE170D1 (Fig.
3B) PSII are complex and are made up of many overlapping positive and negative lines. Therefore, a quantitative comparison of
Fig. 3 (A and B) is necessary. These spectra are
corrected for any differences in protein concentration and path length
(27). A double-difference spectrum, DE170D1 (Fig.
3B)-minus-wild-type (Fig. 3A), will reveal the
structural changes induced by the DE170D1 mutation and associated with
the S1-to-S2 transition (Fig. 3C). Observed spectral alterations will be due to the substitution of a
glutamate for aspartate 170.
1 and negative lines at 1696 and
1669 cm
1. Broad positive spectral features, with center
frequencies at 1570 and 1496 cm
1, and a negative spectral
feature, with a center frequency at 1543 cm
1, are also
observed (Fig. 3C, dotted areas). The narrow line
width of the negative 1543 cm
1 may be the result of
cancellation of intensity in the double-difference spectrum. An
additional broad, less intense negative line may be observed at 1415 cm
1 (Fig. 3C, dotted areas). These
spectral features are above the level of noise and base line variations
observed in Fig. 3D, which shows the averaged results of
wild-type-minus-wild-type and mutant-minus-mutant subtraction.
1 region (see, for
example, Figs. 2 and 5). The EPR control experiments shown in Fig. 4
suggest an increase in Chl+ content in the mutant when
compared with wild-type PSII. (However, the spectrum obtained from
oxygen-evolving preparations of the DE170D1 mutant exhibits spectral
differences when compared with such a
Chl+QA
-minus-ChlQA
spectrum (Fig. 5).) Lines assignable to Chl+ will increase
in intensity upon manganese depletion.
1 Spectral
Region--
Spectral features with frequencies of 1719 cm
1 (positive), 1696 cm
1 (negative), 1683 cm
1 (positive), and 1669 cm
1 (negative) are
observed in Fig. 3C (striped areas), derived from a comparison of wild-type and DE170D1 PSII. Furthermore, lines with
similar frequencies (1719, 1699, 1684, and 1668 cm
1) and
line widths are observed in Fig. 5C (striped
areas), derived from a comparison of oxygen-evolving and
manganese-depleted DE170D1 mutants. These spectral features have the
same sign in both double-difference spectra. Therefore, we assign these
spectral features to amino acid residues in the DE170D1 mutant that are
perturbed by the S1-to-S2 transition.
1
Spectral Region Assigned to the DE170D1 Mutant--
The only amino
acid residues with fundamental vibrational transitions in the 1750 to
1670 cm
1 region are glutamic and aspartic acid (34-37).
The observation of two different, derivative-shaped lines (1719 (positive)/1696 (negative) cm
1 and 1683 (positive)/1669
(negative) cm
1) suggests that at least two, and possibly
more, glutamic or aspartic acid residues are perturbed in the DE170D1
mutant during the S1-to-S2 transition. One of
these perturbed residues may be glutamic acid 170. Because these amino
acid residues are perturbed by the S1-to-S2 transition, these amino acid residues must be close to or ligating to
the metal cluster.
1 region (35). The second type of structural alteration
that could give rise to such a spectral contribution is a protonation change (35-37). The C=O and C-O stretches of the carboxylate are intermediate in frequency between the C=O and C-O stretching
vibrations of the carboxylic acid (35, 38). The third explanation is that these groups are ligating manganese and that the oxidation of
metal causes a frequency shift of vibrational modes, assignable to the
ligand (38, 39). On the basis of the pattern of observed frequencies,
we favor the third possible explanation of these spectra, as described below.
1 (35). Therefore, a
frequency of 1719 cm
1, as observed in the DE170D1 mutant
(Figs. 3C and 5C), can be regarded as typical of
the C=O vibration of uncoordinated carboxylic acid groups. Free
carboxylic acids give rise to C-O frequencies in the 1300 to 1200 cm
1 range2; high absorption makes it
difficult to interpret this spectral region.
1 (negative), 1683 cm
1 (positive), and 1669 cm
1 (negative),
observed in the DE170D1 mutant (Figs. 3C and 5C) are out of the range expected for the C=O vibration of free carboxylic acid residues in proteins. Because of their low frequency, we attribute
these lines to carboxylate ligands to manganese or calcium. Carboxylate
groups may ligate metals with unidentate, bidentate, or bridging
geometries. An asymmetric CO frequency in the range from 1629 to 1604 cm
1 is consistent with unidentate ligation to metals in
mononuclear clusters (38, 40). However, the nuclearity and the
oxidation state of the metal are expected to influence these
frequencies (38-40). The energy gap between the asymmetric and
symmetric stretching vibrations of carboxylates is also influenced by
metal binding (38, 40). The
for free aspartate in aqueous
solution is 183 cm
1, and that for free glutamate is 236 cm
1.2 Unidentate ligands are expected to have
energy gaps larger than these values (38, 40).
1 (Fig. 3C, striped
areas) are assigned to the asymmetric CO vibration of unidentate
carboxylate ligands to the DE170D1 manganese cluster (Fig.
6A). Unidentate ligands to
mononuclear clusters are expected to give rise to symmetric CO
stretching frequencies in the 1300 to 1200 cm
1 range (38,
40).
View larger version (16K):
[in a new window]
Fig. 6.
Speculative model of the manganese cluster in
the DE170D1 mutant (A) and in wild-type PSII
(B) showing carboxylate shifts. The
dimer-of-dimers model for the manganese cluster was developed based on
x-ray absorption studies of PSII (reviewed in 11). Only two ligands to
one manganese are shown explicitly.
1. The fact that derivative-shaped lines are observed
is consistent with a perturbation of frequency upon manganese oxidation
and the assignment to unidentate manganese ligands. In addition, the direction of the frequency shift observed upon oxidation of manganese during the S1-to-S2 transition supports the
assignment of these vibrational features to manganese ligands (Fig.
3A). These lines are assigned to the DE170D1 mutant;
therefore, the double-difference spectrum (Fig. 3C) is
associated with S2-minus-S1. For a unidentate carboxylate ligand, oxidation of bound manganese should result in a
shortening of the O-Mn bond and a corresponding increase in the
frequency of the asymmetric stretching vibration (Fig. 6A).
The pairing of 1683 cm
1 (positive, S2 state)
and 1669 cm
1 (negative, S1 state) lines is
consistent with such an upshift in CO frequency upon manganese
oxidation (Fig. 3C, striped areas). The pairing
of the 1719 cm
1 (positive, S2 state) and 1696 cm
1 (negative, S1 state) lines is also
consistent with such an upshift (Fig. 3C, striped
areas). However, as discussed above, the
S1-to-S2 transition-induced change from 1696 to
1719 cm
1 is also consistent with an oxidation-induced
change from unidentate ligation to free carboxylic acid residue (Fig.
6A).
1 Spectral
Region--
Spectral features with center frequencies of 1570 cm
1 (positive), 1543 cm
1 (negative), 1496 cm
1 (positive), and, possibly, 1415 cm
1
(negative) are observed in Fig. 3C (dotted
areas), derived from a comparison of wild-type and DE170D1 PSII.
Furthermore, the same pattern of frequencies and intensities is
not observed in Fig. 5C, derived from a
comparison of oxygen-evolving and manganese-depleted DE170D1 mutants.
Therefore, the simplest interpretation of the spectrum is that these
spectral features arise from amino acid residues in wild-type PSII that
are perturbed by the S1-to-S2 transition. Note
the unusually large line width of these lines, which can be attributed
to homogeneous or inhomogeneous broadening mechanisms (see Refs. 12-14
and "Discussion").
1
Spectral Region Assigned to Wild-type PSII--
Positive spectral
features, observed at 1570 and 1496 cm
1, and negative
spectral features, observed at 1543 cm
1 and, possibly,
1415 cm
1, have been assigned to amino acid residues in
wild-type PSII (Fig. 3C, dotted areas). The
frequencies of these lines provide information concerning
their origin. Frequencies of lines assigned to wild-type PSII
(double-difference spectrum; Fig. 3C, dotted areas), 1570 cm
1 (positive), 1543 cm
1
(negative), 1496 cm
1 (positive), and 1415 cm
1 (negative), are in the range of frequencies expected
for carboxylate anions that are ligating to high valence manganese. For
example, bridging carboxylate ligands to Mn3+ dinuclear
complexes gave rise to bands in the 1570 to 1415 cm
1
region, and Mn3+·Mn4+ dinuclear complexes
gave rise to bands in the 1540 to 1440 cm
1 region (39,
41). By contrast, free aspartate has asymmetric and symmetric
stretching vibrations at 1574 and 1391 cm
1, respectively,
and free glutamate has asymmetric and symmetric stretching vibrations
at 1555 and 1399 cm
1, respectively.2 On the
basis of this comparison and consideration of 15N labeling
experiments (13), we favor the assignment of these lines to carboxylate
ligands. It is reasonable to expect that a component of or all of these
broad spectral features in the double-difference spectrum (Fig.
3C, dotted areas) may arise from aspartate 170 (see "Discussion").
for free aspartate in aqueous solution is 183 cm
1, and that for free glutamate is 236 cm
1.2 As discussed above, unidentate ligands
are expected to have energy gaps larger than these values (38). When
carboxylates bind to metals with bridging ligation, the energy gap
between the asymmetric and symmetric stretching vibrations may be
similar to that of the free carboxylate (38, 40). In recent studies,
was also found to be dependent on the oxidation state of the
manganese, (39, 40). When carboxylates bind to metals with bidentate ligation, the characteristic spectral change is a decrease in
between the asymmetric and symmetric modes of the carboxylate (38,
40).
equal to 74 cm
1 for the positive lines and 128 cm
1 for
the negative lines. Although these values can be influenced by the
oxidation state of the metal and by cancellation of intensity in the
difference spectra (13, 39, 40), these numbers are far smaller than the
expected energy gap between asymmetric and symmetric stretching
vibrations of free carboxylates that are not ligating metals. These
numbers are smaller or on the same order of magnitude as the expected
energy gap for bridging carboxylate ligands. For example, in a study of
trimeric and dimeric manganese compounds, bridging carboxylates gave
rise to energy splitting of 220 to 100 cm
1, with the
smaller values of
arising from
Mn3+·Mn4+ dinuclear complexes (39, 41). We
conclude that the broad lines in Fig. 3C (dotted
areas) may arise from bidentate or bridging carboxylate ligands to
the manganese cluster (Fig. 6B).
1 in comparing Mn2+ and
Mn4+ dinuclear complexes (39, 41). We expect similar
behavior for bidentate ligands. The pairing of 1570 cm
1
(positive, S1 state) and 1543 cm
1 (negative,
S2 state) lines is consistent with such a downshift in the
C-O frequency upon manganese oxidation (Fig. 3C,
dotted areas). The pairing of the 1496 cm
1
(positive, S1 state) and 1415 cm
1 (negative,
S2 state) lines is also consistent with such a downshift (Fig. 3C, dotted areas). Thus, both the direction
of the shift and the frequency of the lines are consistent with
assignment to bridging or bidentate carboxylate ligands of the
manganese cluster (Fig. 6B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minus-S1QA
FT-IR spectrum has been obtained under these conditions (12-14). In
these studies, we have used the
S2QA
-minus-S1QA
FT-IR spectrum to detect a structural change, involving carboxylate
coordination of manganese or calcium, in DE170D1 PSII. The frequencies,
direction of the frequency shift, and observed splittings of observed
spectral features support the interpretation that the mode of
manganese/calcium ligation is altered from bridging/bidentate to
unidentate when aspartate 170 is changed to a glutamate. The exact
number of ligands altered is not known, but because two different, new
derivative-shaped lines (1719/1696 and 1683/1669 cm
1) are
observed in the DE170D1 spectrum, we deduce that at least two different
coordinating carboxylates are affected. The ~1.5-Å increase in side
chain length is presumed to be the cause of these alterations in metal
coordination. The frequencies of the observed vibrational lines and the
direction of the observed shifts are consistent with the assignment of
spectral lines to high valence manganese ligands. Significantly, the
altered active site is still active in oxygen evolution.
-minus-ChlQA
spectrum upon illumination at 200 K (14). This work is consistent with
a high affinity binding site for manganese on the donor side of
wild-type PSII. On the other hand, hydroxylamine treatment of DE170D1
results in a PSII preparation that gives rise to a characteristic
Chl+QA
-minus-ChlQA
spectrum upon 200 K illumination without treatment with EDTA
(Fig. 5). This result is consistent with a change in binding affinity
for manganese in this mutant. A change in the Km for
manganese oxidation has been observed previously in non-oxygen-evolving
core preparations from the DE170D1 mutant (21).
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant MCB-9808934.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Howard Hughes Medical Inst., Stanford Medical
School, Stanford, CA 94305.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 1479 Gortner Ave., St. Paul, MN 55108-1022. Tel.: 612-624-6732; Fax: 612-625-5780; E-mail: barry{at}biosci.cbs.umn.edu.
2 R. S. Hutchison, J. J. Steenhuis, C. F. Yocum, R. M. Razeghifard, and B. A. Barry, submitted for publication.
3 M. R. Razeghifard, J. J. Steenhuis, S. Kim, J. Patzlaff, R. S. Hutchison, I. Ayala, and B. A. Barry, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PSII, photosystem II; FT, Fourier transform; Chl, chlorophyll; QA, plastoquinone acceptor.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|