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Alteration of the O2‑Producing Mn4Ca Cluster in Photosystem II by

the Mutation of a Metal Ligand
Richard J. Debus*

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ABSTRACT: The O2-evolving Mn4Ca cluster in photosystem II (PSII) is arranged as a

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distorted Mn3Ca cube that is linked to a fourth Mn ion (denoted as Mn4) by two oxo bridges.
The Mn4 and Ca ions are bridged by residue D1-D170. This is also the only residue known to
participate in the high-affinity Mn(II) site that participates in the light-driven assembly of the
Mn4Ca cluster. In this study, we use Fourier transform infrared difference spectroscopy to
characterize the impact of the D1-D170E mutation. On the basis of analyses of carboxylate and
carbonyl stretching modes and the O−H stretching modes of hydrogen-bonded water
molecules, we show that this mutation alters the extensive network of hydrogen bonds that
surrounds the Mn4Ca cluster in the same manner as that of many other mutations. It also alters
the equilibrium between conformers of the Mn4Ca cluster in the dark-stable S1 state so that a
high-spin form of the S2 state is produced during the S1-to-S2 transition instead of the low-spin
form that gives rise to the S2 state multiline electron paramagnetic resonance signal. The mutation may also change the coordination
mode of the carboxylate group at position 170 to unidentate ligation of Mn4. This is the first mutation of a metal ligand in PSII that
substantially impacts the spectroscopic signatures of the Mn4Ca cluster without substantially eliminating O2 evolution. The results
have significant implications for our understanding of the roles of alternate active/inactive conformers of the Mn4Ca cluster in the
mechanism of O2 formation.

■ INTRODUCTION
Plants, algae, and cyanobacteria use sunlight to oxidize water,
releasing O2 that sustains all aerobic life and providing the
electrons and protons that drive the production of earth’s
biomass from atmospheric CO2. Water oxidation is catalyzed by
the Mn4Ca cluster in photosystem II (PSII). Our understanding
of water oxidation by the Mn4Ca cluster has progressed rapidly
in recent years because of advances in structural, computational,
and biophysical methods (for review, see refs 1−5). PSII is a
large dimeric protein complex that is integral to the thylakoid
membrane and is surrounded by a light-harvesting apparatus
that increases the light-absorbing cross-section of PSII and
transfers excitation energy to the PSII core. Each monomer of
the PSII core contains approximately 20 subunits and has a
molecular weight of approximately 350 kDa. High-resolution
(1.85−1.95 Å) structures of PSII from cyanobacteria have been
determined using X-ray crystallography6−8 and cryogenic
electron microscopy (cryo-EM).9 Moderate-resolution (2.7−
5.3 Å) structures from plants, algae, and a diatom have been
determined using X-ray crystallography10 and cryo-EM.11−18
Structures of intermediates of water oxidation by the Mn4Ca
cluster have been determined using serial femtosecond X-ray
crystallography.19−23
Each monomer of PSII contains a heterodimer of the two
subunits D1 and D2. Light-induced charge separation between
P680 (a chlorophyll multimer that serves as the light-induced
electron donor in PSII) and a pheophytin molecule in the D1/
D2 heterodimer drives the oxidation of the Mn4Ca cluster, with
redox-active tyrosine YZ serving as an electron transfer
intermediate between the Mn4Ca cluster and P680•+. Four
electrons are removed from the Mn4Ca cluster during each
catalytic cycle, advancing the cluster through five oxidation
states termed Sn, where “n” denotes the number of oxidizing
equivalents stored (n = 0−4). Dark-adapted PSII is poised in the
S1 state. Both substrate water molecules are bound to the Mn4Ca
cluster by at least the S2 state.24−26 The S4 state is a transient
intermediate whose formation triggers the utilization of the four
stored oxidizing equivalents to form O2 from two substrate-
water-derived metal ligands. This process regenerates the S0
state and involves the binding of at least one of the two new
substrate water molecules. Because of its transient nature, the S4
state remains uncharacterized.
In the most supported model for the Mn4Ca cluster’s
oxidation states,27−34 the S0-to-S1, S1-to-S2, and S2-to-S3
transitions each corresponds to the oxidation of one Mn(III)
ion to Mn(IV), where the S1 state consists of two Mn(III) and
Received: July 27, 2021
Revised: September 27, 2021
Published: December 13, 2021

© 2021 American Chemical Society https://doi.org/10.1021/acs.biochem.1c00504

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two Mn(IV) ions, the S2 state contains a single five-coordinate
Mn(III) ion, and the S3 state consists of four octahedrally
coordinated Mn(IV) ions, with a water-derived ligand relocating
to the empty coordination position during the S2-to-S3 transition
(see below).29,32,33,35 The S3-to-S4 transition has been proposed
to correspond to the formation of an oxyl radical on Mn1,36,37 an
oxyl radical on Mn4,26,38,39 a Mn(V) ion at Mn4,40−42 or a
Mn(VII) ion at Mn4.43 The oxidations of the Mn4Ca cluster are
coupled to the release of protons, with electrons and protons
being removed alternatively as the S state cycle proceeds.44−46
This alternation prevents the redox potential of the Mn4Ca
cluster from increasing to levels that prevent its subsequent
oxidation by YZ•. During the S2-to-S3 and S3-to-S4 transitions,
proton deprotonation precedes the oxidation of the Mn4Ca
cluster.44−49 The cluster is surrounded by an extensive network
of H-bonds that merge into three well-defined pathways that
lead to the thylakoid lumen. These pathways facilitate proton
egress and/or substrate water access.6,20−22,50−58
The Mn4Ca cluster’s five metal ions are connected by oxo
bridges and are ligated by six bridging carboxylate groups and
one histidine side chain. All but one of these protein ligands are
provided by the D1 polypeptide (see Figure 1). Numerous
Figure 1. Water molecules and selected residues in the vicinity of the
Mn4Ca cluster and D1-D170 based on 4UB6.7 All residues shown are
from the D1 subunit, unless indicated otherwise. The salmon-colored
spheres are the manganese ions. The yellow sphere is the Ca ion. The
red spheres are the oxygen atoms of water molecules and μ-oxo bridges.
Water molecules are labeled as in ref 22.
immobilized water molecules are located on or near the Mn4Ca
cluster, including two (W1 and W2) that are bound to Mn4 and
two (W3 and W4) that are bound to the Ca ion. In the S1 state,
the Mn4Ca cluster is arranged as a distorted Mn3CaO4 cube that
is linked to a fourth Mn ion (denoted Mn4) by two oxo bridges
denoted as O4 and O5, with O5 located between Mn4 and Mn1.
Although not universally accepted, considerable evidence
supports the identification of O5 as the deprotonated form of
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one of the two substrate water molecules that ultimately form
the O−O bond.59−63 In at least the S1,64−67 S2,65,66,68−71 and
S335,42,66,70−75 states, the Mn4Ca cluster exists in multiple
conformers. In each S state, the different conformers differ
slightly in structure, Mn-oxidation state distribution, proto-
nation, and/or water ligation. In the S2 state, a low-spin
conformer gives rise to the multiline electron paramagnetic
resonance (EPR) signal centered at g = 2, whereas high-spin
conformers give rise to broad featureless signals appearing at g =
4.1 to g = 4.7.65,66,68−71,76 In the S3 state, multiple conformers
exist that have S = 3,35,42,66,70−75 and a conformer having S = 6
has been proposed to correspond to a precursor form of the S3
state having a five-coordinate Mn(IV) ion,74 although in an
alternate proposal, it corresponds to the glycerol-induced
distortion of a six-coordinate Mn(IV) ion.75 There is
considerable evidence that a high-spin conformer of the S2
state is required for the advancement to the S 3
state,1,35,42,66,70−72,74,77−80 but the nature of this high-spin
state remains under debate (e.g., see refs 63, 67, 70, 71, and 81−
84). The prevailing view is that the low-spin conformer of the S2
state has O5 bound to Mn4 (the “open” conformation) and that
the high-spin conformer has O5 bound to Mn1 (giving rise to a
cubane-like “closed” conformation).85−87 In this view, the
toggling of O5 between Mn4 and Mn1 is linked to redox
isomerization, with the five-coordinate Mn(III) ion being
located at the Mn1 position in the “open” conformer and at
the Mn4 position in the “closed” conformer. However, several
alternate models for high-spin conformers of the S2 state having
“open” configurations have been proposed recently.63,70,71,81−84
During the S2-to-S3 transition, a water molecule, or a water-
derived metal ligand, deprotonates and moves to a position on
Mn1 or Mn4 in close proximity to O5, completing the empty
coordination position of the S 2 state’s Mn(III)
ion.19−22,29,32,33,36,46,47,88 This oxygen has been denoted
O619,21 or Ox20,22 and forms an oxo or hydroxo bridge
connecting Mn1 to the Ca ion. The origin of O6/Ox has been
debated. In some models (e.g., the “carousel”79,89 and
“pivot”78,90 models), O6/Ox originates from the Mn4-bound
W2. In competing models,81,91−96 O6/Ox originates from the
Ca-bound W3. Serial femtosecond X-ray crystallographic data
are more compatible with O6/Ox originating from W3.20−22 If
O6/Ox originates from W3, then this oxygen could be a
deprotonated form of the second substrate water molecule that
combines with O5 in a radical-coupling mechanism to form the
O−O bond.29,36,39,47,78,81,88,90−92,97−100 However, if W2 is the
second substrate water molecule and O6/Ox originates from
W3, then O6/Ox may simply be positioned on Mn1 during the
S2-to-S3 transition to replace O5 during the S4-to-S0 transition,
thereby becoming a substrate during the next cycle of O2
formation.20,63 The details of the events comprising the S2-to-
S3 transition and the nature of the various forms of the S3 state
are currently under intense scrutiny because of their impact on
the mechanism of O−O bond formation during the S3-to-S4-to-
S0 transition.
Attempts to identify the Mn4Ca cluster’s ligands with site-
directed mutagenesis began long before X-ray crystallographic
structures were available. The first residue targeted was D1-
D170 because of its proximity in the amino acid sequence to YZ
(D1-Y161). Of the 19 mutations constructed at this position,
only D1-D170E, D1-D170H, and D1-D170V cells are photo-
autotrophic, with D1-D170H and D1-D170V cells being only
weakly photoautotrophic.101−106 In contrast, nonphotoauto-
trophic mutants D1-D170N, D1-D170A, and D1-D170S abolish
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the high-affinity binding site for Mn(II) in apo-PSII and
assemble no Mn4Ca clusters.101,103 Characterization of O2-
evolving mutants initially focused on D1-D170E. Cells and PSII
core complexes of this mutant were found to have a slightly
stabilized S2 state,101,102,104 and the high-affinity Mn(II) site on
the D1-D170E apo-protein was sufficiently altered that freezing
under illumination in the presence of Mn(II) ions produced a
Mn(IV) ion rather than Mn(III).107 However, no S2 state
multiline EPR signal was detected,108,109 and initial attempts to
record a mid-frequency S2−S1 Fourier transform infrared
(FTIR) difference spectrum were unsuccessful. Consequently,
characterization shifted to D1-D170H PSII core complexes.
These exhibit S1 and S2 state multiline EPR signals,110 S2-state
electron-spin echo envelope modulation spectra,110,111 and mid-
frequency FTIR difference spectra112,113 that are unchanged
from wild-type, although the low-frequency S2−S1 FTIR
difference spectrum shows that the D1-D170H mutation shifts
the 606 cm−1 Mn−O−Mn cluster mode to 612 cm−1.112 The
side chain of D1-D170 bridges Mn4 with the Ca ion and forms
multiple H-bonds with CP43-R357. Consequently, the muta-
tion of D1-D170 would be expected to cause alterations in the
network of H-bonds that surrounds the Mn4Ca cluster. Because
FTIR difference spectroscopy is highly sensitive to changes in
bonding,114−116 the absence of mutation-induced changes in
any of the mid-frequency Sn+1−Sn FTIR difference spectra of
D1-D170H112,113 has been extremely puzzling. However, a
recent mass spectrometry analysis of D1-D170H PSII core
complexes showed that the mass of the polypeptide fragment
including position 170 was heterogeneous, with 70% of the mass
implying the presence of D170 despite there being no trace of
the wild-type codon in the genome.117 Approximately 7% of the
mass implied the presence of N170, and only 23% implied the
presence of the expected H170. Although no mechanism was
identified to explain this apparent posttranslational modifica-
tion, there is precedent for histidine being oxidized to aspartate
and asparagine in proteins.118 Furthermore, because many
current models for the O2 formation place the most powerful
oxidizing component of the S4 state at the Mn4 position [i.e.,
oxyl radical, Mn(V), or Mn(VII)], Mn4Ca clusters in D1-
D170H might preferentially oxidize D1-H170 to D1-D170
without oxidizing either D1-H332 or D1-H337 (this possibility
was suggested to us by J. Wang, W. H. Armstrong, G. W.
Brudvig, and V. S. Batista). Because of the new uncertainty about
the D1-D170H mutation, we have characterized D1-D170E
PSII core complexes in the current study with FTIR difference
spectroscopy. This method has been employed extensively to
study the S state cycle in PSII119−123 and is particularly suited for
probing changes in H-bonding. On the basis of the analysis of
FTIR difference spectra, we conclude that the D1-D170E
mutation alters (1) the network of H-bonds surrounding the
Mn4Ca cluster in much the same manner as that of other
mutations constructed in this network, (2) the equilibrium
between conformers of the Mn4Ca cluster in the S1 state so that a
high-spin form of the S2 state is produced during the S1-to-S2
transition, and (3) possibly the coordination mode of the
carboxylate group at position 170.
■ MATERIALS AND METHODS
Construction of Mutants and Purification of PSII Core
Complexes. The D1-D170E mutation was constructed
previously in the psbA-2 gene of Synechocystis sp. PCC 6803104
and transformed into a host strain of Synechocystis that lacks all
three psbA genes and contains a hexahistidine tag fused to the C-
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terminus of CP47.124 The wild-type strain described in this
article was constructed in an identical manner as that of the D1-
D170E strain but with a transforming plasmid that carried no
mutation. Large-scale cultures of Synechocystis cells were
propagated as described previously.125 PSII core complexes
were purified under dim green light at 4 °C with a Ni-NTA
superflow affinity resin (Qiagen, Valencia, CA), as described
previously.125 The purification buffer consisted of 1.2 M betaine,
10% (v/v) glycerol, 50 mM 2-(N-morpholino)-ethanesulfonic
acid (MES)−NaOH (pH 6.0), 20 mM CaCl2, 5 mM MgCl2, 50
mM histidine, 1 mM ethylenediaminetetraacetic acid (EDTA),
and 0.03% (w/v) n-dodecyl β-D-maltoside. The purified PSII
core complexes were concentrated to approx. 1 mg of Chl per
mL, flash-frozen in liquid N2, and stored at −80 °C. An aliquot of
each large-scale culture of D1-D170E was set aside, and the
sequence of the relevant portion of the psbA-2 gene was obtained
after the PCR amplification of genomic DNA.104 No trace of the
wild-type codon was detected in any of the mutant cultures.
Preparation of FTIR Samples. PSII core complexes were
transferred into 40 mM sucrose, 10 mM MES−NaOH (pH 6.0),
5 mM CaCl2, 5 mM NaCl, and 0.06% (w/v) n-dodecyl β-D-
maltoside, concentrated, mixed with 1/10 volume of fresh 100
mM potassium ferricyanide, spread in the center 13 mm of a 25
mm BaF2 window, and then dried lightly with N2 gas, as
described previously.93 The lightly dried samples were
rehydrated with six 1 μL drops of a solution of 20% (v/v)
glycerol placed around the periphery of the lightly dried sample,
though without touching it. This concentration of glycerol
maintains the sample humidity at 99% RH.126 A second window
was placed over the first with a thin O-ring spacer in between
.93,95,125,127,128 Sealed samples were equilibrated in the FTIR
sample compartment at 0 °C in darkness for 1.5 h, illuminated
with six preflashes, and then dark-adapted for 30
min.93,95,125,127,128 For each sample, the absorbance at 1657
cm−1 (amide I band) was 0.6−1.1.
FTIR Spectroscopy. Spectra were recorded using a Bruker
Vertex 70 spectrometer (Bruker Optics, Billerica, MA) outfitted
with a preamplified, midrange D317 photovoltaic MCT detector
(Kolmar Technologies, Inc., Newburyport, MA), as described in
ref 93. Actinic flashes (∼20 mJ/flash, ∼7 ns fwhm) were
provided by a frequency-doubled Q-switched Nd:YAG laser
(BRIO, Quantel USA, Bozeman, MT). After the dark
adaptation, samples were given six flashes at 13 s intervals.
Two transmission spectra were recorded before the first flash,
and one transmission spectrum was recorded starting at 0.33 s
after the first and subsequent flashes (each transmission
spectrum consisted of 100 scans). The 0.33 s delay was included
to allow the oxidation of QA•− (primary plastoquinone electron
acceptor) by ferricyanide. Difference spectra of the successive S-
state transitions (e.g., Sn+1−minus−Sn difference spectra, written
as Sn+1−Sn in this study) were obtained by dividing the
transmission spectrum obtained after the nth flash by the
transmission spectrum obtained before the nth flash and then
converting the ratio to units of absorption. The background
noise level and the stability of the baseline were obtained by
dividing the second preflash transmission spectrum by the first
and converting the ratio to units of absorption (these spectra are
labeled dark−dark in each figurethese are control difference
spectra obtained in the presence of the sample but without a flash
being given). The sample was then dark-adapted for 30 min, and
the cycle was repeated. For each sample, the illumination cycle
was repeated 19 times. The spectra of 12−26 samples were
averaged (see figure legends). The amplitudes of the D1-D170E
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difference spectra were multiplied by factors of 1.1−1.4 to

normalize the amplitudes of the peaks corresponding to the
reduction of ferricyanide to ferrocyanide by QA•− (at 2115 and
2037 cm−1, respectively) to those in the corresponding wild-type
spectra (this procedure normalizes the spectra to the extent of
the flash-induced formation of QA•−).
Other Procedures. Chlorophyll concentrations and initial
light-saturated rates of O2 evolution were measured as described
previously.129

■ RESULTS
The D1-D170E cells employed for this study evolved O2 at a rate
of 320 ± 12 μmol O2 (mg of Chl)−1 h−1 under light-saturating
conditions compared to 635 ± 50 μmol O2 (mg of Chl)−1 h−1 for
the wild-type cells. This activity (51 ± 5% compared to the wild-
type) corresponds to the activity of D1-D170E cells reported
previously (55 ± 5% compared to the wild-type).102,104
Previously, 75−80% of D1-D170E PSII centers were estimated
to contain Mn4Ca clusters in vivo.104 Notably, because the
activity is substantially less than the fraction of PSII centers
containing Mn4Ca clusters, the efficiency of O2 formation in D1-
D170E PSII centers is less than that in the wild-type. The D1-
D170E PSII core complexes examined in this study evolved O2
at a rate of 3.1 ± 0.3 mmol O2 (mg of Chl)−1 h−1 under light-
saturated conditions compared to 5.8 ± 0.4 mmol O2 (mg of
Chl)−1 h−1 for the wild-type PSII core complexes. This activity
(53 ± 6% compared to the wild-type) correlates with the O2-
evolving activity of the D1-D170E cells, showing that the D1-
D170E PSII core complexes were not damaged during their
purification.
Mid-Frequency Region. The mid-frequency FTIR differ-
ence spectra produced by the first through sixth actinic flashes
given to wild-type and D1-D170E PSII core complexes are Figure 2. Mid-frequency FTIR difference spectra of wild-type (black)
and D1-D170E (red) PSII core complexes in response to six successive
compared in Figure 2. In wild-type, the spectra produced by the flash illuminations applied at 0 °C. The wild-type data represent the
first and fifth flashes correspond predominantly to the S1-to-S2 averages of 24 samples (42,600 scans for each trace). The D1-D170E
transition, the spectra produced by the second and sixth flashes data represent the averages of 26 samples (40,800 scans for each trace).
correspond predominantly to the S2-to-S3 transition, the The D1-D170E spectra were multiplied by factors of 1.1−1.4 to
spectrum produced by the third flash corresponds predom- normalize the wild-type and mutant spectra to the extent of the flash-
inantly to the S3-to-S0 transition, and the spectrum produced by induced formation of QA•−. The dark−dark control traces show the
the fourth flash corresponds predominantly to the S0-to-S1 noise level and the stability of the baseline (lower traces). The dark−
transition.119−123 dark control trace of the mutant was multiplied by a factor of 1.3.
Despite being normalized to the extent of the flash-induced
formation of QA•−, the features in most of the D1-D170E spectra 1509(+) cm−1 feature was sharply diminished. The 1586(+)
are substantially less intense than those in the corresponding and 1561(−) cm−1 features correspond to νasym(COO−) modes
wild-type spectra. Indeed, the spectra produced by the third and because they shift in globally 13C-labeled PSII130−132 but not in
fourth flashes applied to D1-D170E PSII core complexes are globally 15N-labeled PSII.130−134 The 1552(+), 1543(−),
nearly featureless. Nevertheless, the features in the D1-D170E 1531(+), and 1522(−) cm−1 features correspond to amide II
spectra produced by the fifth and sixth flashes resemble those modes because they shift in both 13C-labeled130−132 and 15N-
produced by the first and second flashes, showing that the D1- labeled130−134 PSII. The 1509(+) cm−1 feature consists of
D170E PSII core complexes advanced through the S state cycle overlapping amide II and νasym(COO−) modes.133 In the
in response to the six applied flashes. symmetric carboxylate stretching [νsym(COO−)] region, the
The D1-D170E spectrum produced by the first flash shows 1434(+) cm−1 feature was replaced by two less intense positive
that the mutation produces substantial alterations to the S2−S1 features at 1443 and 1427 cm−1, the prominent 1400 (−) and
spectrum. In the carbonyl stretching [ν(CO)] region, the 1364(+) cm−1 features were eliminated, a negative feature
negative feature at 1747 cm−1 was substantially diminished. In appeared at 1355 cm−1, and a positive feature appeared at 1341
the amide I region, the 1680(−) cm−1 feature was eliminated, cm−1.
the 1651(+) cm−1 feature was diminished, and the 1640(−) and The D1-D170E spectrum produced by the second flash shows
1623(+) cm−1 features were eliminated or substantially that the mutation also produces substantial alterations to the
diminished. In the overlapping asymmetric carboxylate stretch- S3−S2 spectrum. In the amide I region, the 1675(−)/1668(+)
ing [νasym(COO−)] and amide II region, the large 1586(+) cm−1 cm−1 derivative-shaped feature was eliminated and the 1633(+)
feature was almost entirely eliminated, the 1561(−), 1552(+), cm−1 feature was diminished. In the overlapping asymmetric
and 1543(−) cm−1 features were diminished, the 1531(+)/ carboxylate stretching [νasym(COO−)] and amide II region, the
1522(−) cm−1 derivative feature was abolished, and the 1596(+), 1569(−), and 1545(−) cm −1 features were
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diminished, and the 1524(+), 1507(+), and 1495(−) cm−1

features were replaced by a 1519(−)/1503(+) cm−1 derivative-
shaped feature. The 1596(+) and 1569(−) cm−1 features
correspond to νasym(COO−) modes because they shift in
globally 13C-labeled PSII130,132 but not in globally 15N-labeled
PSII.130,132 The 1524(+) and 1495(−) cm−1 features corre-
spond to amide II modes because they shift in both 13C-
labeled130,132 and 15N-labeled130,132 PSII. In the symmetric
carboxylate stretching [νsym(COO−)] region, the large 1445(+)
feature was substantially diminished, the negative features at
1422, 1396, and 1362 cm−1 were eliminated, and the positive
features at 1344 and 1326 cm−1 were abolished.
The D1-D170E spectra produced by the third and fourth
flashes show few features above the noise level. Typically,
spectral features that appear in the FTIR difference spectra
during the S1-to-S2 and S2-to-S3 transitions are reversed during
the S3-to-S0 and S0-to-S1 transitions.119−123 The general lack of
features in the third- and fourth-flash D1-D170E spectra,
especially in the in the νsym(COO−) region, may reflect the
diminished amplitudes of most of the features in the mutant S2−
S1 and S3−S2 spectra. As noted above, the features in the fifth-
and six-flash D1-D170E spectra resemble those produced by the
first and second flashes, demonstrating the flash-induced
advancement through the S state cycle by the mutant PSII
core complexes.
To isolate the vibrational features in the S2−S1 spectrum that
are altered by the D1-D170E mutation and to display them more
clearly, a WT-minus-mutant double-difference spectrum was
calculated for the spectrum produced by the first flash (Figure
3). The largest features in this spectrum emphasize the

Figure 4. FTIR difference spectra of wild-type (black) and D1-D170E

(red) PSII core complexes in the weakly H-bonded O−H stretching
region in response to four successive flash illuminations applied at 0 °C.
The data were collected simultaneously with those shown in Figure 2.
Dark−dark control traces show the noise level and the stability of the
baseline (lower traces).

by the first, second, third, and fourth flashes correspond

Figure 3. Double-difference spectrum (WT-minus-mutant) obtained
by subtracting the FTIR difference spectra of D1-D170E PSII core
complexes produced by the first flash from the corresponding spectrum
of wild-type PSII core complexes. The first-flash data in Figure 2 were
subtracted directly.
mutation-induced elimination of νsym(COO−) modes at
1398(−) and 1361(+) cm−1 and the elimination of the
νasym(COO−) mode at 1587(+) cm−1. The double-difference
spectrum also shows the mutation-induced elimination of the
νasym(COO−) mode at 1562(−) cm−1.
Weakly H-Bonded O−H Stretching Region. Changes in
the O−H stretching vibrations of weakly H-bonded O−H
groups of water molecules in PSII can be observed as mostly
negative features between 3700 and 3500 cm−1.120−123 These
regions of the FTIR difference spectra of D1-D170E PSII core
complexes are compared with those of the wild-type complexes
in Figure 4. For the wild-type complexes, the spectra produced
3845
predominantly to the S1-to-S2, S2-to-S3, S3-to-S0, and S0-to-S1
transitions, respectively. The spectrum produced by the first
flash shows that in the S2−S1 spectrum, the D1-D170E mutation
eliminates the small negative feature at 3663 cm−1 and the broad
positive feature that is centered at 3617 cm−1, replacing the latter
with a broad negative feature at 3604 cm−1. The spectra
produced by the second, third, and fourth flashes show that the
differences between D1-D1-D170E and wild-type complexes in
the S3−S2, S0−S3, and S1−S0 spectra are minor. These
differences could reflect baseline shifts, especially in the S3−S2
and S1−S0 spectra.
Strongly Hydrogen-Bonded O−H Stretching Region.
Changes in the O−H stretching vibrations of strongly H-bonded
O−H groups of water molecules in PSII can be observed as very
broad positive features between 3200 and 2300 cm−1.120−123
These regions of the FTIR difference spectra of D1-D170E PSII
core complexes are compared with those of the wild-type
complexes in Figure 5. For the wild-type complexes, the spectra
produced by the first, second, third, and fourth flashes
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Figure 5. FTIR difference spectra of wild-type (black) and D1-D170E
(red) PSII core complexes in the strongly H-bonded O−H stretching
region in response to four successive flash illuminations applied at 0 °C.
The data were collected simultaneously with those shown in Figure 2.
Dark−dark control traces show the noise level and the stability of the
baseline (lower traces). The sharp feature at 2350 cm−1 is caused by the
residual atmospheric CO2.
correspond predominantly to the S1-to-S2, S2-to-S3, S3-to-S0, and
S0-to-S1 transitions, respectively. The wild-type S2−S1 spectrum
(upper traces, black) shows a broad feature extending from 3100
cm−1 to approximately 2600 cm−1. The intensity of this broad
feature is diminished substantially in the corresponding D1-
D170E spectrum (upper traces, red). The wild-type S2−S1
spectrum is overlain with numerous sharp, positive peaks that
correspond to Fermi resonance peaks arising from the coupling
between the H-bonded His N−H stretching modes and
combinations/overtones of imidazole ring modes.135,136 These
arise from D1-H337136 and possibly D1-H332.128 In the
corresponding D1-D170E spectrum, the intensities of the
sharp features at 2914, 2838, and 2805 cm−1 are diminished
substantially. In the wild-type spectrum, the sharp features that
form during the S1-to-S2 transition are reversed during the S3-to-
S0 transition (third flash traces, black). These features are also
substantially diminished or absent in the D1-D170E spectrum
corresponding to the S3-to-S0 transition (third flash traces, red).
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Aside from diminishing/eliminating the sharp features in the
wild-type S0−S3 spectrum, the D1-D170E mutation did not alter
the broad features corresponding to the S3−S2, S0−S3, or S1−S0
transitions (second, third, and fourth flash traces).
α-COO− Group of D1-A344. To determine if the D1-
D170E mutation perturbs the α-COO− group of D1-A344 at the
C-terminus of the D1 polypeptide, the frequency of this mode
was determined by specifically labeling wild-type and D1-D170E
PSII core complexes with L-[1-13C]alanine.137,138 A comparison
of the unlabeled and L-[1-13C]alanine-labeled S2−S1 FTIR
difference spectra of wild-type and D1-D170E PSII core
complexes is shown in Figure 6 (left panels). To isolate the L-
[1-13C]alanine-shifted νsym(COO−) modes and to display them
more clearly, the 12C−minus−13C double-difference spectra of
the region between 1380 and 1280 cm−1 in the wild-type and
mutant samples are also presented (Figure 6, right panel). The
data show that while the mutation did not alter the modes at
1354(−) and 1340(+) cm−1 significantly, the mutation shifted
the 1321(+) and 1305(−) cm−1 modes to higher frequencies by
5−7 cm−1 compared to their positions in the wild-type
spectrum. Because the νsym(COO−) mode of L-[1-13C]alanine
downshifts be approximately 18 cm−1 in the solution,137 the
1354(−) and 1321(+) cm−1 features in the wild-type 12C−
minus−13C double-difference spectrum have been assigned to
the unlabeled νsym(COO−) mode of D1-A344 in the S1 and S2
states, respectively, and the 1340(+) and 1305(−) cm−1 modes
have been assigned to the 13C-labeled νsym(COO−) mode of D1-
A344 in the S1 and S2 states, respectively.137,138 However, a
quantum mechanics/molecular mechanics (QM/MM) analysis
has assigned the 1340(+) and 1321(+) cm−1 features to the
unlabeled mode in the S2 state and the labeled mode in the S1
state, respectively.139
■ DISCUSSION
The side chain of D1-D170 bridges Mn4 and the Ca ion, so
mutations of this residue would be expected to perturb the
coordination environments of one or both of these metal ions. In
addition, D1-D170 interacts with both η nitrogens of CP43-
R357, whose ε and η nitrogens interact with D1-N87 and W19 at
the start of the O4 pathway. Consequently, the mutation of D1-
D170 would be expected to perturb the network of H-bonds that
surrounds the Mn4Ca cluster. Our analysis of the FTIR
difference spectra of D1-D170E confirms these expectations.
Network of H-BondsMid-Frequency Region. The D1-
D170E-induced alterations to the S2−S1 spectrum in the ν(C
O) and νasym(COO−)/amide II regions (Figure 2) resemble
those produced by the mutation of many other residues in the
H-bond network that surrounds the Mn4Ca cluster (See Table
1). This includes the alterations to the ν(CO) feature at
1747(−) cm−1, the νasym(COO−) feature at 1586(+) cm−1, the
amide II features at 1552(+), 1543(−), 1531(+), and 1522(−)
cm−1, and the νasym(COO−)/amide II feature at 1509(+) cm−1.
The negative feature at 1747 cm−1 corresponds to a protonated
carboxylate group whose pKa decreases during the S1-to-S2
transition.140,141 That the D1-D170E mutation alters the
ν(CO) and νasym(COO−)/amide II regions of the mid-
frequency S2−S1 FTIR difference spectrum in the same manner
as that of the many other mutations shows that it perturbs the
same extensive network of H-bonds.
Network of H-BondsWeakly H-Bonded O−H
Stretching Region. Features between 3700 and 3500 cm−1
in the FTIR difference spectra correspond to the weakly H-
bonded O−H stretching modes of the water molecules that are
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Figure 6. Comparison of the mid-frequency S2−S1 FTIR difference spectra of unlabeled (black traces) and L-[1-13C]alanine-labeled (red traces) wild-
type (upper left) or D1-D170E (lower left) PSII core complexes. The spectra of the unlabeled wild-type and D1-D170E PSII core complexes are
reproduced from Figure 2. The L-[1-13C]alanine-labeled wild-type and D1-D170E spectra represent the averages of 12 samples (19,100 scans) and 15
samples (23,800 scans), respectively. Within each panel, the spectra have been normalized to maximize their overlap between 1450 and 1360 cm−1.
The right panel shows the double-difference spectra, 12C−minus−13C, of wild-type (black trace) and D1-D170E (red trace) PSII core complexes that
were obtained by subtracting the S2−S1 FTIR difference spectra of the L-[1-13C]alanine-labeled PSII core complexes from the S2−S1 FTIR difference
spectra of the unlabeled PSII core complexes (the spectra shown in the left panels were subtracted directly). Only the regions between 1380 and 1270
cm−1 are shown.

Table 1. Features in the S2−S1 FTIR Difference Spectrum That Are Altered by a Specific Treatment or Specific Mutationsa in the
H-Bond Networks near the Mn4Ca Cluster (in cm−1)
1747(−) 1586(+) 1544(−) 1531(+)/1522(−) 1509(+) 3663(−) 3617(+)
D61A125 D61A125,140 D61A125,140 D61A125,140 D61A125,140 D61A125 D61A125
E65A140 Q165E141 E65A140 S169A96,117,142 Q165E141 S169A142 N87A,D143
S169A142 S169A96,117,142 S169A96,142 D170Eb S169A96,117,142 D170Eb S169A142
D170Eb D170Eb D170Eb N181A,S144 D170Eb N181A144 D170Eb
V185N127 N181A,S144 V185N127 V185N127 E189G,S128 N298A145 N181A,S144
E189G,S128 V185N127 E189G,S128 E189G,S128 N298A145 Sr/Ca exchange93 V185N127
N289A145 E189G,S128 N298A145 N298A145 R334A141 N298A145
E329Q,A128,140 N298A145 E329Q,A128,140 R334A141 D2-K317A134 Sr/Ca exchange93
R334A141 R334A141 R334A141 D2-E312A140
D2-E312A140 D2-K317A134 D2-E312A140 D2-K317A134
sample dehydration140 Sr/Ca exchange93,146,147 Sr/Ca exchange93,146
a
Residues are from the D1 subunit unless indicated otherwise. bThis work.

coupled to the Mn4Ca cluster.122,123,148,149 Negative features
correspond to water molecules that either deprotonate or form
stronger hydrogen bonds (i.e., weakly H-bonded O−H groups
become strongly H-bonded). Unlike the features in the mid-
frequency region, the features in the weakly H-bonded O−H
stretching region do not oscillate during the S state cycle. The
lack of oscillations has been attributed, at least in part, to the
consumption of the two substrate water molecules during the S
state cycle.122,123,148,149 The O−H stretching modes that
correspond to the features in the S2−S1 spectrum in this region
are highly coupled.149 The analysis of numerous mutations
shows that the water molecules whose weakly H-bonded O−H
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groups give rise to these features are located in a region that
extends at least to a volume bound approximately by D1-N87,
D1-N298, and D2-K317. The D1-D170E-induced elimination
of the 3663(−) and 3617(+) cm−1 features in the S2−S1
spectrum (Figure 4) resembles the alterations produced by
the mutation of numerous other residues in the H-bond network
that surrounds the Mn4Ca cluster (see Table 1). The elimination
of the same features in D1-D170E PSII core complexes (Figure
4) confirms that this mutation perturbs the same extensive
network of H-bonds that is perturbed by many other mutations.
None of the mutations mentioned (except D1-D61A during the
S2-to-S3 transition125) alter the features in this region of the S3−
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S2, S0−S3, or S1−S0 spectra. The same appears to be true for the
D1-D170E mutation.
Network of H-BondsStrongly H-Bonded O−H
Stretching Region. Features between 3200 and 2200 cm−1
in the FTIR difference spectra correspond to strongly H-bonded
O−H stretching modes of the water molecules that are coupled
to the Mn4Ca cluster.122,123,148,149 In the S2−S1 spectrum, the
broad feature is dominated by the O−H stretching modes of W1
and W2.149 This feature is eliminated by the D1-D61A
mutation,125 consistent with the H-bond that exists between
this residue and W1. Other mutations that alter this feature to a
substantial degree are D1-S169A,142 D1-N181A,144 and D1-
N298A. 145 The feature’s positive amplitude has been
attributed149 to the strengthening of the H-bonds in which
W1 and W2 participate during the S1-to-S2 transition, a
strengthening caused by the increased charge that develops on
the Mn4Ca cluster during this transition. The broad feature’s
lower amplitude in D1-D170E PSII core complexes (Figure 5)
implies that these H-bonds are weaker in the mutant. Because
both W1 and W2 ligate the same Mn ion as D1-D170, the lower
amplitude suggests that the magnitude of the charge increase on
Mn4 during the S1-to-S2 transition is less in the mutant than that
in the wild-type complexes. A smaller charge increase on Mn4
would be consistent with a D1-D170E-induced change in the
valence distribution of the Mn ions in the Mn4Ca cluster in the
S2 state (see below).
The features at 2914, 2838, and 2805 cm−1 in the wild-type
S2−S1 spectrum correspond to the Nτ-H group of D1-H337136
and possibly the Nπ-H group of D1-H332.128 The former
donates an H-bond to O3, whereas the latter donates an H-bond
to the backbone carbonyl of D1-E329. The substantial decrease
in the amplitudes of these features in the D1-D170E S2−S1
spectrum suggests alterations to the strengths of the H-bonds
involving these N−H groups during the S1-to-S2 transition. Such
alterations could be caused by a change in the S-state-dependent
charge on O3136 or by mutation-induced alterations to the
polypeptide backbone that are manifested in part by the changes
to the amide I region that are observed between 1690 and 1620
cm−1 in the D1-D170E S2−S1 spectrum (Figure 2).
Metal CoordinationCarboxylate Stretching Re-
gions. The similar frequencies of the νsym(COO−) modes of
D1-A344 in wild-type and D1-D170E PSII core complexes in
the S1 and S2 states (Figure 6, right panel) show that the
mutation does not substantially perturb the overall structure of
the Mn4Ca cluster. Nevertheless, one striking consequence of
the D1-D170E mutation is the elimination of four carboxylate
stretching modes from the S2−S1 FTIR difference spectrum: the
prominent νsym(COO−) modes at 1398(−) and 1361(+) cm−1,
the prominent νasym(COO−) mode at 1587(+) cm−1, and the
νasym(COO−) mode at 1562(−) cm−1 (Figure 3). The same four
modes are also eliminated in Ca-depleted PSII prepara-
tions.150,151 However, the D1-D170E PSII core complexes are
not depleted of Ca ions: Ca-depleted PSII preparations fail to
advance beyond the S2Y•Z state,152,153 whereas D1-D170E PSII
core complexes advance through the complete S state cycle
(Figure 2) and evolve O2 at 53 ± 6% the rate of wild-type PSII
core complexes. Furthermore, Ca depletion eliminates the
features in the weakly and strongly H-bonded O−H stretching
regions,149 whereas the D1-D170E mutation merely alters these
features. The mutation-induced elimination of the four
carboxylate stretching modes shows that they correspond to
the νasym(COO−) and νsym(COO−) modes of D1-D170 in the S1
and S2 states. The identification of the νsym(COO−) modes of
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D1-D170 at 1398 and 1361 cm−1 in the S1 and S2 states,
respectively, agrees well with a QM/MM analysis of the Mn4Ca
cluster’s ligand vibrations.139 Originally, the frequencies of the
four carboxylate modes eliminated in Ca-depleted samples were
interpreted as showing that a carboxylate residue (now known to
be D1-Asp170) in native PSII centers changes its coordination
mode from bridging to unidentate during the S1 -to-S 2
transition.150,151 However, recent serial femtosecond X-ray
crystallographic studies have shown that D1-Asp170 does not
change its coordination mode during the S1-to-S2 transi-
tion.20−23 More recently, the substantial downshift of the
νsym(COO−) mode of D1-D170 during the S1-to-S2 transition
(from 1398 to 1361 cm−1) has been attributed primarily to the
loss of Jahn−Teller elongation along the axis D170-Mn4-E333
when Mn4 undergoes oxidation from Mn(III) to Mn(IV).30,67
The loss of Jahn−Teller elongation would bring the ligating
oxygens of D1-D170 and D1-E333 closer to Mn4, thereby
weakening and downshifting the νsym(COO−) modes of both
D1-D170 and D1-E333. Substantial downshifts of the
νsym(COO−) modes of both residues were predicted in the
earlier QM/MM analysis,139 and a 0.2 Å shortening of the
distance between Mn4 and D1-E333 during the S1-to-S2
transition was reported in a recent femtosecond X-ray
crystallographic study.22
The mutation-induced elimination of the carboxylate
stretching modes of D1-D170 from the S2−S1 spectrum implies
that the carboxylate stretching modes of D1-E170 are not altered
substantially during the S1-to-S2 transition, in contrast to those
of D1-D170. One possibility is that D1-E170 does not ligate the
Mn4Ca cluster. This possibility seems unlikely because the high-
affinity Mn(II) binding site on apo-PSII is retained in D1-
D170E PSII core complexes with minor modification.101
Another possibility is that the mutation alters the equilibrium
between two Jahn−Teller conformers of the S1 state. In a recent
computational study, one S1 state conformer (S1A) was
determined to have a Jahn−Teller elongation axis on Mn4
oriented such that the ligating oxygens of D1-D170 and D1-
Glu333 are collinear with Mn4.67 The second conformer (S1B)
was determined to have a Jahn−Teller elongation axis on Mn4
oriented such that O5 and the ligating oxygen of W1 are
collinear with Mn4. The SA1 conformer was calculated to be
lower in energy and to promote Mn4 oxidation during the S1-to-
S2 transition, thereby producing the low-spin conformer of the
S2 state with its characteristic multiline EPR signal.67 The SB1
conformer was calculated to promote Mn1 oxidation during the
S1-to-S2 transition, thereby producing a high-spin conformer of
the S2 state. If the D1-D170E substitution alters the environ-
ment of the Mn4Ca cluster sufficiently to stabilize SB1 relative to
SA1 , the oxidation of Mn1 during the S1-to-S2 transition would
leave the Jahn−Teller elongation axis on Mn4 unchanged (and
oriented with O5 and W1 collinear with Mn4). Consequently,
the substantial downshift of the νsym(COO−) mode from 1398
to 1361 cm−1 that occurs in the wild-type complexes would not
occur in D1-D170E. The oxidation of Mn1 instead of Mn4
would also result in a smaller increase in the charge on Mn4,
consistent with the lower amplitude of the broad feature
observed in the S2−S1 spectrum of D1-D170E PSII core
complexes in the strongly H-bonded stretching region (Figure
5). The preferential formation of a high-spin isomer of the S2
state in D1-D170E PSII core complexes would also account for
the inability to generate the S2 state multiline EPR signal in the
earlier studies of D1-D170E PSII core complexes.108,109 A
D170E-induced alteration of the equilibrium between the
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different conformers of the S2 and/or S3 states may also underlie
the alterations to the S3−S2 FTIR difference spectrum that are
observed especially in the νsym(COO−) region (Figure 2, second
flash traces).
Although not considered previously, the stabilization of the SB1
conformer also would account for the inability to generate the S2
state multiline EPR signal in non-O2-evolving mutant D1-E189
PSII core complexes.154 The D1-E189 residue ligates Mn1, so
mutations of this residue might alter the equilibrium between SA1
and SB1 in the same manner that the binding of ammonia to Mn4
alters the equilibrium between the low- and high-spin con-
formers of the S2 state at elevated pH values.70 The same
νsym(COO−) and νasym(COO−) modes in the S2−S1 spectrum
that are altered by D1-D170E are also altered by the D1-E189G
and D1-E189S mutations:128 the 1586(+) and 1561(−) cm−1
features are diminished substantially, the 1364(+) cm−1 feature
is eliminated, and the 1401(−) cm−1 feature is sharply
diminished by D1-E189S and possibly by D1-E189G [the
elimination of this feature by D1-E189G might be masked by the
downshift of the wild-type 1416(−) cm−1 feature].
It should be noted that an early study reported that the wild-
type S2−S1 FTIR difference spectrum is unaltered when the
equilibrium between the low- and high-spin conformers of the S2
state is changed.155 However, this equilibrium was altered by
changing the cryoprotectant present in the samples. Con-
sequently, samples enriched in the high-spin conformer also
contained a substantial population (approximately 50%) of the
low-spin conformer. The S2−S1 FTIR difference spectra of
samples enriched in the high-spin S2 state conformer should be
reexamined at elevated pH values, where higher enrichment has
been reported recently.70
In Ca-depleted PSII preparations,150,151 D1-D170 becomes a
unidentate ligand of Mn4. In the earlier QM/MM study,139 the
νsym(COO−) mode of D1-D170 in Ca-depleted PSII prepara-
tions was calculated to appear at 1350 cm−1 in the S1 state and
1330 cm−1 in the S2 state, consistent with the unidentate
ligation. Therefore, the 1355(−) and 1341(+) cm−1 features in
our D1-D170E S2−S1 difference spectrum (Figure 2) are
consistent with the D1-D170E mutation changing the
coordination mode of this carboxylate group from bridging
Mn4 and Ca in the wild-type complex to unidentate in D1-
D170E, ligating only Mn4. The smaller downshift of this mode
during the S1-to-S2 transition (from 1355 to 1341 cm−1)
compared to that in the wild-type complex would reflect the
absence of the loss of Jahn−Teller distortion on Mn4. Instead,
the downshift would reflect the oxidation of Mn1 and the
increased charge on the Mn4Ca cluster during the S1-to-S2
transition. Because the electrons removed from the Mn4Ca
cluster during the individual S state transitions originate from
highly delocalized orbitals,156,157 the electrostatic charges on the
individual Mn ions do not correlate with their individual
oxidation states.139,158,159 Indeed, the oxidation of Mn1 during
the S1-to-S2 transition has been calculated to increase the
electrostatic charge on Mn4 as well as that on Mn1.139 An
increased charge on the metal ion weakens the ligating C−O
bond, leading to a downshift of the νsym(COO−) mode. It should
be noted that an early study of D1-D170E PSII core complexes
also concluded that the D1-D170E mutation changes the
coordination mode of this residue, plus one other residue, from
bidentate or bridging to unidentate.108 However, the wild-type
FTIR difference spectra published in ref 108 are unlike those
published by any other research group (see footnote 2 in ref
137).
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In the wild-type spectra, the oxidation of Mn4 during the S1-
to-S2 transition lengthens D1-D170’s Mn4-ligating C−O bond,
thereby shortening the C−O bond that ligates the Ca ion.139
The shortening of the latter partly increases the electrostatic
charge on the Ca ion during the S1-to-S2 transition, decreasing
the pKA values of W3 and W4.139 The changes induced by the
D1-D170E mutation proposed here, that is, the mutation-
induced oxidation of Mn1 instead of Mn4 during the S1-to-S2
transition and the lack of D1-E170 ligation to Ca, would cause
the accumulation of less positive charge on the Ca ion during the
S1-to-S2 transition, thereby decreasing the pKA values of W3 and
W4 to lesser extents. These altered pKA values may contribute to
the decreased efficiency of O2 formation observed in D1-D170E
cells and may underlie the 4−5 cm−1 upshifts of the 1321(+) and
1305(−) cm−1 features of D1-A344 in the 12C−minus−13C
double-difference spectrum of L-[1-13C]alanine-labeled D1-
D170E PSII core complexes during the S1-to-S2 transition
(Figure 6).
In the mass spectrometry study that revealed the posttransla-
tional modification of D1-H170 to D1-D170 and D1-N170 in
D1-D170H cells,117 it was suggested that Mn4Ca clusters do not
assemble in D1-H170 PSII centers and that D1-H170 becomes
oxidized in the apo-protein. This suggestion would be consistent
with the slightly elevated Km for binding Mn(II) to the high-
affinity Mn site in apo-D170H101 and would explain the
observations that (1) approximately 50% of the D1-D170H PSII
centers lack Mn4Ca clusters in vivo,104 (2) the illumination of
Mn-depleted D1-D170H PSII core complexes in the presence of
Mn(II) ions produces a Mn(III) ion having parallel polarization
EPR properties that are altered compared to that of the wild-
type,107 and (3) the photoactivation of Mn-depleted D1-D170H
results in the accumulation of nonfunctional high-valency Mn
ions in approximately 50% of the D1-D170H PSII centers.160
However, it does not explain how D1-H332 and D1-H337
escape a similar modification in the apo-protein, especially in
light of the recent proposals that the first Mn(III) ion produced
at the high-affinity Mn4 site during the photoactivation migrates
to the Mn1 site prior to the oxidation of the second Mn(II)
ion.161−163 It also does not explain the alteration of the 606 cm−1
Mn−O−Mn cluster mode in the low-frequency S2−S1 FTIR
difference spectrum of D1-D170H PSII core complexes.112 On
the other hand, the mechanism suggested in the Introduction
section of this article, that a powerful oxidation equivalent on
Mn4 in the S4 state oxidizes D1-H170 in the assembled Mn4Ca
clusters, does not explain the observation that approximately
50% of the D1-D170H PSII centers lack Mn4Ca clusters in
vivo.104 Understanding how D1-H170 becomes changed to D1-
D170 and D1-N170 in D1-D170H cells will require further
investigation.
■
SUMMARY AND CONCLUSIONS
On the basis of the analysis of the high- and mid-frequency FTIR
difference spectra of D1-D170E PSII core complexes, we
conclude that the D1-D170E mutation alters (1) the network of
H-bonds surrounding the Mn4Ca cluster in much the same
manner as that of other mutations constructed in this network
and (2) the equilibrium between the S1 state conformers of the
Mn4Ca cluster so that a high-spin form of the S2 state is
produced during the S1-to-S2 transition instead of the low-spin
form that gives rise to the S2 state multiline EPR signal. In
addition, our data are consistent with the coordination mode of
D1-D170 changing from bridging Mn4 and Ca in the wild-type
complex to unidentate ligation of Mn4 in D1-D170E. This is the
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first mutation of a metal ligand in PSII that substantially impacts
the spectroscopic signatures of the Mn4Ca cluster without
substantially eliminating the O2 evolution. The mutation’s
apparent alteration of the equilibria between different isomers of
the S1 and S2 states without substantially eliminating the O2
evolution has significant implications for our understanding of
the roles of alternate S state conformers in the mechanism of O2
formation in PSII.
■
ASSOCIATED CONTENT
Accession Codes
D1 protein was obtained from Synechocystis sp. PCC 6803,
UniProtKB P16033.
■
AUTHOR INFORMATION
Corresponding Author
Richard J. Debus − Department of Biochemistry, University of
California, Riverside, California 92521, United States;
orcid.org/0000-0003-1321-8730; Phone: +1 (951) 941-
8647; Email: richard.debus@ucr.edu
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biochem.1c00504
Funding
This work was supported by the Department of Energy, Office of
Basic Energy Sciences, Division of Chemical Sciences (grant
DE-SC0005291).
Notes
The author declares no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Anh P. Nguyen for maintaining the mutant and wild-
type cultures of Synechocystis sp. PCC 6803 and for purifying the
thylakoid membranes that were used for the isolation of PSII
core complexes. This work was supported by the Department of
Energy, Office of Basic Energy Sciences, Division of Chemical
Sciences, grant DE-SC0005291.
■
ABBREVIATIONS
Chl, chlorophyll; EDTA, ethylenediaminetetraacetic acid;
FTIR, Fourier transform infrared; MES, 2-(N-morpholino)-
ethanesulfonic acid; P680, chlorophyll multimer that serves as the
light-induced electron donor in PSII; PSII, photosystem II; QA,
primary plastoquinone electron acceptor; YZ, tyrosine residue
that mediates electron transfer between the Mn4Ca cluster and
P680•+; YD, tyrosine residue that acts as an additional electron
donor to P680•+
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