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The low spin - high spin equilibrium in the S2-state of the water oxidizing enzyme
Alain Boussac1*, Ilke Ugur2, Antoine Marion2, Miwa Sugiura3, Ville R. I. Kaila2, A. William
Rutherford4
1CNRS UMR 9198, CEA Saclay, 91191 Gif-sur-Yvette, France.2 Department Chemie, Technische Universität München, Lichtenbergstraße 4, D-85748
Garching, Germany.3Proteo-Science Research Center, Ehime University, Bunkyo-cho, Matsuyama, Ehime, 790-
8577, Japan.4Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom.
*Corresponding author: [email protected]
Keywords: Photosystem II, Oxygen evolution, Mn4CaO5 cluster, spin state, DFT, EPR
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Abbreviations
PSII, Photosystem II; Chl, chlorophyll; MES, 2-(N-morpholino) ethanesulfonic acid; HEPES,
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; TAPS, N-
[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, [(2-Hydroxy-1,1-
bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid; CHES, 2-
(Cyclohexylamino)ethanesulfonic acid; P680, primary electron donor; PD1 and PD2, Chl
monomer of P680 on the D1 or D2 side, respectively; QA, primary quinone acceptor; QB,
secondary quinone acceptor; Phe, pheophytin; EPR, Electron Paramagnetic Resonance;
ENDOR Electron Nuclear Double Resonance; HS, high spin; LS, low spin; DFT, density
functional theory; XFEL, X-ray free-electron laser.
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Abstract
In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five
sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. Here, we
have studied the transition between the low spin (LS) and high spin (HS) configurations of S2 using EPR spectroscopy, quantum chemical calculations using
Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy.
The EPR experiments show that the equilibrium between S2LS and S2
HS is pH dependent, with
a pKa 8.3 (n 4) for the native Mn4CaO5 and pKa 7.5 (n 1) for Mn4SrO5. The DFT
results suggest that exchanging Ca with Sr modifies the electronic structure of several
titratable groups within the active site, including groups that are not direct ligands to Ca/Sr,
e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of
the pKa upon the Ca/Sr exchange. EPR also showed that NH3 addition reversed the effect of
high pH, NH3-S2LS being present at all pH values studied. Absorption spectroscopy indicates
that NH3 is no longer bound in the S3TyrZ state, consistent with EPR data showing minor or
no NH3-induced modification of S3 and S0. In both Ca-PSII and Sr-PSII, S2HS was capable
of advancing to S3 at low temperature (198 K). This is an experimental demonstration that the
S2LS is formed first and advances to S3 via the S2
HS state without detectable intermediates. We
discuss the nature of the changes occurring in the S2LS to S2
HS transition which allow the S2HS
to S3 transition to occur below 200 K. This work also provides a protocol for generating S 3 in
concentrated samples without the need for saturating flashes.
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Introduction
Oxygenic photosynthesis provides the main input of energy into biology. This process
produces food, fibers and fossil fuels, and energizes the atmosphere with O2. Photosystem II
(PSII) is the unique water-oxidizing enzyme of the cyanobacteria, algae and higher plants that
is at the heart of oxygenic photosynthesis. In cyanobacteria, the PSII complex comprises 17
trans-membrane and 3 extrinsic membrane proteins [1,2]. These 20 subunits bind 35
chlorophylls, 2 pheophytins (Phe), 2 hemes, 1 non-heme iron, 2 plastoquinones (QA and QB),
the Mn4CaO5 cluster, 2 Cl-, 12 carotenoids and 25 lipids [1-3].
The conversion of solar energy into chemical energy starts with the absorption of a
photon by one of the chlorophylls. The resulting excitation is transferred to the photochemical
trap, P680, which is composed of four chlorophyll a molecules, PD1/PD2 and ChlD1/ChlD2, and
two pheophytin a molecules, PheD1/PheD2. Here, the excitation energy induces a charge
separation, and after a few picoseconds following the light-absorption process, the positive
charge is mainly stabilized on PD1 and the negative charge is on PheD1. This state is often
referred to as P680+PheD1
-, e.g. [4-6] and references therein. P680+ then oxidizes TyrZ, the Tyr161
of the D1 polypeptide, which in turn oxidizes the Mn4CaO5 cluster [4-7]. On the acceptor side,
the electron is transferred to the primary quinone electron acceptor, QA, and then to the second
quinone, QB. In contrast to QA, QB accepts two-electrons and two-protons and then leaves its
binding site [3,4].
Sequential excitations of PSII allow the Mn4CaO5 cluster to accumulate oxidizing
equivalents. It thus cycles through five redox states denoted Sn, where n stands for the number
of stored oxidizing equivalents. Upon formation of the S4 state, two molecules of water are
rapidly oxidized, the S0 state is regenerated and O2 is released [8-12].
The S1-state is the dark-stable state, and the S2-state can be formed from the S1-state
either by illumination with a single flash of light at room temperature or by continuous
illumination between 140 K and 230 K. In this temperature interval, the S1 to S2 transition
occurs, while the electron transfer from QA- to QB is blocked thus preventing a second
turnover [13,14].
The dark-stable S1-state is silent in conventional EPR and only visible by parallel mode
detection [15,16]. The existence of two different isomers of S1 has been suggested based on
EPR [17] and theoretical studies [18,19].
The S2-state is EPR-detectable and exhibits two different ground state configurations
depending on the experimental conditions, reviewed in [20,21] (see Fig.1). The first S2
configuration has a low spin S = 1/2 state, S2LS, exhibits a multiline signal centered at g 2.0
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and spreads over roughly 1800 gauss. The signal is made up of at least 20 lines, separated by
approximately 80 gauss [22]. The second S2-state configuration exhibits either a derivative-
like signal centered at g 4.1 [23,24] or more complex signals at lower magnetic fields (i.e.
at higher g values) attributed to high spin states, S2HS, with S ≥ 5/2 [25,26]. ENDOR analysis
and X-ray spectroscopy of the S2LS state indicate that the Mn4CaO5 contains one MnIII ion and
three MnIV ions [12, 27-30] in a configuration in which the net oxidation states of the four
manganese ions are generally considered to be Mn4IV, Mn3IV, Mn2IV, Mn1III, with the Mn4
and Mn3 being connected by a di--oxo bridge involving O4 and O5, while the Mn1, Mn2,
Mn3 Ca and O1, O2 and O3 form an open cubane structure (i.e. there is no connection
between O5 and Mn1), see Fig. 1.
Using the high-resolution crystal structure of the S1-state [1,2], computational studies
[31-33] gave a rationale for the inter-conversion between the S2LS and S2
HS states: the two
almost isoenergetic structures share the same coordination environment but the MnIII ion is
located on Mn1 in the S2LS state and on the Mn4 in the S2
HS state. The dynamic S2LS S2
HS
equilibrium is considered to be associated with a small change in the structure of the cluster
and with the position of the O5 oxygen bridge linking Mn4, Mn3 and the Ca in the S2LS state
shifting to a position at the corner of the cubane, where it bridges Mn1, Mn3 and Ca ions in
the S2HS state. This dynamic switching of the oxidation states between the Mn4 and Mn1 ions
(see Fig. 1) has also been proposed to explain the unusually rapid exchange of water into the
oxo-bridges compared to chemical models [11].
The S3-state is generated by giving 2 flashes at room temperature to dark-adapted PSII.
The complete X-band EPR spectrum [34] of this state was simulated assuming a spin S = 3
ground state [34,35]. A multi-frequency, multi-dimensional magnetic resonance spectroscopy
study of this state showed that all four Mn ions are structurally and electronically similar: all
exhibiting a +4 formal oxidation state with an octahedral ligation sphere in an open cubane
configuration [36].
The current view of the structures of the Mn4O5Ca in the S2-state and S3-state currently
rely on DFT calculations that are based on the high-resolution X-ray structure of the S1-state
structure and further restrained by data from X-ray spectroscopy and EPR spectroscopy
[28,36-49]. While X-ray free-electron laser (XFEL) crystallography experiments are expected
to resolve the structures of S2 and S3 soon, the published XFEL studies require improvements
in resolution and in S-state homogeneity and/or deconvolution before the conflicting results
and anomalies can be understood [50-53]. One complicating factor is that both experimental,
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e.g. [10,17,54,55], and computational studies, e.g. [10,56], suggest the existence of more than
one structural form of S3.
The structure and mechanism of the Mn4CaO5 cluster during O=O bond formation are
the subject of investigation using both experimental and theoretical approaches, e.g. [36-
38].The suggestion from DFT calculations that O=O bond formation is based on an oxo-oxyl
coupling mechanism [36,37,49] shares some features of an earlier proposal based on substrate
water exchange, EPR and EXAFS data [56]. An alternative mechanism, involving a
nucleophilic attack from either a free or Ca-bound water molecule on a MnV=O, appears less
likely based on substrate water exchange experiments [11,56] and theoretical studies
[43,47,57]. An 17O EPR study [58] showed that labelling of O5 occurred in the time-scale of
the exchange of Ws, the slower of the exchangeable water substrate. Regarding Wf, the faster
exchangeable water substrate, experiments have shown that it is exchangeable in the S2 and S3
states. Recently, Wf was proposed to be either the terminal water ligand W2 on Mn4
[10,58,59] or W3 bound to the Ca2+ in the S2-state [32,48,60,61]. The situation is further
complicated by the possibility that the binding sites of Wf and Ws may change during the
S2S3 transition. It also seems likely that the S2S3 transition involves the uptake of an
additional water molecule (Wx) [11,37,62].
To explain how the open coordination site on Mn1 can be filled by a terminal water-
derived ligand without Wx directly binding to this site, it was proposed [44] that the
formation of TyrZ in the S2
LS state leads to the reorientation of the dipole moment of the
Mn4CaO5 cluster such that the locus of the negative charge becomes directed towards W1, a
H2O molecule bound to Mn4, and its hydrogen bonding partner, D1-Asp61 [44]. This leads to
the deprotonation of W1 and a shift of the equilibrium S2LS S2
HS in favor of the high spin
configuration. In this model, only the S2HS state would be able to progress to the S3-state, and
thus S2LS needs to convert to S2
HS in order to advance [44-46]. In an alternative suggestion, the
formation of TyrZ was proposed to affect the pKa of W3 triggering its movement onto either
Mn4 or Mn1 depending on whether the cube is in the closed (HS) or open (LS) configuration
[48]. With the HS route being the lower energy option, the route to S3 via the S2HS
intermediate was favored. In contrast to these models, other mechanisms suggest that the S2-
state advances to S3 from its low spin, open cube structure [37] (see also [29,30]).
The proposition that the structural change from S2LS to S2
HS is an obligate [44,45] or
favored [48] intermediate step in S2 to S3 transition was based on computational chemistry,
however it recently received experimental support [17]. In Sr-PSII, in which Sr2+ was
substituted for Ca2+, a proportion of the centers in the S2-state exhibited a high spin form.
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These S2HS centers were able to progress to the EPR-detectable S3-state when illuminated at
temperatures as low as 180 K. In contrast, the S2LS state could only advance to the S3 state at
temperatures ≥ 235 K, the temperature below which the S2LS S2
HS transition is blocked. It
was suggested that in Sr-PSII the S2HS state is capable of advancing to S3 at low temperature
because its formation involves a deprotonation or redistribution of protons prior to the low
temperature illumination [17].
NH3 has long been known to inhibit water oxidation and is considered a water analog.
The inhibition of O2 evolution by NH3, when measured using light intensities near-saturation
for untreated PSII, is due to a slowdown in the rate of S-state cycling [63]. In contrast, under
very high light intensities NH3 is not able to inhibit oxygen evolution, presumably because
NH3 binding is S-state dependent and the susceptible S state (S2) is turned over too quickly.
The period-of-four oscillation seen with saturating flashes remained virtually unaffected by
NH3 indicating it was released in some S states in the time between the flashes [63]. More
recently, it was found that the binding/exchange kinetics of the two substrate water molecules
under flash illumination were only slightly modified in the presence of ammonia [64]. NH3
binds to the Mn4O5Ca cluster in the S2-state S2LS [63,65] by displacing a water ligand on the
outer manganese (Mn4-W1) and elongating the Mn4-O5 bond [64,66-68].
In the present work, the pH dependence of the low spin to high spin transition in the
S2-state has been investigated by EPR spectroscopy using Ca- and Sr-containing PSII. The
ability of these two forms of S2 to advance to S3 has also been investigated. The changes to
the electronic structure resulting from the Ca/Sr-exchange have been probed by quantum
chemical density functional theory (DFT) calculations. Given the potential role of W1
deprotonation associated with the S2LS to S2
HS transition and the reported displacement of W1
by NH3, we also investigated the effect of NH3 on this transition, see [10,64,66-69], and at
which step NH3 is released.
Materials and Methods
Photosystem II preparation
The Thermosynechococcus elongatus strain used was the psbA1psbA2 deletion
mutant [70] constructed from the T. elongatus 43-H strain that had a His6-tag on the carboxy
terminus of CP43 [71]. The biosynthetic Ca/Sr exchange and PSII purification were achieved
as previously described [72,73]. Native PSII is designated Ca-PSII and PSII in which Sr2+ was
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substituted for Ca2+ is designated Sr-PSII. The purified Ca-PSII and Sr-PSII were suspended
in 1 M betaine, 15 mM CaCl2, 15 mM MgCl2, 40 mM MES, pH 6.5, then frozen in liquid
nitrogen until use.
EPR spectroscopy
X-band cw-EPR spectra were recorded with a Bruker Elexsys 500 X-band
spectrometer equipped with a standard ER 4102 (Bruker) X-band resonator, a Bruker
teslameter, an Oxford Instruments cryostat (ESR 900) and an Oxford ITC504 temperature
controller. Flash illumination at room temperature was provided by a neodymium:yttrium–
aluminum garnet (Nd:YAG) laser (532 nm, 550 mJ, 8 ns Spectra Physics GCR-230-10).
Illumination with visible light for approximately 5-10 seconds with a 800 W tungsten lamp
filtered by water and infrared cut-off filters at temperatures close to 200 K were done in a
non-silvered Dewar in ethanol cooled either with liquid nitrogen or with dry ice for
illumination at 198 K. No artificial electron acceptors were added to avoid the oxidation of
the non-heme iron, which gives an EPR signal [74] that overlaps with parts of the high spin
EPR signals in S2 and with the S = 3 S3 signal [34]. In separate experiments, it was verified
that results similar to those presented here were obtained in the presence of the electron
acceptor phenyl-para-benzoquinone (not shown).
For varying the pH values, PSII samples were washed by cycles of dilutions in 1 M
betaine, 15 mM CaCl2, 15 mM MgCl2, followed by concentration using Amicon Ultra-15
centrifugal filter units (cut-off 100 kDa) until the estimated residual MES concentration was
smaller than 1 µM. PSII samples were then loaded in the dark into quartz EPR tubes at 1.1 mg
of Chl mL-1 and dark-adapted for 1 h at room temperature. The samples were then
synchronized in the S1-state with one pre-flash [75]. After a further 1 h dark-adaptation at
room temperature the pH was adjusted by adding concentrated buffers, 100 mM final
concentration, of MES for pH 5.5 and 6.5, HEPES for pH 7.0, 7.5 and 8.0, TAPS for pH 8.3
and 8.6 and CHES for pH 9.0 and 9.3. After incubation in darkness for 1 min at the specific
pH, the samples were frozen in the dark to 198 K and then transferred to 77 K. The samples
were degassed at 198 K prior to recording the spectra. The equilibria between the two S2-
states were studied by a further warming of the S2-samples at room temperature for few
seconds (< 5 s).
Due to the high concentration of the protein originally buffered previously at pH 6.5, it
seemed likely that the actual pH obtained by the procedure described above could result in a
pH slightly lower than that of the concentrated buffer addition. In a separate experiment we
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checked that this treatment (MES removal plus addition of strong buffer) used to generate a
sample at pH 8.3 (a pH value close to the pKa observed for Ca-PSII in Fig. 4C) after the
addition of TAPS, and it was found that the pH was indeed lower by 0.2 pH unit. However,
the pH values plotted in Fig. 4 do not take into account this correction. The effect of the
freezing on the pH value in the same pH range was also tested, in the absence of PSII, as in
[76] by using cresol red as a dye and no pH change was observed. Fig. 2 shows schemes of
the EPR protocols used in this study in which the samples were subjected to a series of
illuminations at 198 K each followed by a warming step to room temperature (see Figs.
3,4,7,8).
Time-resolved absorption change spectroscopy
Absorption changes measurements were measured with a lab–built spectrophotometer
[77], in which the absorption changes are sampled at discrete times by short analytical flashes.
These analytical flashes were provided by an optical parametric oscillator pumped by a
frequency tripled Nd:YAG laser, producing monochromatic flashes (355 nm, 1 nm full-width
at half-maximum) with a duration of 5 ns. Actinic flashes were provided by a second
Nd:YAG laser (532 nm), which pumped an optical parametric oscillator producing
monochromatic saturating flashes at 695 nm with the same flash-length. The path-length of
the cuvette was 2.5 mm. PSII was used at 25 µg of Chl mL–1 in 1 M betaine, 15 mM CaCl2, 15
mM MgCl2, and 40 mM MES (pH 6.5). PSII samples were dark–adapted for ≈ 1 h at room
temperature (20–22°C) before the addition of 0.1 mM phenyl p–benzoquinone (PPBQ)
dissolved in dimethyl sulfoxide.
Quantum Chemical Calculations
Density functional theory (DFT) calculations were performed on the S2 state using the
X-ray structure of PSII from T. vulcanus (PDB ID: 3ARC) [1]. The DFT models comprised
223-225 atoms in addition to the Mn4O5Ca or Mn4O5Sr core, 13 surrounding water molecules,
and residues Asp61, Tyr161 (Yz), Asn181, Asp170, Glu189, His190, His332, Glu333,
His337, Asp342, Ala344, Glu354, Arg357, and Lys317 are included. The DFT models
included Cl1 due it is direct salt-bridging contact with the active site, but not the second
chloride, Cl2, which is ca. 5 Å away from the closest polar side chains of the amino acids of
interest here. The ca. 225 atoms DFT models are likely to have converged electronic
structures, and similar sized models have also been used in previous DFT studies (cf. e.g. [36,
37]). The protein residues were cut at the C positions, except for His332, Glu333, Ala344,
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for which we also modeled parts of the protein backbone. The models were structure
optimized using the TPSS-D3 functional [78,79] together with the multipole accelerated
resolution of identity approximation (MARIJ) [80] and def2-TZVP (for Mn, Ca)/def2-SVP
[81,82] (for C, H, O, N, Cl-) basis sets. Terminal atoms were fixed in the structure
optimizations to account for protein strain, and the protein surroundings were modeled using a
polarizable dielectric medium with = 4 using the conductor-like screening model (COSMO)
[83]. The structures were modeled in the open and closed conformations, with O5 bound to
Mn4 and Mn1, respectively, in the high spin (S = 5/2) and low spin (S = 1/2) states, and by
probing protonation/deprotonation of Asp61 (AspH→Asp-), His337 (HisH+→His), His332
(His→His-), W1 (H2O→OH-), W2 (H2O→OH-), W2 (OH-→O2-), W3 (H2O→OH-), and W4
(H2O→OH-), i.e., modeling in total 96 microstates. The spin energetics were treated using the
broken symmetry spin-flip approach [84] and single point energies were estimated at the
dispersion corrected B3LYP*-D3 level containing 15% exact exchange [85,88]. Bond orders
were computed following Mayer’s definition in its open-shell formulation from Kohn-Sham
orbitals coefficients and overlap matrix elements [86,87]. All calculations were performed
using TURBOMOLE v. 6.6 [89] and Visual Molecular Dynamics (VMD) was used for
visualization [90].
Results
The effects of 198 K illumination on dark-adapted PSII samples at different pH values
prior to freezing are shown in Fig. 3A for native Ca-containing PSII (Ca-PSII) and in Fig. 3B
for Sr-PSII. The EPR spectra shown are the “light”-minus-“dark” difference spectra (for
original traces see Fig. S1). Several observations can be made. Firstly, both the native S 2LS
multiline signal in the Ca-PSII and the modified [28,91] S2LS multiline signal in Sr-PSII
exhibited an amplitude that was little changed in the pH range studied (between 7.5 and 9.0
for Ca-PSII and between 5.5 and 8.6 for Sr-PSII). In Ca-PSII, the spectrum at pH 6.5 (shape
and amplitude) in [17] was similar to that recorded at pH 7.5 here. Secondly, upon the
illumination at 198 K, a large QA-Fe2+QB
- signal at g 1.6 ( 4160 gauss) was detected
[13,14,92]. Indeed, as shown earlier under comparable conditions, in the absence of an added
artificial acceptor, QB- was present in approximately half of centers in the dark-adapted T.
elongatus PSII sample [13]. Thirdly, a very small S2HS signal, S = 5/2, with a turning point at
around 1400 gauss was detected [17], which is unaffected by pH. Lastly, the small negative
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signals at g = 5.65 (≈ 1200 gauss) and g = 7.5 (≈ 900 gauss), which were unaffected by pH,
show that the non-heme iron was oxidized in a minor fraction of PSII in the dark-adapted
samples. These data show that under the conditions used, i.e. a short incubation of PSII in the
S1-state at different pH values, the S1 to S2 transition (generated by illumination at 198 K see
Figure 2) remained essentially unaffected up to pH 9.0 in Ca-PSII and pH 8.6 in Sr-PSII
samples.
Panels A and B in Fig. 4 show the “warmed”-minus-“dark” difference spectra obtained
by warming the same samples to room temperature for ≈ 2-5 s (for original traces see Fig.
S1). The warming resulted in a pH-dependent conversion of the low-spin multiline signal into
a high-spin signal at g = 4.75 (turning point at 1425 gauss) in Ca-PSII (Fig. 4A) and g = 4.9
(turning point at 1383 gauss) in Sr-PSII (Fig. 4B). Fig. 4C shows the effect of pH on the
amplitude of the S2HS signal induced by the warming in Ca-PSII (closed circles) and Sr-PSII
(open circles). The pH dependence for the LS to HS conversion with Sr-PSII was markedly
different from that seen with Ca-PSII: with pKa values 7.5 in Sr-PSII and 8.3 in Ca-PSII.
In addition, the number of protons apparently involved in the S2LS to S2
HS transition differed:
Fig. 4C shows fits of the data assuming a simple equilibrium (LS)Hn (HS)n- + nH+, for
which the best fits were obtained with n 1 for Sr-PSII and n 4.4 in Ca-PSII. Plots of the
amplitude of S2LS vs S3
HS showed a linear relationship without any indication of intermediate
states in both Ca-PSII and Sr-PSII (Fig. S2).
The amplitude of the background S2HS signal present with a similar amplitude at all the
pH values prior to the warming was not included in the plots. In the same way, the fraction of
S2 multiline signal that did not convert into the S2HS signal at high pH was not taken into
account. At present, we cannot offer an explanation for these heterogeneities, however, they
represent less than 20% of the centers and their presence does not affect the main conclusions
drawn below.
In order to probe electronic structure changes caused by the Ca→Sr substitution, we
performed quantum chemical density functional theory (DFT) calculations of S2 in its HS (S =
5/2) and LS (S = 1/2) states, and considered both the open and closed conformational forms of
the cluster. Moreover, to obtain an insight into the identity of potential titratable groups, we
studied the system in several alternative protonation states, considering W1, W2, W3, W4,
O5, O4, His337, His332, Asp61 as putative titratable groups.
Our DFT calculations of the Ca-PSII and the Sr-PSII systems show a preference for the
LS/open and HS/closed forms, which are energetically preferred by 0.8 kcal mol-1 over the
HS/open and LS/closed forms for both Ca-PSII and Sr-PSII, respectively (Table 1). This
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indicates that the LS → HS transition is likely to reflect an open → closed conformational
change in both Ca-PSII and Sr-PSII, as previously reported for the Ca-PSII [31,32].
To obtain an insight into the electronic structure changes induced by the ion
replacement, we further calculated density differences by substituting the Ca by a Sr ion in the
S2-optimized structure of Ca-PSII and subtracted the electron and spin densities between the
two systems. We find that the Ca → Sr substitution leads to both local and non-local changes
in the electronic structure of the Mn4O5Ca/Sr cluster, as shown by the charge (Fig. 5A) and
spin (Fig. 5B) density differences computed upon substituting the Ca by the Sr ion. The 18
additional electrons of Sr in comparison to Ca lead to an increase in the electron density
difference around the Sr/Ca (Fig. 5A, inset: large red sphere) as expected, but in addition this
substitution polarizes bonding molecular orbitals, not only for immediate ligands of the Ca/Sr-
ion (Fig. 5A, inset: W3, W4, O5), but also non-local ligands, most notably on W2 and
Mn1/Mn4 (Fig. 5A, B inset).
We obtain a similar picture from analysis of spin density differences (Fig. 5B), but in
contrast to the charge density distributions, the Ca/Sr-ion and associated ligands (including
W3 and W4) show no significant perturbation in the spin distribution. We obtain large spin
density changes around the Mn1/Mn4 ions and their ligands, in particular on W2 (Fig. 5B
inset). The charge density difference analysis does not account for structural changes induced
by the Ca→Sr substitution, but we obtain matching results by replacing Sr by Ca in the S2-
optimized structure of Sr-PSII.
An assessment of proton affinities in the LS/open and HS/closed conformations (Table
2) suggests that the Ca→Sr substitution induces pKa-shifts on several titratable groups, thus
supporting the qualitative picture derived from the density difference analysis (Fig. 5). We
find that for the Ca-PSII system, His-332, His-337, W2, and Asp-61, have relatively low
proton affinities that could allow them to undergo protonation changes within the 4 pH-unit
(5.7 kcal mol-1) range studied experimentally (Fig. 4), whereas for Sr-PSII, His-337 and W2
have comparable proton affinities (Table 2). In contrast to our EPR data (Fig. 4), however, our
proton affinity calculations suggest that the Ca → Sr substitution leads to shifts towards
higher pKas for most titratable groups (Fig. 5, Table 2). For both Ca-PSII and Sr-PSII, large
proton affinities are predicted for W3 and W4 (Table 2), but removal of protons from W3/W4
is energetically disfavored by 20-40 kcal mol-1 (Table 2) unless Tyr161 is oxidized [48].
In order to obtain insight into electronic structure changes arising from the protonation
changes, we also computed how the spin-splitting energies are affected by the Ca→Sr
substitution. To this end, we estimated the S2HS-S2
LS splitting energies for the protonated and
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deprotonated species, which can be related to the pKa shifts for the S2HS and S2
LS forms
according to Scheme 1 and equation (1):
pKaHS - pKa
LS = (EHS-LS(A
-) - EHS-LS
(AH))/2.303RT Eqn. (1)
where EHS-LS(A
-) andEHS-LS
(AH) are the S2HS-S2
LS energy gap for the deprotonated, A-, and
protonated, AH, species, respectively. We have considered here only the LS/open and
HS/closed forms, but extension to the LS/closed and HS/open forms would also be possible
by extending the derivation to a full thermodynamic cycle.
Scheme 1. Thermodynamic cycle relating the high spin (HS) to low spin (LS) transition with
the proton affinities (pKa values) of the protonated (AH) and deprotonated (A-) species.
Eqn. 1 does not compare absolute DFT energies for systems with a different total
charge, and thus avoids potential artifacts due to, e.g., the DFT self-interaction errors, cf. [93]
and references therein. The spin-splitting calculations suggest that the deprotonation reactions
preserve the energetic ordering of the conformational state, such that the open conformations
prefer the LS state EHS-LS>0, Table 3) and closed conformations preferring the HS form
EHS-LS<0, Table 3), except upon deprotonation of W1, where the LS form is weakly favored
in both conformational states. Derivation of pKa values from the spin-splitting energies (Eqn.
1) suggest that several groups shift towards lower pKa values as a result of the Ca → Sr
substitution (Table 3), except for His-337 (+0.1 pK-units), W2 (+0.1 pK-units), Asp-61 (+0.4
pK-units) that shift towards higher pKa-values, consistent with the EPR data (Fig. 4).
To further analyze electronic structure changes arising from protonation changes of the
different groups, we also computed bond orders (BO) that give insight into covalent bonding
character and bond strengths [94]. The BO-calculations indicate that deprotonation of W1 and
W2 decrease the bond order between Mn4 and O5 (BO=1→0.61 (W1)/0.86(W2)), while
14
somewhat increasing the bond order between Mn1 and O5 (BO=0.04→0.07 (W1, W2)) in the
open form, and vice versa for the closed form (Table S2 As W2 deprotonation in the closed
form is energetically favored in Sr-PSII, this could lead to a higher proportion of S2HS EPR
signals at low pH in Sr-PSII (Figs. 3 and 4). Structural changes upon W2 deprotonation in the
Ca-PSII and Sr-PSII systems are shown in Fig. 5c. For Ca-PSII, this deprotonation increases
the distance between O5 and Mn4 by 0.03 Å in the open/LS and by 0.17 Å in the closed/HS
structure (Fig. 5c). Similarly, we find for Sr-PSII that the O5 and Mn4 distance increases by
0.02 Å in the open/LS and by 0.15 Å in the closed/HS form. Due to these structural changes,
the dangling oxygen (O5) moves away from the Mn4 and gets closer to Mn1 in both Ca-PSII
and Sr-PSII, which are consistent with the BO data, since decreased distances (O5-Mn1) yield
lower BO values and vice versa.
It is well known that increasing the pH in plant PSII has multiple effects, including the
depletion of chloride and the formation of a S2HS
state with a corresponding loss of the S2LS
configuration [95]. By using density functional theory, it was proposed that indeed the S = 5/2
configuration was stabilized upon the removal of the chloride Cl1 [96]. The chloride
requirement reported previously [95] occurred in the S1-state since the g 4 EPR signal was
observed already upon a 198 K illumination, in contrasts to the data reported in Figs. 3 and 4.
This difference is likely due to the presence of 60 mM Cl- in our samples, a concentration
high enough to maintain chloride binding in plant PSII at the 5 min incubation at high pH.
Nevertheless, to exclude completely the possibility of Cl--depletion in the present
experiments, the concentration was raised to 160 mM NaCl in the PSII/Ca sample (which
required the higher pH values to trigger the spin change), and the result is reported in Fig. 6A.
Spectrum a is the difference spectrum “light”-minus-“dark” after the illumination at 198 K at
pH 9.0 in the presence of 160 mM chloride and spectrum b is the difference spectrum
“warmed”-minus-“dark”. The results shown in Fig. 6 are similar to those in Fig. 3 and Fig. 4,
confirming that the S2LS to S2
HS transition seen at high pH is not due to a high pH-induced
chloride-depletion.
The reversibility of the S2LS to S2
HS high pH-induced conversion was tested in the
experiment shown in Fig. 6B using Ca-PSII. A dark-adapted Ca-PSII sample was first
illuminated at 198 K in the presence of 10 mM CHES at pH 9.3 before being warmed and
refrozen prior to recording the EPR spectrum. Then, the sample was thawed again at room
temperature in the dark, 100 mM MES at pH 5.5 was added and the sample was refrozen (this
operation took less than 10 s) and a second EPR spectrum was recorded. Fig. 6B shows the
difference spectrum “warmed at pH 5.5”-minus-“warmed at pH 9.3”. This spectrum exhibits a
15
negative HS signal and a positive LS signal thus confirming that the S2LS to S2
HS transition is a
reversible process. The spectrum in Fig. 6B also shows a negative QA-Fe2+QB
- signal at g = 1.6
due to the electron transfer from QA- to QB
- upon the warming of the sample.
The discovery of the ability of the S2HS to advance to S3 at low temperature was
suggested to be associated with the loss of a proton upon formation of S2HS [17]. Here, we
studied this phenomenon as a function of pH in Sr-PSII and Ca-PSII. For this, the samples at
a range of pH values were first illuminated at 198 K to form S2 (see Fig. 3), warmed briefly to
room temperature to allow formation the high spin form and to allow electron transfer from
QA- to QB (see Fig. 4) and then, re-illuminated at 198 K to test for low temperature formation
of S3 (see Fig. 2 for scheme of the illumination and warming protocol).
Fig. 7 shows the difference spectra obtained after the last 198 K illumination minus the
warmed S2 state spectra. Whatever the pH value, the S2HS signal disappeared when re-
illuminated at 198 K, as manifest by an ‘inverse’ S2HS signal in the difference spectra. In both
the Ca-PSII and Sr-PSII samples this was accompanied by the appearance of the most intense
feature of the S3 signal at g 10 ( 680 gauss). The S3 state signal is poorly resolved in Sr-
PSII at the temperature used for recording the spectra in Fig. 7B (i.e. at 9.5 K, the optimum
temperature for the recording of the S2LS and S2
HS signals in Sr-PSII) whereas the S3 signal is
better resolved at 4.2 K, the optimal temperature for this signal in Sr-PSII (see Fig. S3A).
In both Ca-PSII and Sr-PSII the second illumination at 198 K induced the formation of
i) the QA-Fe2+QB
- signal at g = 1.6 in centers with the QAFe2+QB- state present prior to the
second illumination (n.b.. this kind of PSII preparation contains another plastoquinone in
addition to QA and QB [13]) and ii) the S2TyrZ• split signal in centers that remained in the S2
LS
configuration after the first 198 K illumination and warming step [97,98]. Formation of the
S2TyrZ• split signal resulted in negative S2 multiline signals in the difference spectra in Fig. 7
due to the magnetic interaction of the TyrZ• radical and the Mn4-cluster. This is best seen at the
lowest pH values where the residual S2 multiline signal was the largest (see Fig. S4). The
S2TyrZ• state, which is formed under these conditions, is not stable even at helium temperature
[99] so that its amplitude in Fig. 7 cannot be used reliably.
The S3 state formation upon illumination at 198 K was further investigated in an
experiment shown in Fig. 8 (see Fig. 2B for a scheme of the illumination/warming protocol
used in this experiment). The pH value of a Ca-PSII sample was first adjusted to 8.3, i.e. the
pKa of the S2LS to S2
HS transition, before being illuminated at 198 K. Spectrum a in Fig. 8A
shows the resulting h198K-minus-dark difference spectrum that is similar to that reported in
Fig. 3. Then, the sample was warmed to room temperature to allow the S2LS to S2
HS to
16
equilibrate in about half of the centers (spectrum b). Then, the sample was subjected to a
series of illuminations at 198 K each followed by a warming to room temperature. Fig. 2B
allows us to estimate the expected proportions of S2LS, S2
HS and S3 states at the pH value equal
to the pK value (see legend of Fig. 2). Spectra b, d, f and h in Fig. 8 show the formation of the
S2HS signal accompanied by the loss of the remaining S2
LS upon each warming step and spectra
c, e and g show the disappearance of the S2HS signal and the corresponding formation of the S3
signal upon each of the illuminations at 198 K. More generally, with α the proportion of the S2HS at a given pH value (with 0 < α < 1) and n, the number of cycles (warming at > 230 K followed by illumination at 198 K) after the first illumination at 198 K, the proportion of S3 is 1-(1-α)n and the proportion of the
remaining S2LS is (1-α)n.
Panel B in Fig. 8 compares the 2 flashes-minus-dark difference spectrum at pH 7.5
(spectrum a) to the difference spectrum (spectrum b) obtained by subtracting the dark
spectrum from the spectrum recorded at the end of the 4 cycles of “198 K
illumination/warming in the dark” at pH 8.3 and reported in Panel A. This experiment shows
that the S3 signals formed in the two conditions have a comparable shape and amplitude
despite the absence of an added artificial electron acceptor, which likely resulted in the
closing (i.e. reduction of QA) of a proportion of centers upon several low temperature
illuminations as suggested from the larger signal at g 1.6 seen in spectrum b in Panel B.
Since NH3 is known to displace W1 in S2 [64,66-68] and W1 is thought be deprotonated
in the S2TyrZ• state [44-46], we have tested the influence of NH3 on the S2 to S3 transition in
the light of the new findings here. The results are shown in Fig. 9 and Fig. 8B.
Spectrum a in Fig. 9 is the “light”-minus-“dark” difference spectrum in Ca-PSII after
illumination at 198 K at pH 9.3, i.e. the highest pH value that can be probed without
additional spectroscopic changes occurring (not shown). Spectrum b is the “warmed”-
minus-“dark” difference spectrum, which shows that the formation of the S2HS signal
corresponds to the loss of the S2LS signal, as in Fig. 4. After the recording of spectrum b, the
sample was again warmed and 50 mM NH4Cl was added. Spectrum c is the “after the addition
of NH3”-minus-“dark” difference, it shows that the addition of NH3 resulted in the
disappearance of the S2HS signal and the appearance of the S2
LS signal exhibiting the hyperfine
pattern characteristic of the ammonia-modified [65,100,101] multiline signal. In a separate
experiment (not shown), NH3 was added before the 198 K illumination, i.e. in the S1-state, and
upon the warming of the sample following the 198 K illumination, the formation of the HS
configuration was also inhibited. These data show that NH3 favors the S2LS configuration
17
resulting in no S2HS signal being detectable, effectively reversing the high pH-induced
formation of S2HS.
When the pH 9.3 Ca-PSII, NH3-treated sample was illuminated at 198 K the modified
S2LS did not advance to the S3 state (not shown). This experiment indicates that it is not the
high pH but rather the spin state that is responsible for the ability to advance to S3 at low
temperature.
We went on to investigate in which S-state the removal of NH3 occurs after its binding
in S2 since it is no longer bound in the S1 state formed by 4 flashes [63]. Fig. 10A shows the
S0 multiline signal formed by 3 flashes in the magnetic region where the hyperfine structure
of the multiline signal is better resolved in the absence of methanol in cyanobacterial PSII
[102]. Spectra were recorded either in the absence (spectrum a) of the presence (spectrum b)
of 100 mM NH3. The hyperfine structure of the multiline appeared to be similar in both
conditions suggesting either that ammonia does not modify the S0 multiline signal, in contrast
to the S2-multiline signal, or more likely that NH3 does not bind in the S0 state. Panel B in Fig.
10 shows the kinetics of the S3TyrZ (S3TyrZ
)’ S0TyrZ transitions measured at 292 nm
and the inset shows the amplitude of the absorption changes upon flash illumination at 292
nm [72]. The oscillating pattern observed, i.e. a period of four similar to that in the absence of
ammonia [72], confirms our earlier results [63] that in the conditions where ammonia
modifies S2, it does not inhibit the period 4 oscillation. In Fig. 10B, neither the lag phase (
200 µs), which corresponds to a deprotonation occurring on the S3TyrZ (S3TyrZ
)’ step
[29,103], nor the kinetics of the (S3TyrZ)’ S0TyrZ step (t1/2 of 1-2 ms) [29,72,103-105] were
affected by the presence of ammonia, suggesting that ammonia already left its binding site
prior to formation of the S3TyrZ state.
Fig. 8B shows the S3 signal after two flashes at room temperature at pH 7.5 either in the
absence (spectrum a) or the presence (spectrum c) of ammonia. As reported earlier [106], the
S3 signals were not identical in the presence and in the absence of NH3 but the field position
of the main feature of the signal was only weakly affected, suggesting that NH3 could have
been lost from the site where it binds in S2. Taken together, the data above strongly suggest
that NH3 binds only in the S2 state but could remain close enough to the Mn4CaO5 cluster,
perhaps in a water channel possibly associated with its competition with chloride, see [107]
for a recent discussion, resulting in a slight perturbation of the S3 signal.
Discussion
18
In the present work, we have studied the pH dependence of the equilibrium between the
low spin and high spin configurations of the S2 state (S2LS S2
HS) by using EPR spectroscopy
on both Ca-PSII and Sr-PSII. The S2LS S2
HS equilibrium was found to be pH dependent with
pKa 8.3 in Ca-PSII and pKa 7.5 for Sr-PSII. Surprisingly, the apparent number of protons
involved in the transition derived from the titration curve was different in Ca-PSII (n 4.4)
compared to Sr-PSII (n 1). Above the pKa, where the vast majority of the centres show the
S2HS form, both the Ca-PSII and Sr-PSII are able to advance to form S3 upon illumination at
198 K. This confirms and extends our earlier observation of this effect at pH 6.5 in Sr-PSII
[17] but now, with the pH dependence, this behavior is demonstrated in most of centers in the
native Ca-PSII as well as in Sr-PSII. This is strong experimental support that i) S2HS represents
the favored intermediate through which the S2 to S3 transition occurs and ii) deprotonation is
associated with the formation of the S2HS state. This was originally suggested using
computation chemistry [32,44,48] based on the high resolution crystal structure [1] and on
earlier EPR studies [17,108,109].
The pH-dependence of the S2LS S2
HS equilibrium with a pKa of 8.3 in the native PSII
of T. elongatus means that and under functional, physiological conditions, where the pH of
the lumen is 6.5 or lower, the concentration of S2HP is low while that of S2
LS is high. At lower
pH, light induced formation of TyrZ•(His+) is proposed to electrostatically trigger a
deprotonation event that tips the equilibrium toward the S2HS form [44]. The observations
made here suggest that increasing the pH mimics the electrostatic influence of TyrZ•(His+) on
the S2LS S2
HS equilibrium.
The ability of the S2HS state to progress to S3 at 198 K was previously suggested to be
due to the S2HS state being deprotonated [17]. However, the expected difference in the proton
release pattern between the Sr-PSII and Ca-PSII samples was not observed [110]. The present
study seems to resolve this discrepancy by showing that at pH 6.5 the S2HS state is present
only in a minority of centers. In plant PSII, it was previously reported that the proton release
in the S1 to S2 transition reached a maximum at pH close to 8.0 [111], which is close to the
pKa for Ca-PSII found in the present work. The use of isolated PSII from a thermophilic
cyanobacterium in the present study, rather than plant PSII membranes for the proton release
study [111], may explain the minor differences in the reported pH dependences. Direct
measurements of H+ release are required under the conditions defined here in order to clarify
whether or not proton release actually occurs upon formation of S2HS. It remains possible that
the deprotonation (most likely W1 [44] and see below) on formation of the S2HS state may
reflect the proton movement to an internal base rather than H+ release to bulk.
19
The results described here for T. elongatus also seem to apply, at least in part, to plant
PSII [112,113]. These articles have been overlooked in most of the recent computational
studies dealing with the S2 to S3 transition. In [112,113], after forming an S3-state at pH 6.5, as
manifest by the typical S3 EPR signal of plant PSII, a S2HS signal formed by charge
recombination of S3QA•- by long dark incubation at 77 K. Importantly, this S2
HS EPR signal g
5 was the same as that detected when PSII at pH 8.1 in the S1 state was illuminated at -30
°C. The authors therefore concluded that this S2HS state, while being a transient state at pH 6.5,
was comparatively stable at alkaline pH and could be formed directly by illumination of the
S1 state at elevated temperatures, i.e. a similar to what is observed in the present work. It
seems very likely that the temperature threshold at which the S2LS to S3 transition is blocked,
that is 230-240 K in Ca-PSII from T. elongatus (unpublished data but see [114] for data in
plant PSII), corresponds to the temperature at which the S2LS to S2
HS transition is inhibited
[17]. Since the S2HS to S3 transition can occur at much lower temperatures (< 198 K), this
explains why the S2HS state has never been reported as a trapped intermediate at the low values
of pH generally used for PSII.
Computation studies of the native Ca/Cl-PSII indicated that the S = 1/2 and S = 5/2
configurations were almost isoenergetic and in equilibrium at room temperature [19,31,32].
Due to the slight endergonicity of the S2LS to S2
HS transition (with ΔG ≈ 1.1 kcal mol−1) and the
relatively large activation barrier (with ΔG# ≈ 10.6 kcal mol−1), the half-time of S2LS to S2
HS
conversion is predicted to be very slow at low temperature [32]. Recently, it has been reported
that in plant PSII the S2LS state is approximately 0.7 kcal mol–1 more stable than the S2
HS state
at 298 K [115]. The current results show that this is likely to be pH dependent.
Several experimental and computational studies suggest the existence of more than one
structural form of S3, e.g. [10,17,36,54-56,116]: 1) the dominant S3 state, in which the four
MnIV ions are all 6-coordinated (all-octahedral) resulting in an open cubane structure [36]
giving a S = 3 ground state with a typical EPR signal [34,36]; and 2) a state that does not give
an EPR signal per se but which is manifest as a EPR signal generated by near-IR illumination
at low temperature in approximately 30 to 40 % of the centers [117]. The near-IR radiation is
thought to lead to the formation of an (S2TyrZ•)’ state in which the structure of cluster in the
S2’ state likely reflects the structure of the cluster in S3 state prior to near-IR illumination. A
computational study has suggested that this state, designated S3B [118], contains a square-
pyramidal, 5-coordinate Mn4IV and that S3B is an obligatory intermediate formed upon
oxidation of S2HS by TyrZ• [118]. The final all-octahedral S3 state, is formed from the S3
B
intermediate after a non-substrate water is bound and the cluster readjusts to an open form
20
[118]. In the current work we found no indication of the S3B intermediate being stable between
the S2HS form and the all-octahedral S3 state but this does not argue against its transient
formation. It is not yet clear what differences exist between the S3B state that is proposed to
exist as an intermediate and that which stably coexists with the all-octahedral S3 state after 2
flashes [17,117].
The changes upon the Ca/Sr exchange were also studied recently with QM/MM [120]
and DFT [121] calculations. In [120], the authors found that the Sr-substitution leads to pKa
= +1.0 relative Ca-PSII and that the open cubane form (S2LS) is energetically preferred in all
substituted systems. The data presented here suggest that replacing Ca2+ with Sr2+ induces both local and non-local changes in the electronic structure of the cluster since ≥ 4 protons are involved in the spin transition in Ca-PSII in contrast to only one proton with Sr-PSII, with pKa values in the 7.5-8.3 range..
In the light of these new EPR data, we studied how the Ca-substitution affects the spin- and conformational energetics, as well as proton affinities of the cluster's first-sphere ligands. Our DFT calculations also show that the open cubane form is energetically preferred in the LS state [120] and that the HS state prefers the closed cubane form. In addition to the LS/open → HS/closed transitions, our calculations suggest that replacing the Ca2+
with the Sr2+ not only affects the pKa of direct ligands (W3 and W4) but also the more distant groups, W1, W2, Asp61, and His 332/337. The pKa of His
337, W2, and Asp-61 could shift towards higher pKa's upon replacement of Ca2+
with Sr2+. In addition, the bond order analysis indicated that deprotonation of W1 or W2
might trigger the conformational changes between the open and closed conformations, and
consequently the alterations between the LS-HS configurations. This finding is in agreement
with the earlier computational studies [118].
The calculated pK-shifts, either obtained by the "direct approach" via estimating proton
affinities or the "indirect approach" by computing spin-splitting energies (Table 3), are at the
threshold of DFT's accuracy, and thus quantitative estimates of the exact nature and number
of the groups undergoing protonation changes are difficult to assess. Nevertheless, the
findings suggest that titratable groups beyond the immediate Ca/Sr ligands undergo
protonation changes in pKas as a result of non-local perturbations in the electronic structure of
the oxygen-evolving complex. This is important because a key assumption in assigning
substrate waters in the MIMS-detected water exchange studies rely on the assumption that Sr
21
substitution-induced changes will only affect Sr/Ca bound waters [11]. The present work
implies that this restriction is not necessarily valid, and that these arguments should be
reassessed.
When NH3 is added to PSII at high pH, its binding to the (residual) S2LS configuration is
sufficient to tip equilibrium strongly away from the S2HS configuration, reversing the effect of
increased pH (Fig. 9). This clearly indicates that NH3 binding greatly favors the S2LS
configuration. Earlier EPR studies have indicated that ammonia i) displaces W1 [93]; ii) does
not prevent enzyme turnover [63] and iii) is no longer bound when the S1-state is formed from
the fully NH3-modified S2-state by 3 flashes [63]. Here we provide evidence indicating that
NH3 is very likely no longer bound in the S3 state formed by 2 flashes. Thus it is likely that
the disassociation of NH3 and the rebinding (and deprotonation) of W1 is triggered by the
formation of the S2TyrZ state. In the model in which the S2
HSTyrZ-state is an intermediate
between S2LSTyrZ
and S3TyrZ, the destabilization of the S2HS state by ammonia binding to S2
LS
could explain the 30-fold slowdown, from t1/2 = 400 µs to t1/2 = 13 ms [122], of the rate of the
reduction of TyrZ• by the Mn4CaO5 cluster in the S2 state.
A computational study [123] proposed that the water substrate exchange in the S2 state
occurred in S2HS which is in equilibrium with the S2
LS. In the light of the pH dependent S2LS
S2HS equilibrium reported here, the water exchange from S2
HS would be expected to be
determined by the forward and back reactions rates and to be favored by high pH. Membrane
inlet mass spectroscopy studies can test this model directly. However, while EPR monitors the
steady state concentrations of the S2HS and S2
LS states, the membrane inlet mass spectroscopy
experiments are more complex, in that the observed substrate-water exchange rates depend on
i) the intrinsic exchange rates in each of the two spin states, ii) the steady-state concentrations
of the two spin states and iii) the rate at which S2LS and S2
HS equilibrate. In a recent study [64],
it was observed that the exchange of Ws at pH 7.6 was slower when NH3 was present. This
finding can be seen as supporting the water exchange from S2HS model [123] in the context of
the S2LS S2
HS equilibrium, with NH3 binding to S2LS shifting it to the left.
In conclusion, the new data presented here includes experimental support for several
aspects of the numerous theoretical studies (see e.g. [119]) that have dealt with the events
occurring in the S2 to S3 transition. Here we summarize these events in the context of the
present work. i) The S2 state exists as open cubane S2LS, the favoured state in the S2
LS↔S2HS
equilibrium [31-33]. Here we show that in this equilibrium the closed cubane S2HS state is
favored by increasing the pH, while the open cubane S2LS is favored by ammonia binding. ii)
The pH effect reported here fits with the prediction that a deprotonation occurs, probably from
22
W1, upon S2HS formation [44]. iii) The dynamic structural equilibrium in S2 is considered to
be key importance for substrate exchange. The single MnIII ion present, which is expected a
much more rapid site for water exchange than the three Mn IV ions, shifts its position within
the cluster through electron transfer, with the MnIII jumping between Mn1 and Mn4 in the S2LS
to S2HS equilibrium [11, 31-33] (see Fig. 1). Given the good access for water to Mn4 [1,2], this
suggests that rapid exchange takes place in S2HS, where the non-cubane Mn4III is present, and
this suggestion is backed by modelling [123]. The observation here that S2LS is favored by
NH3 binding, when taken with the literature report of slower water exchange when NH3 is
present [64], provides experimental support for rapid substrate exchange in S2HS [123]. iv)
Theoretical studies have suggested that the S2HS state is an intermediate in S3 formation [32,
44, 48]. This finds strong experimental support from the observation here, extending the
earlier indications [17], that S2HS is able to advance to the S3 state at 198K, whereas the S2
LS
only advances to the S3 state above 240 K, the temperature needed for S2LS to S2
HS conversion.
Future studies may address what kind of changes (e.g, preloading of water and/or
deprotonation steps), have occurred in S2HS that allow it to advance to S3 at this temperature.
v) The S2HS to open cubane S3 transition has been suggested to occur via a closed cubane (S3
B)
intermediate [118]. While this seems very plausible, the data here showed no evidence for
accumulation of such an intermediate but thus could simply be due to its stability under the
conditions used.
Acknowledgements
We dedicate this work to our friend and colleague Fabrice Rappaport who passed away in January 2016 and who participated in this work by providing the time-resolved absorption data. This work was supported in part by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05 and ANR, ANR15PS2FIRX. AWR was supported by the
Biotechnology and Biological Sciences Research Council grant numbers BB/K002627/1 and
BB/L011206/1 and the Royal Society Wolfson Research Merit Award. MS acknowledges
financial support from MEXT (17K07367) and Japan Health Foundation, VRIK is supported
by the German Research Foundation (DFG).
Supplementary Material
23
Additional EPR data. Tables with electronic structure data.
24
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37
Legends of figures
Figure 1:
Structure of the open S2LS state (left) and closed S2HS state (right). Ca is
yellow, O in red, H in white, MnIII in blue and MnIV in green.
Figure 2:
Scheme showing the proportion of the S2LS and S2HS states in EPR experiments in which the PSII samples were subjected, after the first illumination at 198 K, to cycles of warming at room temperature (RT) followed by illumination 198 K. Ideally, at pH = pK, the S2
LS formed in nearly all the
centres (100 % of the centres) upon the first illumination at 198 K, would equilibrate to form
an equal amount of S2LS (50%) and S2
HS (50%) upon warming. Subsequent illuminations at 198
K will advance S2HS to S3 (50%) and while S2
LS is unable to advance to S3 at this temperature.
The next warming will allow the S2LS to re-equilibrate forming equal quantities of S2
LS and
S2HS again, this time 25%/25% of the centers. It can be seen that this illumination and
warming regime would be expected to generate S3 after the 2nd, 3rd, and 4th illuminations in
50%, 25% and 12.5% of the centres and totaling 87.5% of the centres after the 4th
illumination. This would reflect the concentration of the S2HS present prior to the illumination
at 198 K. The ideal situation will be affected by the availability of QB or QB-• as electron
acceptors from QA-•
.limitations on the acceptor side would diminish yields because charge
recombination with QA-• during the warming of the sample or by the presence of QA-•prior to
illumination preventing stable charge separation.
Figure 3:
EPR spectra recorded in Ca-PSII (Panel A) and Sr-PSII (Panel B) versus the pH value. The
spectra are the “light”-minus-“dark” difference spectra i.e. the spectra recorded after
illumination at 198 K minus spectra recorded in dark-adapted PSII [Chl] = 1.1 mg ml-1.
Instrument settings: Temperature, 8.6 K for Ca-PSII and 9.5 K for Sr-PSII; modulation
38
amplitude, 25 G; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation
frequency, 100 kHz. The YD• spectral region at g ≈ 2 was deleted.
Figure 4:
EPR spectra recorded in Ca-PSII (Panel A) and Sr-PSII (Panel B) versus the pH value. The
spectra are the “light + warmed”-minus-“dark” difference spectra i.e. the spectra recorded
after a warming at room temperature following the 198 K illumination minus spectra recorded
in dark-adapted PSII. Same instrument settings as in Fig. 3. Panel C shows the amplitude of
the high spin S2 signal in Panels A and B versus the pH value. Open circles, Sr-PSII; closed
circles, Ca-PSII. The continuous lines are a fit of the experimental points done as explained in
the text.
Figure 5:
A) Charge density difference between Ca-PSII and Sr-PSII systems (Ca-minus-Sr electron
density), and B) spin density differences (Ca-minus-Sr spin density) between Ca-PSII and Sr-
PSII systems. Red (blue) surfaces indicate increased (decreased) electron density/spin density
upon replacement of the Sr with the Ca. Inset: the lower panels are expansions of the upper
panel showing the Ca/Sr environment. The figure shows charge/spin density differences
beyond the local ligands of Ca/Sr. C) DFT-optimized molecular structures of the S2+ in
Ca/PSII (right) and Sr/PSII (left). Only the central catalytic core of the 220 atom models are
shown with Ca (in yellow), Sr (in silver), O (in red), H (in white), N (in blue), C (in gray),
MnIII (in blue), MnIV (in green). All distances are given in Å.
Figure 6:
Spectra in Panels A and B were recorded in PSII/Ca. Panel A: spectrum a is the “light at 198
K”-minus-“dark” difference spectrum recorded at pH 9.0 after the further addition of 100 mM
NaCl and spectrum b is “light at 198 K + warmed”-minus-“dark” difference spectrum. Panel
B: PSII was illuminated at 198 K at pH 9.3 then warmed at room temperature and a first EPR
spectrum was recorded (spectrum 1). Then, the sample was warmed again and the pH
decreased to 5.5 before the recording of the second EPR spectrum (spectrum 2). The
difference spectrum shown in Panel B is the spectrum 2 minus spectrum 1.
39
Figure 7:
EPR spectra recorded in Ca-PSII (Panel A) and Sr-PSII (Panel B) versus the pH value. The
spectra are the “1st light + warmed + 2nd light”-minus-“ 1st light + warmed” difference spectra.
Both illuminations were at 198 K and were separated by warming of the sample in the dark to
room temperature for a few seconds. Same instrument settings as in Fig.3.
Figure 8:
Panel A: A Ca-PSII sample at pH 8.3 was first illuminated at 198 K. Then the
sample was subjected to a series of warmings to room temperature and re-illuminations at 198
K. Spectra b, d, f and h were recorded after the warmings (the spectra recorded before the
warmings were subtracted) and spectra a, c, e and g were recorded after the illuminations at
198 K (the spectra recorded before the illuminations were subtracted). Panel B: Spectrum a is
the “2 flashes”-minus-dark spectrum at pH 7.5 in Ca-PSII and spectrum b is the difference
spectrum recorded at pH 8.3 after the 4 cycles in Panel A. Spectrum c is the “2 flashes”-
minus-dark spectrum at pH 7.5 in Ca-PSII with 50 mM NH4Cl present. Same instrument
settings as in Fig. 1.
Figure 9:
Spectrum a is the “light”-minus-“dark” difference spectrum recorded at pH 9.3. Spectrum b is
the “light + warmed”-minus-“dark” difference spectrum. After a second warming 50 mM
NH4Cl were added and spectrum c is the difference spectrum “after the addition of 50 mM
NH4Cl”-minus-“dark”. Same instrument settings as in Fig. 3.
Figure 10:
Panel A: EPR spectra recorded in Ca-PSII after 3 flashes in the presence of either 100 mM
NaCl (spectrum a) or 100 mM NH4Cl at pH 7.5 in Ca-PSII. Same instrument settings as in
Fig. 3 except 4.2 K for the temperature. Panel B: Kinetics of the absorption changes at 292
nm after the third flash given to dark-adapted Ca-PSII in the presence of 100 mM NH4Cl at
pH 7.5 and 100 µM Phenyl-para-benzoquinone dissolved in dimethyl-sulfoxyde. The inset
40
shows the amplitude of the absorption changes at 292 nm 100 ms after each flash in a series
of saturating flashes (spaced 400 ms apart).
41
Table 1. Energetics (in kcal mol-1) for HS/LS spin states and open/closed conformational
states for S2 Ca-PSII and Sr-PSII. The systems were modeled with the oxo-bridges are in the
O2− form, Asp61 deprotonated, His332 is single protonated (Nδ), His337 is double protonated
(Nδ, Nε) W2 is in the hydroxide (OH−) form, and W1, W3, W4 in their standard protonation
states. The energetically preferred states are highlighted in bold font.
Ca-PSII Sr-PSIIConformation LS HS LS HSOpen 0.0 0.8 0.0 0.8Closed 1.9 1.1 1.4 0.6
42
Table 2. Relative proton affinities (in kcal mol-1) of selected titratable groups in Ca-PSII and
Sr-PSII in the LS/open and HS/closed S2 states (see SI Table 1 for LS/closed and HS/open
states). In the reference S2 model, the oxo-bridges are in the O2− form, Asp61 is deprotonated,
His332 is single protonated (Nδ), His337 is double protonated (Nδ, Nε), W2 is in the
hydroxide (OH−) form, and W1, W3, W4 are in their standard protonation states. ∆pKa values
are calculated with respect to this reference structure.
Protonation site Spin/conformation
Proton affinity Ca-PSII[kcal mol-1]
Proton affinity Sr-PSII[kcal mol-1]
∆pKa
[pK-units]
Asp61 LS/open 5.9 9.8 -2.7Asp61 HS/closed 7.5 7.9 -0.3W1: OH- LS/open 9.5 14.2 -3.3W1: OH- HS/closed 12.5 14.6 -1.5W2: H2O LS/open 5.2 8.0 -2.0W2: H2O HS/closed 0.0 0.0 0.0W2: O2- LS/open 25.7 28.2 -1.8W2: O2- HS/closed 26.5 28.4 -2.0His332: δN LS/open 0.0 5.2 -3.7His332: δN HS/closed 1.9 3.0 -1.4His337: δN LS/open 0.0 0.0 0.0His337: δN HS/closed 1.7 2.1 -0.3W3: OH- LS/open 41.2 42.1 -0.6W3: OH- HS/closed 18.8 18.7 0.1W4: OH- LS/open 40.1 32.0 +5.7W4: OH- HS/closed 18.2 19.0 -0.6
43
Table 3. Spin splitting energies (in kcal mol-1) for deprotonated (A-) and protonated (AH) selected titratable groups in Ca-PSII and Sr-PSII in the LS/open and HS/closed S2 states. The difference spin splitting energies upon deprotonation (A-AH; EHS-LS
(A-) - EHS-LS
(AH)) are related to pKa shift (in pK-units, E/2.303RT, with T=310 K, EHS-LS=EHS-LS(Ca-PSII)-EHS-LS(Sr-PSII)) of titratable groups according to Eqn. 1. pKa values which are higher in Sr-PSII
than in Ca-PSII are highlighted in bold.
Protonation site EHS-LS/Ca-PSII(A-)
EHS-LS/Ca-PSII (AH)
EHS-LS/Sr-PSII(A-)
EHS-LS/Sr-PSII(AH)
EHS-LS/ (A-/AH)Ca-PSII
EHS-LS/(A-/AH)Sr-PSII
EHS-LS/ 1.42=∆pKa
Asp61 Open 0.8 0.8 0.8 0.8 0 0 0Asp61 Closed -0.8 -1.2 -0.8 -0.7 0.4 -0.1 +0.4W2: H2O Open 0.8 0.7 0.8 0.7 0.1 0.1 0W2: H2O Closed -0.8 -0.8 -0.8 -0.9 0 0.1 -0.1W1: OH- Open 0.5 0.8 0.6 0.8 -0.3 -0.2 -0.1W1: OH- Closed 0.8 -0.8 0.8 -0.8 1.6 1.6 0W2: O2- Open 0.9 0.8 0.8 0.8 0.1 0 +0.1W2: O2- Closed 0.1 -0.8 0.4 -0.8 0.9 1.2 -0.2His332: δN Open 0.9 0.8 1.1 0.8 0.1 0.3 -0.1His332: δN Closed -0.7 -0.8 -0.6 -0.8 0.1 0.2 -0.1His337: δN Open 0.8 0.8 1 0.8 0 0.2 -0.1His337: δN Closed -0.5 -0.8 -0.7 -0.8 0.3 0.1 +0.1W3: OH- Open 0.8 0.8 0.9 0.8 0 0.1 -0.1W3: OH- Closed -1.4 -0.8 -1.6 -0.8 -0.8 -0.8 0W4: OH- Open 1 0.8 0.9 0.8 0.2 0.1 +0.1W4: OH- Closed -1.3 -0.8 -1.7 -0.8 -0.5 -0.9 +0.3