The Mn4CaO5-cluster in Photosystem II advances through five oxidation states, S0 to S4, before water is oxidized and O2 is generated. The S2-state exhibits either a low-spin, S = 1/2 (S2LS), or a ...high-spin state, S = 5/2 (S2HS). Increasing the pH favors the S2HS configuration and mimics the formation of TyrZ in the S2LS-state at lower pH values (Boussac et al. Biochim. Biophys. Acta 1859 (2018) 342). Here, the temperature dependence of the S2HS to S3 transition was studied by EPR spectroscopy at pH 8.6. The present data strengthened the involvement of S2HS as a transient state in the S2LSTyrZ → S2HSTyrZ → S3TyrZ transition. Depending on the temperature, the S2HS progresses to S3 states exhibiting different EPR properties. One S3-state with a S = 3 signal, supposed to have a structure with the water molecule normally inserted in S2 to S3 transition, can be formed at temperatures as low as 77 K. This suggests that this water molecule is already bound in the S2HS state at pH 8.6. The nature of the EPR invisible S3 state, formed down to 4.2 K from a S2HS state, and that of the EPR detectable S3 state formed down to 77 K are discussed. It is proposed that in the S2LS to S3 transition, at pH < 8.6, the proton release (Sugiura et al. Biochim. Biophys. Acta 1859 (2018) 1259), the S2LS to S2HS conversion and the binding of the water molecule are all triggered by the formation of TyrZ.
•The results support the involvement of S2HS in the S2LSTyrZ to S3 transition.•The present data do not argue against a closed cubane structure for the S2HS state.•The data support the heterogeneity in S3 also found in computational works.•Formation of S2TyrZ likely triggers the H+ release and the S2LS to S2HS conversion.•Formation of S2TyrZ likely also triggers the insertion of the new H2O in S2LS to S3.
Photosystems I and II convert solar energy into the chemical energy that powers life. Chlorophyll a photochemistry, using red light (680 to 700 nm), is near universal and is considered to define the ...energy "red limit" of oxygenic photosynthesis. We present biophysical studies on the photosystems from a cyanobacterium grown in far-red light (750 nm). The few long-wavelength chlorophylls present are well resolved from each other and from the majority pigment, chlorophyll a. Charge separation in photosystem I and II uses chlorophyll f at 745 nm and chlorophyll f (or d) at 727 nm, respectively. Each photosystem has a few even longer-wavelength chlorophylls f that collect light and pass excitation energy uphill to the photochemically active pigments. These photosystems function beyond the red limit using far-red pigments in only a few key positions.
Light-driven oxidation of water into dioxygen, catalysed by the oxygen-evolving complex (OEC) in photosystem II, is essential for life on Earth and provides the blueprint for devices for producing ...fuel from sunlight. Although the structure of the OEC is known at atomic level for its dark-stable state, the mechanism by which water is oxidized remains unsettled. Important mechanistic information was gained in the past two decades by mass spectrometric studies of the H2(18)O/H2(16)O substrate-water exchange in the four (semi) stable redox states of the OEC. However, until now such data were not attainable in the transient states formed immediately before the O-O bond formation. Using modified photosystem II complexes displaying up to 40-fold slower O2 production rates, we show here that in the transient S3YZ state the substrate-water exchange is dramatically slowed as compared with the earlier S states. This further constrains the possible sites for substrate-water binding in photosystem II.
The photosynthetic protein complex photosystem II oxidizes water to molecular oxygen at an embedded tetramanganese-calcium cluster. Resolving the geometric and electronic structure of this cluster in ...its highest metastable catalytic state (designated S₃) is a prerequisite for understanding the mechanism of O-O bond formation. Here, multifrequency, multidimensional magnetic resonance spectroscopy reveals that all four manganese ions of the catalyst are structurally and electronically similar immediately before the final oxygen evolution step; they all exhibit a 4+ formal oxidation state and octahedral local geometry. Only one structural model derived from quantum chemical modeling is consistent with all magnetic resonance data; its formation requires the binding of an additional water molecule. O-O bond formation would then proceed by the coupling of two proximal manganese-bound oxygens in the transition state of the cofactor.
The stoichiometry and kinetics of the proton release were investigated during each transition of the S-state cycle in Photosystem II (PSII) from Thermosynechococcus elongatus containing either a ...Mn4CaO5 (PSII/Ca) or a Mn4SrO5 (PSII/Sr) cluster. The measurements were done at pH 6.0 and pH 7.0 knowing that, in PSII/Ca at pH 6.0 and pH 7.0 and in PSII/Sr at pH 6.0, the flash-induced S2-state is in a low-spin configuration (S2LS) whereas in PSII/Sr at pH 7.0, the S2-state is in a high-spin configuration (S2HS) in half of the centers. Two measurements were done; the time-resolved flash dependent i) absorption of either bromocresol purple at pH 6.0 or neutral red at pH 7.0 and ii) electrochromism in the Soret band of PD1 at 440 nm. The fittings of the oscillations with a period of four indicate that one proton is released in the S1 to S2HS transition in PSII/Sr at pH 7.0. It has previously been suggested that the proton released in the S2LS to S3 transition would be released in a S2LSTyrZ• → S2HSTyrZ• transition before the electron transfer from the cluster to TyrZ• occurs. The release of a proton in the S1TyrZ• → S2HSTyrZ transition would logically imply that this proton release is missing in the S2HSTyrZ• to S3TyrZ transition. Instead, the proton release in the S1 to S2HS transition in PSII/Sr at pH 7.0 was mainly done at the expense of the proton release in the S3 to S0 and S0 to S1 transitions. However, at pH 7.0, the electrochromism of PD1 seems larger in PSII/Sr when compared to PSII/Ca in the S3 state. This points to the complex link between proton movements in and immediately around the Mn4 cluster and the mechanism leading to the release of protons into the bulk.
•No proton is released in the S1TyrZ• → S2LS transition in PSII/Ca at pH 6.0 and 7.0.•No proton is released in the S1TyrZ• → S2LS transition in PSII/Sr at pH 6.0.•A proton is released in the S1TyrZ• → S2HS transition in PSII/Sr at pH 7.0.•The Ca2+/Sr2+ exchange slows down the S0TyrZ• → S1 and S1TyrZ• → S2 steps at pH 6 and 7.•In PSII/Sr, at pH 6, the H+ release and the S0TyrZ• → S1 step are kinetically coupled.
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 S2HS 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 S3via the S2HS state without detectable intermediates. We discuss the nature of the changes occurring in the S2LS to S2HS transition which allow the S2HS to S3 transition to occur below 200 K. This work also provides a protocol for generating S3 in concentrated samples without the need for saturating flashes.
•The equilibrium between S2LS and S2HS is pH dependent.•pKa ≈ 8.3 (n ≈ 4) for the native Mn4CaO5 and pKa ≈ 7.5 (n ≈ 1) for Mn4SrO5.•From DFT analysis the Ca/Sr exchange affects several titratable groups.•Along titrable groups there W1/W2, Asp61, His332 and His337.
Electrochromic band-shifts have been investigated in Photosystem II (PSII) from Thermosynechoccocus elongatus. Firstly, by using Mn-depleted PsbA1-PSII and PsbA3-PSII in which the QX absorption of ...PheD1 differs, a band-shift in the QX region of PheD2 centered at ~ 544 nm has been identified upon the oxidation, at pH 8.6, of TyrD. In contrast, a band-shift due to the formation of either QA•- or TyrZ• is observed in PsbA3-PSII at ~ 546 nm, as expected with E130 H-bonded to PheD1 and at ~ 544 nm as expected with Q130 H-bonded to PheD1. Secondly, electrochromic band-shifts in the Chla Soret region have been measured in O2-evolving PSII in PsbA3-PSII, in the PsbA3/H198Q mutant in which the Soret band of PD1 is blue shifted and in the PsbA3/T179H mutant. Upon TyrZ•QA•- formation the Soret band of PD1 is red shifted and the Soret band of ChlD1 is blue shifted. In contrast, only PD1 undergoes a detectable S-state dependent electrochromism. Thirdly, the time resolved S-state dependent electrochromism attributed to PD1 is biphasic for all the S-state transitions except for S1 to S2, and shows that: i) the proton release in S0 to S1 occurs after the electron transfer and ii) the proton release and the electron transfer kinetics in S2 to S3, in T. elongatus, are significantly faster than often considered. The nature of S2TyrZ• is discussed in view of the models in the literature involving intermediate states in the S2 to S3 transition.
•Electrochromic band-shifts in Photosystem II from Thermosynechoccocus elongatus•A protocol for detecting a band shift of the QX absorption band of PheD2 is described.•The time-resolved electrochromism is reported for all the S-state transitions.•The lifetime of intermediate states in S2 to S3 is shorter than previously considered.
The Mn
CaO
-cluster in Photosystem II advances through five oxidation states, S
to S
, before water is oxidized and O
is generated. The S
-state exhibits either a low-spin, S = 1/2 (S
), or a ...high-spin state, S = 5/2 (S
). Increasing the pH favors the S
configuration and mimics the formation of Tyr
in the S
-state at lower pH values (Boussac et al. Biochim. Biophys. Acta 1859 (2018) 342). Here, the temperature dependence of the S
to S
transition was studied by EPR spectroscopy at pH 8.6. The present data strengthened the involvement of S
as a transient state in the S
Tyr
→ S
Tyr
→ S
Tyr
transition. Depending on the temperature, the S
progresses to S
states exhibiting different EPR properties. One S
-state with a S = 3 signal, supposed to have a structure with the water molecule normally inserted in S
to S
transition, can be formed at temperatures as low as 77 K. This suggests that this water molecule is already bound in the S
state at pH 8.6. The nature of the EPR invisible S
state, formed down to 4.2 K from a S
state, and that of the EPR detectable S
state formed down to 77 K are discussed. It is proposed that in the S
to S
transition, at pH < 8.6, the proton release (Sugiura et al. Biochim. Biophys. Acta 1859 (2018) 1259), the S
to S
conversion and the binding of the water molecule are all triggered by the formation of Tyr
.
Photosystem II (PSII) uses the energy from red light to split water and reduce quinone, an energy-demanding process based on chlorophyll a (Chl-a) photochemistry. Two types of cyanobacterial PSII can ...use chlorophyll d (Chl-d) and chlorophyll f (Chl-f) to perform the same reactions using lower energy, far-red light. PSII from
has Chl-d replacing all but one of its 35 Chl-a, while PSII from
, a facultative far-red species, has just 4 Chl-f and 1 Chl-d and 30 Chl-a. From bioenergetic considerations, the far-red PSII were predicted to lose photochemical efficiency and/or resilience to photodamage. Here, we compare enzyme turnover efficiency, forward electron transfer, back-reactions and photodamage in Chl-f-PSII, Chl-d-PSII, and Chl-a-PSII. We show that: (i) all types of PSII have a comparable efficiency in enzyme turnover; (ii) the modified energy gaps on the acceptor side of Chl-d-PSII favour recombination via P
Phe
repopulation, leading to increased singlet oxygen production and greater sensitivity to high-light damage compared to Chl-a-PSII and Chl-f-PSII; (iii) the acceptor-side energy gaps in Chl-f-PSII are tuned to avoid harmful back reactions, favouring resilience to photodamage over efficiency of light usage. The results are explained by the differences in the redox tuning of the electron transfer cofactors Phe and Q
and in the number and layout of the chlorophylls that share the excitation energy with the primary electron donor. PSII has adapted to lower energy in two distinct ways, each appropriate for its specific environment but with different functional penalties.
The Mn4CaO5 cluster of photosystem II (PSII) advances sequentially through five oxidation states (S0 to S4). Under the enzyme cycle, two water molecules are oxidized, O2 is generated and four protons ...are released into the lumen. Umena et al. (2011) have proposed that, with other charged amino acids, the R323 residue of the D1 protein could contribute to regulate a proton egress pathway from the Mn4CaO5 cluster and TyrZ via a proton channel identified from the 3D structure. To test this suggestion, a PsbA3/R323E site‐directed mutant has been constructed and the properties of its PSII have been compared to those of the PsbA3‐PSII by using EPR spectroscopy, polarography, thermoluminescence and time‐resolved UV–visible absorption spectroscopy. Neither the oscillations with a period four nor the kinetics and S‐state‐dependent stoichiometry of the proton release were affected. However, several differences have been found: (1) the P680+ decay in the hundreds of ns time domain was much slower in the mutant, (2) the S2QA−/DCMU and S3QA−/DCMU radiative charge recombination occurred at higher temperatures and (3) the S0TyrZ•, S1TyrZ•, S2TyrZ• split EPR signals induced at 4.2 K by visible light from the S0TyrZ, S1TyrZ, S2TyrZ, respectively, and the (S2TyrZ•)' induced by NIR illumination at 4.2 K of the S3TyrZ state differed. It is proposed that the R323 residue of the D1 protein interacts with TyrZ likely via the H‐bond network previously proposed to be a proton channel. Therefore, rather than participating in the egress of protons to the lumen, this channel could be involved in the relaxations of the H‐bonds around TyrZ by interacting with the bulk, thus tuning the driving force required for TyrZ oxidation.
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