Environment of Tyr_Z in Photosystem II from Thermosynechococcus elongatus in which PsbA2 Is the D1 Protein

Background: Photosystem II, the water-splitting enzyme, includes a protein, D1, which can be coded by three different psbA genes in Thermosynechococcus elongatus.

Results: In PsbA2-PSII, the environment of Tyr_Z is different from that in PsbA1-PSII and PsbA3-PSII.

Conclusion: The geometry of the Tyr_Z-O···H···Nϵ-His-190 bonding is an important parameter for PSII activity.

Significance: The environment of the cofactors is involved in the tuning of the electron transfer efficiency.

Abstract

The main cofactors that determine the photosystem II (PSII) oxygen evolution activity are borne by the D1 and D2 subunits. In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for D1. Among the 344 residues constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption spectroscopy, we show that: (i) the time-resolved EPR spectrum of Tyr_Z^• in the (S₃Tyr_Z^•)′ is slightly modified; (ii) the split EPR signal arising from Tyr_Z^• in the (S₂Tyr_Z^•)′ state induced by near-infrared illumination at 4.2 K of the S₃Tyr_Z state is significantly modified; and (iii) the slow phases of P₆₈₀^+⋅ reduction by Tyr_Z are slowed down from the hundreds of μs time range to the ms time range, whereas both the S₁Tyr_Z^• → S₂Tyr_Z and the S₃Tyr_Z^• → S₀Tyr_Z + O₂ transition kinetics remained similar to those in PsbA(1/3)-PSII. These results show that the geometry of the Tyr_Z phenol and its environment, likely the Tyr-O···H···Nϵ-His bonding, are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to the dynamics of the proton-coupled electron transfer processes associated with the oxidation of Tyr_Z being affected. From sequence comparison, we propose that the C144P and P173M substitutions in PsbA2-PSII versus PsbA(1/3)-PSII, respectively located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, His-190, are responsible for these changes.

Introduction

Light-driven water oxidation catalyzed by photosystem II (PSII)³ is the first step in the photosynthetic production of biomass, fossil fuels, and O₂ on earth. Refined three-dimensional x-ray structures from 3.5 to 2.9 Å resolution have been obtained using PSII isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus (1, 2). More recently, an x-ray structure at 1.9 Å resolution has been obtained using PSII isolated from the thermophilic cyanobacterium Thermosynechococcus vulcanus (3). PSII core complex is made up of 17 transmembrane protein subunits and 3 extrinsic proteins. In total, it contains 35 chlorophylls, 2 pheophytins, 2 hemes, 1 non heme iron, 2 quinones, 3–4 calcium ions, one of which is part of the Mn₄CaO₅ cluster, 3 chloride ions, two of which are at 7 Å from the Mn₄CaO₅ cluster, 11–12 carotenoid molecules, more than 20 lipids, and over 1300 water molecules (3).

The main cofactors involved in the function of PSII are integral components of the D1 and D2 proteins. The exciton resulting from the absorption of a photon is transferred to the photochemical trap, which undergoes a charge separation. The positive charge is then stabilized on P₆₈₀, a weakly coupled chlorophyll dimer (P_D1 and P_D2). Then, P₆₈₀^+⋅ oxidizes a tyrosine residue of the D1 polypeptide, Tyr_Z, which in turn oxidizes the Mn₄CaO₅ cluster. On the acceptor side, the pheophytin anion (Pheo_D1^˙̄) transfers the electron to the primary quinone electron acceptor, Q_A, which in turn reduces the second quinone, Q_B. Q_A is tightly bound and acts as a one-electron carrier, whereas Q_B acts as a two-electron and two-proton acceptor with a stable semiquinone intermediate, Q_B^˙̄. Although the Q_B^˙̄ semiquinone state is tightly bound, the quinone and quinol forms are exchangeable with the quinone pool in the thylakoid membrane (e.g. Refs. 4–9).

The Mn₄CaO₅ cluster acts both as a device accumulating oxidizing equivalents and as the catalytic site for water oxidation. The enzyme cycles sequentially through five redox states, denoted S_n, where n stands for the number of oxidizing equivalents stored. Upon formation of the S₄ state, two molecules of water are rapidly oxidized, the S₀ state is regenerated, and O₂ is released (10, 11).

Cyanobacterial species have multiple psbA variants coding for the D1 protein (e.g. Refs. 12–20). These different genes are known to be differentially expressed depending on the environmental conditions (e.g. Refs. 12–18). In particular, specific up/down-regulations of one of these genes under high light conditions is indicative of a photo-protection mechanism. For example, the mesophilic cyanobacterium, Synechocystis PCC 6803, has three psbA genes. Two of these (psbAII and psbAIII) produce an identical D1. Nevertheless, although psbAII is expressed under the “normal” cultivation conditions, transcription of psbAIII is induced by high light or UV light (15), and that of psbAI seems triggered by microaerobic conditions (18). T. elongatus also has three different psbA genes in its genome (20). The comparison of the mature D1 amino acid sequence deduced from the psbA₃ gene with those of the psbA₁ and psbA₂ genes points to a difference of 21 and 31 residues, respectively (see supplemental Figs. S1 and S2). It has been reported that in T. elongatus, psbA₁ is constitutively expressed under normal laboratory conditions, whereas the transcription of psbA₃ occurred under high light or UV light conditions (16, 21, 22).

In T. elongatus, the change of Q130 in PsbA1-PSII to E130 in PsbA3-PSII has been shown to increase the midpoint potential of Pheo_D1/Pheo_D1^˙̄ by 17 mV from −522 mV in PsbA1-PSII (23) to −505 mV in PsbA3-PSII (19). Because this increase was half the one observed upon single site-directed mutagenesis in Synechocystis PCC 6803 (24, 25), this led us to propose that the effects of the D1-Q130E substitution could be, at least partly, compensated for by some of the additional amino acid changes associated with the PsbA3 for PsbA1 substitution (19). For manganese-depleted PSII and above pH 7.5, where the phenolic hydroxyl group of Tyr_Z hydrogen-bonds H190, the proton acceptor, Tyr_Z oxidation by P₆₈₀^+⋅ is controlled by its prior deprotonation (26), and the electron transfer rate between P₆₈₀^+⋅ and Tyr_Z has been found to be slightly faster in PsbA3-PSII (global t½ ∼100 μs) than in PsbA1-PSII (global t½ ∼200 μs (19). The temperature dependences of the S₂Q_A^˙̄ charge recombination in PsbA1 and PsbA3 have shown that the environments of Q_A and, as a consequence, its redox potential, are likely to be different (19). The exchange of S270 in PsbA1 for A270 in PsbA3 has been suggested to influence the stabilization of the sulfoquinovosyl-diacylglycerol molecule that lies between Q_B and nonheme iron (27). Maybe as a consequence, the binding of bromoxynil in PsbA3-PSII and in PsbA1-PSII has been found to differ, suggesting that the Q_B pocket had different properties. It has been also found that the midpoint potential of the Fe^III/Fe^II couple was likely higher in PsbA1-PSII than in PsbA3-PSII (28). In addition, under photo-inhibitory conditions, the accelerated decrease in O₂ evolution in WT*1 ⁴ (producing PsbA1-PSII) cells was found to correlate with a much faster inhibition of the S₂ state formation than in WT*3 (producing PsbA3-PSII) cells (29).

Although there have been an increasing number of studies aimed at characterizing the properties of PsbA1-PSII when compared with those of PsbA3-PSII, those of PsbA2-PSII have not yet been reported. In the present work, we describe the first construction of a T. elongatus deletion mutant lacking both the psbA₁ and the psbA₃ genes and expressing only psbA₂. We focused our first characterization of PsbA2-PSII on the electron transfer reactions involving Tyr_Z. Using continuous wave EPR at helium temperature, time-resolved EPR at room temperature, and time-resolved UV-visible absorption spectroscopy, it is shown that the properties of Tyr_Z are modified in PsbA2-PSII when compared with those in Psb(A1/3)-PSII.

EXPERIMENTAL PROCEDURES

Construction of T. elongatus Mutants

The construction of the ΔpsbA₁ΔpsbA₂ T. elongatus deletion mutant (WT*3) from a T. elongatus 43-H strain that had a His₆ tag on the C terminus of CP43 (32) has been previously described in Ref. 33.

For making the ΔpsbA₁ΔpsbA₃ T. elongatus deletion mutant (WT*2) (Fig. 1), first, the psbA₁ gene and its promoter region (∼180 bp) were substituted together from the 43-H strain with a chloramphenicol-resistant cassette (∼1300 bp) by using the plasmid vector pBΔpsbA₂. Then, the psbA₃ gene was substituted with a spectinomycin/streptomycin resistance gene cassette (∼2100 bp) by using the plasmid pBΔpsbA₃. For construction of pBΔpsbA₁, a DNA fragment of ∼2300 bp of the psbA₂ gene (tlr1844) including its promoter region (∼180 bp) and the 3′-flanking region of psbA₂ (∼1000 bp) was cloned from T. elongatus wild-type genomic DNA by PCR amplification and subcloned into a plasmid vector pBluescript II SK+ at EcoRV and XhoI sites. Next, a chloramphenicol resistance gene cassette (∼1300 bp) was ligated to the upstream of the psbA₂ gene at BamHI and EcoRV of the plasmid DNA. Then, a separately amplified ∼900-bp DNA fragment of the 3′-flanking region of psbA₁ (but without the psbA₁ promoter region) was ligated to the subcloned plasmid vector at SacI and BamHI. For the construction of pBΔpsbA₃, a DNA fragment of ∼900 bp of the 3′-flanking region of the psbA₃ (tlr1477) was cloned from T. elongatus wild-type genomic DNA by PCR amplification and subcloned into a plasmid vector pBluescript II SK+ between SacI and EcoRI sites. Then, a spectinomycin/streptomycin resistance gene cassette (∼2100 bp) was inserted at PstI and SacI. Then, a separately amplified ∼1100-bp DNA fragment of the 5′-flanking region of psbA₃ was ligated to the subcloned plasmid vector at SphI and PstI.

FIGURE 1.

Map around psbA₁ and psbA₂ and around psbA₃ in T. elongatus genome (A–C) and agarose gel electrophoresis of amplified products by PCR (D). A, wild-type, all three psbA genes are intact. In B, for making WT*2, psbA₁ with 180 bp of the promoter region was deleted by substitution of a chloramphenicol-resistant (Cm^R) cassette, and psbA₃ was substituted by a spectinomycin (Sp)/streptomycin-resistant (Sm^R) cassette. In C, for making WT*3, both psbA₁ and psbA₂ were substituted by a chloramphenicol-resistant cassette. Primers are shown as short arrows, and P1, P2, P3, and P4 indicate annealing position on their T. elongatus genome DNA. The double-pointed arrows show the length of the DNA amplified by PCR with using the appropriate primers. D, agarose gel (1%) electrophoresis of amplified products by PCR using P1 and P2 primers (lanes 1–3) and using P3 and P4 primers (lanes 5–7). Lanes 1 and 5 correspond to the wild type; lanes 2 and 6 correspond to the WT*2 strain; lanes 3 and 7 correspond to the WT*3 strain; and lane 4 corresponds to a 1-kb ladder marker (Toyobo).

The T. elongatus transformants were selected as single colonies on DTN agar plate containing appropriate antibiotics (25 μg ml⁻¹ spectinomycin, 10 μg ml⁻¹ streptomycin, 40 μg ml⁻¹ kanamycin, and 5 μg ml⁻¹ chloramphenicol). Segregation of all genome copies was confirmed by difference in length of amplified DNA by PCR using the P1 primer (5′-GCTGTACTGGCCATCGCTGGGCACCACTCG-3′) and P2 primer (5′-GGACTTATCACTACTTATCACTAGAGAGGT-3′) for psbA₁–psbA₂ region, and using the P3 primer (5′-GGTTGGATCCCCAGCCAGCGATCGCGGGAG-3′) and P4 primer (5′-CCATGCCCGCAAAACAGC-3′) for psbA₃ region as shown in Fig. 1. Complete segregation of the deletion mutants was confirmed by PCR amplification as shown in Fig. 1D. In the wild type, a 3900-bp DNA fragment including both psbA₁ and psbA₂ was amplified by P1 and P2 primers (lane 1). In contrast, a 4000-bp fragment (lane 2) and a 2850-bp fragment (lane 3) were amplified with using the same combination of the primers in WT*1 and WT*3, respectively. In the region of psbA₃, a 2200-bp fragment was amplified with P3 and P4 primers in both wild-type and WT*3 genomes (lanes 5 and 7), whereas a 3100-bp fragment was amplified in WT*2 genome (lane 6).

Purification of PSII

PSII were purified with the protocol already described (28). PSII samples were suspended in 1 m betaine, 10% glycerol, 15 mm CaCl₂, 15 mm MgCl₂, 40 mm MES, pH 6.5 adjusted with NaOH. For the low temperature X-band EPR experiments, glycerol was omitted because its presence decreases the yield of the near-infrared induced split EPR signal in the S₃ state.

For manganese depletion, PSII samples were diluted ∼10-fold in a medium containing 1.2 m Tris-HCl (pH 9.2) and were incubated under room light at 4 °C for 1 h. The samples were collected by centrifugation (15 min at 170, 000 × g) after the addition of 1.2 m Tris (pH 9.2) containing 50% (w/v) polyethylene glycol 8000 so that the final PEG concentration was 12%. The pellet was resuspended in a medium containing 1 m betaine, 10% glycerol, 15 mm CaCl₂, 15 mm MgCl₂, 40 mm MES, pH 6.5 adjusted with NaOH, and 12% PEG. After a new centrifugation, the pellet was resuspended in 1 m betaine, 10% glycerol, 15 mm CaCl₂, 15 mm MgCl₂, 40 mm MES, pH 6.5 adjusted with NaOH.

Oxygen Evolution Measurements

Oxygen-evolving activity of purified PSII (5 μg of Chl ml⁻¹) was measured under continuous saturating white light at 25 °C by polarography using a Clark type oxygen electrode (Hansatech). A total of 0.5 mm 2,6-dichloro-p-benzoquinone (dissolved in dimethyl sulfoxide) was added as an electron acceptor.

EPR Spectroscopy

For helium temperature measurements, continuous wave 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 laser (532 nm, 550 mJ, 8-ns Spectra Physics GCR-230–10). PSII samples at 1.1 mg of Chl ml⁻¹ were loaded in the dark into quartz EPR tubes and dark-adapted for 1 h at room temperature. Then, the samples were synchronized in the S₁ state with one pre-flash (34). After a further 1-h dark adaptation at room temperature and the addition of 0.5 mm PPBQ dissolved in dimethylsulfoxide, the samples were either frozen immediately to 198 K in a solid CO₂/ethanol bath or illuminated by one or two additional flashes to generate the S₂ and S₃ states before being frozen in the dark to 198 K and then transferred to 77 K. In both cases, the samples were degassed at 198 K prior to the recording of the spectra.

For time-resolved measurements at room temperature, the spectrometer was equipped with a Super High Quality Bruker cavity. Saturating laser flash illumination at room temperature was provided by the laser described above. PSII at 1.1 mg of Chl ml⁻¹ was loaded into a small volume flat cell (100 μl) in the presence of 0.5 mm phenyl-p-benzoquinone (PPBQ) and 1 mm potassium ferricyanide. Ferricyanide was added to avoid any contamination from the PPBQ^˙̄ signal, which is detectable in the hundred μs time range after the flash illumination in the absence of ferricyanide. Formation and decay of the signal following laser flash illumination were measured at 32 magnetic field positions spread over 50 G and centered on the Tyr_Z^• EPR signal. For each of the 32 magnetic field values, 16 scans were averaged. The two-dimensional spectra (time versus field) of ∼12–16 samples were averaged. Half of the two-dimensional spectra were obtained by increasing the magnetic field, and the other half were obtained by decreasing the magnetic field. When indicated, near-IR illumination of the samples was done directly in the EPR cavity and was provided by a laser diode emitting at 820 nm (Coherent, diode S-81-1000C) with a power of 600–700 milliwatts at the level of the sample.

The High Field-EPR measurements were taken on a locally built spectrometer described previously (35). Using a manganese-doped magnesium oxide sample, we verified that the relative accuracy of the magnetic field was better than 1 millitesla in the field ranges used in this study. The microwave frequency was accurate to better than 1 MHz. Hence, the measurement accuracy in g was expected to be 1 × 10⁻⁴.

UV-visible Absorption Change Spectroscopy

Absorption changes were measured with a lab-built spectrophotometer (36) where the absorption changes are sampled at discrete times by short flashes. These flashes were provided by a neodymium:yttrium-aluminum garnet (355 nm) pumped optical parametric oscillator, which produces monochromatic flashes (1 nm full-width at half-maximum) with a duration of 5 ns. Excitation was provided by a second neodymium:yttrium-aluminum garnet (532 nm) pumped optical parametric oscillator, which produces monochromatic saturating flashes at 700 nm (1 nm full-width at half-maximum) with a duration of 5 ns. The path length of the cuvette was 2.5 mm. PSII was used at 25 μg of Chl ml⁻¹ in 10% glycerol, 1 m betaine, 15 mm CaCl₂, 15 mm MgCl₂, and 40 mm MES (pH 6.5). PSIIs were dark-adapted for ∼1 h at room temperature (20–22 °C) before the additions of 0.1 mm PPBQ dissolved in dimethyl sulfoxide. For kinetic measurements, the time delay between the actinic flash and the detector flash was first increased from the smaller value to the larger value and then varied in the opposite direction. For each time delay, the measurements were repeated four times so that each data point is the average of eight measurements. The traces shown are typical of those obtained with at least three different PSII preparations.

RESULTS

The oxygen evolution activity of purified PsbA2-PSII was 3000–3500 μmol of O₂ (mg of Chl)⁻¹ ml⁻¹. This activity is close to that found for PsbA1-PSII and about half of that commonly found for PsbA3-PSII (33).

Fig. 2 shows the amplitude of the absorption changes associated with each flash in a series with PsbA3-PSII (circles) and with PsbA2-PSII (squares). Measurements were done at 292 nm (37–39) and at 200 ms after the flashes, i.e. after completion of the reduction of Tyr_Z by the water-oxidizing complex. At this wavelength, the reduction of PPBQ does not lead to any absorption changes, and the successive oxidation steps of the water-oxidizing complex have significant extinction coefficients (38). The pattern, oscillating with a period of four, is clearly observed for both types of PSII preparations with very similar amplitude on the first flash. However, the damping is larger in PsbA2-PSII than in PsbA3-PSII. Indeed, the maxima are clearly shifted from the 5th, 9th, 13th, etc., flashes in PsbA3-PSII to the 6th, 10th, 14th, etc., flashes in PsbA2-PSII. This is at variance with the PsbA(1/3) cases, which displayed similar period four oscillation characteristics like the miss parameter and S₁/S₀ ratio in dark-adapted material (33, 39, 40). This suggests that functional differences exist between PsbA2-PSII and PsbA(1/3)-PSII.

FIGURE 2.

Sequence of amplitude of absorption changes at 292 nm. The measurements were done during a series of saturating flashes (spaced 200 ms apart) given to dark-adapted PsbA3-PSII (black circles) or PsbA2-PSII (red squares). The samples ([Chl] = 25 μg ml⁻¹) were dark-adapted for 1 h at room temperature before the addition of 100 μm PPBQ. The measurements were done 200 ms after each flash.

To determine which step(s) is(are) kinetically affected and therefore responsible for the larger miss parameter in PsbA2-PSII, we first measured the absorption changes at 292 nm in the 10 μs to ms time ranges after the first three flashes in a series to assess the kinetics of electron transfer associated with the S₁Tyr_Z^• → S₂Tyr_Z, S₂Tyr_Z^• → S₃Tyr_Z and S₃Tyr_Z^• → S₀Tyr_Z transitions in both the PsbA2-PSII and the PsbA3-PSII. At 292 nm, the absorption changes associated with the S₂Tyr_Z^• → S₃Tyr_Z transition are small and preclude a reliable kinetic analysis. As shown in Fig. 3, we did not observe any significant differences for the kinetics of the absorption changes associated with the S₁Tyr_Z^• → S₂Tyr_Z and S₃Tyr_Z^• → S₀Tyr_Z transitions in PsbA2-PSII (squares) when compared with PsbA3-PSII (circles). Thus, the larger miss parameter in PsbA2-PSII does not originate from a longer lifetime of the S_iTyr_Z^•. In addition, we note that the light-induced absorption changes associated with the formation of the S₂ state were similar in PsbA2-PSII and PsbA3-PSII. This suggests a similar efficiency for the S₁ to S₂ transition. We aimed at verifying this conclusion by EPR spectroscopy that provides an exquisitely specific spectrum for the S₁ to S₂ transition.

FIGURE 3.

Kinetics of absorption changes at 292 nm after first flash (red), second flash (blue), and third flash (black) given to dark-adapted PsbA3-PSII (circles and continuous lines) or PsbA2-PSII (squares and dashed lines). Other experimental conditions were similar to those in Fig. 2.

Fig. 4A shows the difference EPR spectra “after-minus-before” flash illumination in PsbA3-PSII (spectrum a) and PsbA2-PSII (spectrum b). After one flash, the characteristic S₂ multiline signal centered at g ∼2 and arising from the Mn₄CaO₅ cluster in the Mn^IV₃Mn^III redox state with a spin state S = 1/2 (see Ref. 41 and references therein for a recent discussion) had an identical shape and a similar amplitude in PsbA3-PSII and PsbA2-PSII. In contrast, after two flashes (Fig. 4B), the amplitude of the S₂ multiline signal reflecting the fraction of water-oxidizing complex that did not undergo the S₂ → S₃ transition was larger in PsbA2-PSII (spectrum b) than in PsbA3-PSII (spectrum a). The amplitude of the positive feature seen between 0 and 1000 G and assigned to the spin S = 3 S₃ signal (42) was smaller in PsbA2-PSII than in PsbA3-PSII. The temperature used (8.5 K) was not ideal to measure the S₃ EPR signal but was chosen to allow the detection of both the S₃ and the S₂ signals in the same spectrum. The two observations made above point to a lower yield of the S₂ to S₃ transition in PsbA2-PSII. Although the spectrum in PsbA2-PSII exhibits a larger negative signal in the nonheme iron magnetic field region at ∼1100 G (e.g. Ref. 28), the shape of the signal assigned to S₃ was similar in both samples. This, together with the identical multiline signal observed in both samples, shows that the magnetic structure of the Mn₄CaO₅ cluster is likely unaffected by the substitution of PsbA3 by PsbA2.

FIGURE 4.

Light-minus-dark EPR spectra induced by either one flash (A) or two flashes (B) at room temperature in presence of 0.5 mm PPBQ and recorded on PsbA3-PSII (spectrum a, black) or PsbA2-PSII (spectrum b, red). Sample concentration was 1.1 mg of Chl ml⁻¹. Instrument settings were: modulation amplitude, 25 G; microwave power, 20 milliwatt; microwave frequency, 9.5 GHz; modulation frequency, 100 kHz; and temperature, 8.5 K. The central part of the spectra corresponding to the Tyr_D^• region was deleted.

We conclude from the above data that the larger miss parameter is not due to a longer lifetime of any of the S_iTyr_Z^• states that would result in a larger charge recombination probability. This makes the electron transfer step between P₆₈₀^+⋅ and Tyr_Z the next candidate. Fig. 5A shows the time-resolved flash-induced absorption changes around 433 nm after each of the first five flashes applied to dark-adapted PsbA2-PSII (first flash, black; second flash, blue; third flash, red; fourth flash, green; fifth flash, orange). The spectra were recorded 20 ns after the flashes to avoid any spectral distortions due to the short lived excited state, like uncoupled Chl*, for example. In this spectral region, the redox changes of several species, such as the Chls, cytochromes, Tyr_Z, and Q_A, could potentially contribute to the absorption changes. The most prominent ones, however, are those associated with the formation of P₆₈₀^+⋅. The P₆₈₀^+⋅/P₆₈₀ difference spectrum is characterized by a strong Soret band bleaching. After one flash, i.e. in the S₁P₆₈₀^+⋅ state, the maximum of the bleaching was observed at 433 nm. After the second flash and third flash, i.e. in the S₂P₆₈₀^+⋅ state and S₃P₆₈₀^+⋅ state, the width of the bleaching increased, and the red-most parts of the spectra were slightly red-shifted when compared with the spectrum of the S₁P₆₈₀^+⋅ state. The red-shift was reversed after the fourth and fifth flashes, i.e. in the S₀P₆₈₀^+⋅ and S₁P₆₈₀^+⋅ states. This period four oscillation pattern in the P₆₈₀^+⋅/P₆₈₀ spectrum likely originates from an electrostatic effect on the P_D1^+⋅P_D2⇔P_D1P_D2^+⋅ equilibrium due to the charge(s) stored on/around the Mn₄CaO₅ cluster (e.g. Ref. 4). Irrespective of the flash number, the difference spectra were similar to those in PsbA1-PSII (40) and PsbA3-PSII (33), thus showing that the distribution of the cation over the P_D1 and P_D2 chlorophylls is similar in both cases.

FIGURE 5.

A, difference spectra around 430 nm. The flash-induced absorption changes were measured at 20 ns in PsbA2-PSII after the first five flashes given on dark-adapted PsbA2-PSII (first flash, black; second flash, blue; third flash, red; fourth flash, green; fifth flash, orange). [Chl] = 25 μg ml⁻¹. B, kinetics of P₆₈₀^+⋅ reduction measured at 433 nm after the first three flashes in PsbA3-PSII (filled circles) and PsbA2-PSII (open circles). Black circles, first flash; blue circles, second flash; red circles, third flash.

Fig. 5B shows the decay of P₆₈₀^+⋅ measured at 433 nm after each of the first three flashes in PsbA3-PSII (filled circles) and PsbA2-PSII (open circles). After the first flash (black symbols), both the tens of ns and the tens of μs phases were found comparable in PsbA3-PSII and PsbA2-PSII in terms of amplitude and t½. After the second flash (blue symbols) and the third flash (red symbols), the P₆₈₀^+⋅ decay was much slower in PsbA2-PSII, particularly in the hundreds of μs time domain. This shows up even more clearly after averaging the decay traces from the 1st flash to the 20th flash (supplemental Fig. S6).

The finding that the reduction kinetics of P₆₈₀^+⋅ is hardly affected on the first flash shows that possible changes of the properties of the electron acceptor side originating from the PsbA(1/3) to PsbA2 substitution did not significantly increase the percentage of centers in which the P₆₈₀^+⋅Q_A^˙̄ charge recombination occurred, at least with Q_B in the oxidized state. In addition, the presence of PPBQ prevents the formation of stable Q_B^˙̄ so that the acceptor side is in the same redox state after each flash in the series. Therefore, it is very unlikely that the slower P₆₈₀^+⋅ reduction detected on the second and following flashes originates from a more efficient charge recombination in PsbA2-PSII.

According to the current understanding of the multiphasicity of the reduction of P₆₈₀^+⋅, the ns components are kinetically limited by the electron transfer process, whereas the μs phases involve proton-coupled transfer reactions (e.g. Refs. 43–45). In this framework, the present data would thus point to a slower proton transfer process in PsbA2-PSII. Two amino acid substitutions on the electron donor side of PsbA2-PSII may affect the orientation of the helices, which respectively bear His-190 and Tyr_Z^•: the C144P and P173M exchanges. These two substitutions may impact the hydrogen bond between Tyr_Z and His-190 and/or the H-bond network in which these two residues are involved. If such is indeed the case, this would be expected to affect the rates of the proton transfer steps associated with the oxidation of Tyr_Z. This was assessed using EPR spectroscopy, which has been shown to probe the geometry and the environment of the Tyr_Z phenol ring (e.g. Refs. 46 and 47).

In the S₃ state, NIR illumination at ∼4 K results in the formation of a split EPR signal (48, 49), attributed to a (S₂Tyr_Z^•)′ state formed by NIR-induced conversion of the manganese cluster into an “activated” state able to oxidize Tyr_Z and thus leading to the formation of (S₂Tyr_Z^•)′ at the expense of the S₃Tyr_Z state (50). This split signal is attributed to the magnetic interaction between Tyr_Z^•, with a spin state S = 1/2 and the Mn₄CaO₅ cluster possibly in a S = 7/2 spin state (51), and as such is very sensitive to the geometry of the Tyr_Z^•/Mn₄CaO₅ ensemble. As an example of this sensitivity, the split EPR spectrum is significantly modified upon the Ca²⁺/Sr²⁺ exchange (52). Importantly, these modifications can be reliably ascribed to changes in the geometry of the bridge between Tyr_Z and Ca²⁺/Sr²⁺ via a water molecule (3) rather than to the alteration of the Mn₄CaO₅ magnetic structure, which has been independently shown to be only slightly affected by the Ca²⁺/Sr²⁺ exchange (41).

Fig. 6 shows the EPR difference spectra after-minus-before near-infrared illumination in PsbA2-PSII (red spectrum), which are compared with that recorded in PsbA3-PSII (black spectrum). Notably, either PsbA1-PSII or PsbA3-PSII can be used as control samples because their split signals are identical (40, 49, 52). Fig. 6 evidences manifest differences between the two samples. Because the PsbA exchange does not modify the EPR properties of the Mn₄CaO₅ cluster, at least in the S₂ and S₃ states, the changes in the split signal likely arise from a change in the EPR properties of Tyr_Z^• or in the magnetic interaction between Tyr_Z^• and the Mn₄CaO₅ cluster. The EPR properties of Tyr_Z^• that may be modified are: (i) the values and localization of the spin densities on the carbons and oxygen bearing this spin density; (ii) the orientation of the β-methylene group versus the plan of the phenol ring; (iii) the g_x, g_y, and g_z values; (iv) the relative orientation of Tyr_Z^• versus the Mn₄CaO₅ cluster; and (v) the distance between Tyr_Z^• and the Mn₄CaO₅ cluster. The changes in the magnetic interaction between Tyr_Z^• and the Mn₄CaO₅ cluster could be assessed by a theoretical approach. Nevertheless, although some of the split signals, in acetate-treated PSII or in the S₁Tyr_Z^• generated at 4 K, have been successfully simulated (53, 54), the magnetic properties of the Mn₄ moiety are poorly understood in the S₃ state, which precludes here a reliable simulation. Thus, to gain further insights into the structural reasons underlying these spectroscopic changes, we attempted to measure directly the EPR spectrum or Tyr_Z^•.

FIGURE 6.

NIR-induced split EPR spectra in PsbA3-PSII (black spectrum) and PsbA2-PSII (red spectrum). For the two samples, a spectrum was first recorded after two flashes given at room temperature, and a second spectrum was recorded after a further NIR illumination given in the EPR cavity at 4.2 K. Instrument settings were: modulation amplitude, 25 G; microwave power, 20 milliwatt; microwave frequency, 9.5 GHz; modulation frequency, 100 kHz; and temperature, 4.2 K. The chlorophyll concentration was 1.1 mg ml⁻¹. The center part corresponding to the Tyr_D^• spectrum was deleted.

Figs. 7–9 report the results of time-resolved EPR experiments performed at room temperature. The time resolution of our EPR spectrometer is in the same time range as the lifetime of S₃Tyr_Z^• in WT T. elongatus PSII (t½ ∼1 ms). To circumvent this limitation, Ca²⁺ and Cl⁻ were substituted by Sr²⁺ and Br⁻ in PsbA3-PSII because it has been shown that this markedly increases the lifetime of S₃Tyr_Z^• (39). In such conditions, the S₃Tyr_Z^• to S₀Tyr_Z transition occurs with a t½ close to 7 ms (39), i.e. in a time domain compatible with the reliable detection of Tyr_Z^• decay with our EPR set-up. Because in separate experiments (not shown) we checked that the Cl⁻/Br⁻ exchange had no effect on the Tyr_Z^• spectrum and because the Ca²⁺/Sr²⁺ exchange alone proved sufficient to allow us the full detection of the Tyr_Z^• signal, the formation and decay of the Tyr_Z^• signals following laser flash illumination were done in Sr²⁺-containing PsbA2-PSII. These measurements were done at 32 magnetic field positions spread over 50 G and centered on the Tyr_Z^• EPR spectrum.

FIGURE 7.

A, formation and decay of the Tyr^• signal following laser flash illumination of manganese-depleted PsbA3-PSII measured at 32 magnetic field positions spread over 50 G from 3486 to 3536 G. For each of the 32 magnetic field values, 16 scans were averaged. The two-dimensional spectra (time versus field) of ∼12–16 samples were averaged. Half of the two-dimensional spectra was obtained by increasing the magnetic field, and the other half was obtained by decreasing the magnetic field. Other instrument settings; modulation amplitude, 4 G; microwave power, 20 milliwatt; microwave frequency, 9.7 GHz; modulation frequency, 100 kHz; and temperature, 293 K. The chlorophyll concentration was 1.1 mg ml⁻¹. Sampling time was 500 μs. B, Tyr_D^• (black) and Tyr_Z^• (red) spectra extracted from the two-dimensional spectrum in panel A. The Tyr_D^• spectrum is the envelope of the baseline before the flash, and the Tyr_Z^• spectrum was obtained by extracting the first slice after the flash (i.e. ∼1 ms) after subtraction of the baseline before the flash, which corresponds to the Tyr_D^• spectrum.

FIGURE 8.

A, formation and decay of the Tyr^• signal following laser flash illumination of Sr/Br-PsbA3-PSII. The same protocol as in panel A of Fig. 7 was followed. B, spectra extracted from the two-dimensional spectra as explained for panel B of Fig. 7. The black spectrum corresponds to the Tyr_Z^• spectrum of manganese-depleted PsbA3-PSII, the red spectrum with a continuous line corresponds to the Tyr_Z^• spectrum of Sr/Br-PsbA3-PSII, and the red spectrum with a dashed line corresponds to the Tyr_Z^• spectrum of Sr/Br-PsbA3-PSII with an amplitude multiplied by four.

FIGURE 9.

Black spectrum corresponds to the Tyr_Z^• spectrum of Sr/Br-PsbA3-PSII, and red spectrum with a dashed line corresponds to the Tyr_Z^• spectrum of Sr-PsbA2-PSII. Both spectra were extracted from a two-dimensional spectrum as explained above and correspond to the same Chl concentration and Tyr_D^• signal amplitude.

To validate the approach, we first applied the method to manganese-depleted PSII in which the lifetime of Tyr_Z^• is much longer. Fig. 7A shows the results of such experiments, i.e. a two-dimensional spectrum (time versus field). Fig. 7B shows two slices extracted from the two-dimensional spectrum. The first one (black spectrum), before the flash, corresponds to the Tyr_D^• spectrum, and the second one (red spectrum), immediately after the flash and after subtraction of the baseline for each magnetic field value, corresponds to the Tyr_Z^• spectrum. Although the magnetic field resolution is here limited to 50/31 ∼1.6 G, the Tyr_D^• and Tyr_Z^• spectra thus obtained are similar to those reported in the literature for manganese-depleted PSII (e.g. Refs. 46 and 55).

Fig. 8A shows the results of a similar two-dimensional experiment performed in Sr/Br-containing PsbA3-PSII. Here, in contrast to the situation prevailing in manganese-depleted PSII, the Tyr_Z^• signal was kinetically detectable only in the S₃Tyr_Z^• to S₀Tyr_Z transition. In addition, because 16 consecutive flashes were averaged for each sample, the amplitude of the Tyr_Z^• signal should be at most one-fourth of that detected in manganese-depleted PSII in which almost one Tyr_Z^• species is detectable upon each flash. Fig. 8B shows the Tyr_Z^• spectrum recorded in manganese-depleted PSII in Fig. 7 (black spectrum) and the Tyr_Z^• spectrum recorded immediately after the flash in Sr/Br-containing PsbA3-PSII (red spectrum, solid line). The latter has been multiplied 4-fold (dashed red line) to ease its comparison with the manganese-depleted case. Within experimental accuracy, the Tyr_Z^• spectrum in the (S₃Tyr_Z^•)′ state was found identical to the Tyr_Z^• spectrum in manganese-depleted PSII. In addition, we did not find any significant changes in the shape of the Tyr_Z^• spectrum occurring while it decays (see supplemental Figs. S3 and S4).

Finally, the two-dimensional EPR experiment was performed in Sr-containing PsbA2-PSII. Fig. 9 compares the Tyr_Z^• spectra detected in PsbA3-PSII (black spectrum) and that recorded in PsbA2-PSII (red spectrum) and evidences small but significant differences between the PsbA2-PSII and PsbA3-PSII Tyr_Z^• spectra in the (S₃Tyr_Z^•)′.

DISCUSSION

In this study, we describe for the first time the construction of a T. elongatus mutant in which both the psbA₁ and the psbA₃ genes have been deleted, so thus the mutant expresses only the psbA₂ gene. The O₂-evolving activity of PSII purified from this strain is close to that measured with PsbA1-PSII (∼3500 μmol of O₂ (mg of Chl)⁻¹ h⁻¹) and approximately half of that found for PsbA3-PSII (∼6000 μmol of O₂ (mg of Chl)⁻¹ h⁻¹). In the current understanding, the limiting step of the overall oxygen evolution in vitro is the exchange of the doubly reduced Q_BH₂ molecule by an oxidized one. Thus, these different oxygen-evolving activities may stem from amino acid substitutions located close to the quinone binding sites. For example, in the x-ray three-dimensional structure of PsbA1-PSII⁵ (3), S270 and C212 locate close to the Q_B binding site (supplemental Fig. S1). S270 is replaced by an alanine in both PsbA2-PSII and PsbA3-PSII, and C212 is by replaced by an alanine in PsbA2-PSII and a serine in PsbA3-PSII. However, sequence alignment does not permit the identification of other amino acids that would account for, on the one hand, the similarity between PsbA1-PSI and PsbA2-PSII and, on the other hand, the dissimilarities between these two and PsbA3-PSII. Probably, several amino acid substitutions between PsbA1-PSII, PsbA2-PSII, and PsbA3-PSII could potentially induce long distance effects acting on the hydrogen bond network, the water molecule network, the binding of lipids, and possibly the fine tuning of the interactions between the D1/D2 α-helices.

The C144P and P173M exchanges are two of the less conservative differences between PsbA2 and PsbA(1/3). In PsbA1 and PsbA3, the C144 is located in helix C on the periplasmic side just before the α-helix bearing Tyr_Z (the Y161), and the P173 is located in the loop between the C and D helices bearing Tyr_Z and the H190 (3) (see supplemental data). Many studies have documented the role and importance of this H-bond in determining the reaction pathway as well as the rate of the proton-coupled electron transfer process involved associated with the oxidation of Tyr_Z (e.g. Refs. 26, 30, and 31). Because its ϕ backbone dihedral angle is locked, proline is a singularly rigid amino acid. Its substitution for and by other residues in the vicinities of Tyr_Z and H190 is expected to have significant structural consequences on the local environment of Tyr_Z and therefore on this proton-coupled electron transfer. Therefore, it seems very likely that these substitutions have some consequences on the relative orientation of the Tyr_Z versus H190 and are therefore responsible for the changes reported in this work.

The period four oscillating pattern in PsbA2-PSII indicates that the miss parameter is larger than in either PsbA3-PSII (33, 39) or PsbA1-PSII (40) (Fig. 2). However, the yield of S₂ formation estimated from the amplitude of the S₂ EPR multiline signal was identical in PsbA2-PSII and PsbA3-PSII, thus showing that the yield of the S₁ to S₂ transition is not significantly affected (Fig. 4). In contrast, the smaller amplitude of the absorption change detected upon the second flash and the smaller S₃ EPR signal and the larger fraction of remaining S₂ multiline signal detected after two flashes applied to PSII centers synchronized in the S₁ state prior to the flash illumination suggest that in PsbA2-PSII, the miss parameter strongly increases in the S₂ to S₃ transition. The fact that the miss parameter depends on the S state transitions decreases the accuracy of a fitting of the period four oscillations in Fig. 2 (nevertheless, see supplemental Fig. S7 for a tentative fitting procedure). Based on the fraction of the remaining S₂ multiline signal, we estimate this increase to be 2-fold (i.e. ∼20%).

The S₂ EPR multiline spectra and S₃ EPR spectra are known as being exquisitely sensitive to the structure and spin distribution within the cluster (41, 42). On the contrary, the absence of significant differences between these spectroscopic features obtained in the PsbA2-PSII and PsbA3-PSII show that the structure of the Mn₄CaO₅ is very likely not affected by the PsbA exchange. In addition, the P₆₈₀^+⋅/P₆₈₀ difference spectra versus the flash number in PsbA2-PSII do not reveal any significant changes in the charge distribution over the P_D1P_D2 chlorophyll dimer because the spectra after one, two, or three flashes have a maximum bleaching at 433 nm as in PsbA1-PSII and PsbA3-PSII. Therefore, it seems likely that the increase of the miss parameter reported above originates from a modification of Tyr_Z itself. Indeed, several authors have pointed to the electron transfer between Tyr_Z and P₆₈₀ as being an important contributor to the miss parameter (for example, see Refs. 56–58 for discussions).

The similar kinetics observed for the S₁Tyr_Z^• to S₂Tyr_Z transition (t½ ∼50 μs), for whichever PsbA protein is involved, is consistent with an unmodified miss parameter on this transition. In the S₃Tyr_Z^• to S₀Tyr_Z transition, the decay of the ΔI/I at 292 nm is biphasic (Fig. 3). The fast phase (t½ ∼100 μs) seen as a lag phase at 292 nm has been interpreted as reflecting the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge near/on Tyr_Z^• (59). The slow phase, attested by an absorption decay with t½ ∼1 ms, corresponds to the formation of S₀ and O₂ and to an additional proton release (Refs. 59–63), and see also Ref. 64 for a recent work dealing with this these two phases in T. elongatus). From data in Fig. 3, neither the lag phase S₃Tyr_Z^• to (S₃Tyr_Z^•)′ nor the (S₃Tyr_Z^•)′ to S₀Tyr_Z transition seems affected by the PsbA exchange.

For the S₂Tyr_Z^• to S₃Tyr_Z transition, in which EPR measurements clearly indicate an increase of the miss parameter, the measurements at 292 nm are unfortunately not very informative. Thus, to probe further the Tyr_Z environment, we measured the reduction of P₆₈₀^+⋅. As previously observed by many groups (see below), this kinetics has multiple components. The fast ones have been interpreted as being kinetically limited by the electron transfer process and were similar in rates in the various samples. The phases developing in the μs to tens of μs time range have been interpreted as being kinetically limited by proton transfer, and these were markedly affected by the PsbA3 to PsbA2 exchange, in particular after the second and third flashes in the series. These components were slower, and their amplitudes were larger in PsbA2-PSII. This, together with the modified split EPR signal detected upon NIR illumination in PsbA2-PSII, shows that Tyr_Z is indeed the cofactor with modified properties in PsbA2-PSII. Before discussing these structural issues, we would like to note that as a side result of the present study, we report here the first Tyr_Z^• EPR spectrum in the (S₃Tyr_Z^•)′ state in active PSII. We show that it is similar to that in manganese-depleted PSII. From W-band EPR, the geometry of Tyr_Z^• in manganese-depleted PSII crystals was found to be similar to that expected from the geometry of Tyr_Z in the dark-adapted state of Mn₄CaO₅-containing crystal (65). Thus, the geometry of Tyr_Z^• in the (S₃Tyr_Z^•)′ state is also likely similar to that of Tyr_Z in the S₁ state.

Several structural reasons may be considered to account for the different properties of Tyr_Z. One is a change in the orientation of the Tyr_Z^• radical with respect to the Mn₄CaO₅ cluster that would modify the magnetic interaction between Tyr_Z^• and the Mn₄CaO₅ cluster. To further assess this, we attempted to characterize the g-tensor of Tyr_Z^• by high field EPR. We note, however, that this study was performed with manganese-depleted PsbA2-PSII (supplemental Fig. S5), so its conclusions cannot be straightforwardly extrapolated to oxygen-evolving PSII. Although small, the changes indicate without ambiguities that the g_x resonance of the Tyr_Z^• spectrum was broader and likely up-shifted. This could explain the modified X-band EPR spectrum measured in the (S₃Tyr_Z^•)′ state. An up-shift of the g_x value is indicative of a less positive electrostatic environment for Tyr_Z^• (47). The changes in the hyperfine structure of the time-resolved Tyr_Z^• spectrum in the (S₃Tyr_Z^•)′ state are also reminiscent of that observed in the Tyr_D^• spectrum in the D2-H189L mutant that disrupts the H-bond in which Tyr_D^• is involved (47). Altogether these data thus point to a weaker H-bond between Tyr_Z and H190.

The data above indicate that the main modifications on the electron donor side of PsbA2-PSII occur at the level of Tyr_Z. This could be a consequence of the C144P and P173M exchanges, which in turn would modify the H-bond between Tyr_Z and H190. It is indeed widely agreed that the proton-coupled electron transfer rates for the Tyr oxidation depend on the properties of the Tyr-O···H···N-His bonding (e.g. Refs. 26, 31, and 66). It has indeed been shown that, in model compounds, the proton-coupled electron transfer rate from a tyrosine to an oxidant was strongly dependent on the intramolecular distance between the tyrosine and the base that accepts the proton (e.g. Ref. 67). Interestingly, the hydrogen bond between the phenol group of Tyr_Z and the Nϵ of H190 in PSII is very short (2.46 Å in Ref. 3). Recently, the rationale underlying this short H-bond has been investigated by a quantum mechanical-molecular mechanical approach (68), and the cluster of four water molecules involved in the Mn₄CaO₅-Tyr_Z motif has been shown to play an important role in the stabilization of such a short distance. In this framework, it would not be surprising that a small distortion of such a delicate scaffold would have important consequences on the oxidation of Tyr_Z by P₆₈₀^+⋅ and in particular on those steps that are kinetically limited by the proton transfer within the H-bond network in which Tyr_Z and His-190 are involved.

In contrast to the marked kinetic effects that we observed on the slow components of the oxidation of Tyr_Z by P₆₈₀^+⋅, the reduction rates of the various S_iTyr_Z^• states were unaffected by the PsbA3 to PsbA2 exchange. As regards the S₁ to S₂ transition, this is expected because it is only accompanied by substoichiometric proton release (69–71). However, the subsequent S₂ to S₃ and S₃ to S₀ electron transfer steps are chemically coupled to proton release (e.g. Refs. 72 and 73) and might be affected by the changes in the H-bond network around Tyr_Z and His-190 discussed above. To our knowledge, the present study is the only one reporting a slowdown of the μs components in the oxidation of Tyr_Z besides, of course, the H/D experiments (43, 63, 74). Interestingly, kinetic isotope effects have also been reported for the following electron transfer step, i.e. the reduction of Tyr_Z^•, at least in the presence of the S₂ and S₃ states. Notably, the most pronounced kinetic isotope effect has been reported to occur during the S₃Tyr_Z^• to (S₃Tyr_Z^•)′ (63), which is assigned to an electrostatically triggered proton release (59, 63). The present observation that the PsbA3 to PsbA2 exchange affects the μs components in the oxidation of Tyr_Z^• while keeping unaffected the proton release associated with the S₃Tyr_Z^• to (S₃Tyr_Z^•)′ transition suggests that this particular proton release does not originate from the same H-bond network as the one involved in the proton transfer triggered by the formation of Tyr_Z^•. The latter has been described as a sequence of push-pull steps that would be initiated by the transfer of the phenolic proton from Tyr_Z to Nϵ of H190. The identity of the “proton releaser” during the S₃Tyr_Z^• to (S₃Tyr_Z^•)′ is not known, and several candidates have been considered. A substrate water molecule is an obvious one (for example, see Ref. 76 for a model in which both the proton and the electron originate from the substrate water molecule). Alternatively, it could be a protonated base, proposed to be CP43-R357 (77), that would undergo a pK_a shift upon the formation of the S₃Tyr_Z^•⋯HNϵ(H190)⁺ state and would, by acting as a proton acceptor from water, promote water splitting. These different proton transfer events thus have essentially different mechanistic implications. Although one mainly reflects electrostatic relaxation, the other sets the stage for all the players in the water-splitting process. In such a framework, it is not surprising that they involve different molecular actors, and the present results support this expectation. Notably, they also point to a necessary conformational change to account for the fact that a new proton releaser that had stayed inactive until the formation of S₃ would come into play when S₃Tyr_Z^•⋯HNϵ(H190)⁺ is formed (for example, see Refs. 73 and 75 for experimental evidences of structural changes in the S state cycle).