Environment of TyrZ in photosystem II from Thermosynechococcus elongatus in which PsbA2 is the D1 protein

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 (S(3)Tyr(Z)(•))' is slightly modified; (ii) the split EPR signal arising from Tyr(Z)(•) in the (S(2)Tyr(Z)(•))' state induced by near-infrared illumination at 4.2 K of the S(3)Tyr(Z) state is significantly modified; and (iii) the slow phases of P(680)(+) reduction by Tyr(Z) are slowed down from the hundreds of μs time range to the ms time range, whereas both the S(1)Tyr(Z)(•) → S(2)Tyr(Z) and the S(3)Tyr(Z)(•) → S(0)Tyr(Z) + O(2) 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.

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).

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.

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.

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.

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.

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.

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.