New insights on ChlD1 function in Photosystem II from site-directed mutants of D1/T179 in Thermosynechococcus elongatus

The monomeric chlorophyll, Chl_D1, which is located between the P_D1P_D2 chlorophyll pair and the pheophytin, Pheo_D1, is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg²⁺ is liganded to a water molecule that is H-bonded to D1/T179. Here, two site-directed mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn₄CaO₅ cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *Chl_D1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of P_D1P_D2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the Chl_D1⁺Pheo_D1^? radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of ¹O₂ generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to Chl_D1 upon Q_A^? formation, indicates that in the T179H-PSII and in the WT*3-PSII, the Chl_D1 itself is the chlorophyll that is first damaged by ¹O₂, whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, Chl_D1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of Chl_D1 very likely contribute to the well-known electrochromic shifts observed at ~430?nm during the S-state cycle.     

The primary donor of far-red photosystem II: ChlD1 or PD2?

Far-red light (FRL) Photosystem II (PSII) isolated from Chroococcidiopsis thermalis is studied using parallel analyses of low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies in conjunction with fluorescence measurements. This extends earlier studies (Nurnberg et al 2018 Science 360 (2018) 1210–1213). We confirm that the chlorophyll absorbing at 726 nm is the primary electron donor. At 1.8 K efficient photochemistry occurs when exciting at 726 nm and shorter wavelengths; but not at wavelengths longer than 726 nm. The 726 nm absorption peak exhibits a 21 ± 4 cm⁻¹ electrochromic shift due to formation of the semiquinone anion, Q_A⁻. Modelling indicates that no other FRL pigment is located among the 6 central reaction center chlorins: P_D1, P_D2 Chl_D1, Chl_D2, Pheo_D1 and Pheo_D2. Two of these chlorins, Chl_D1 and P_D2, are located at a distance and orientation relative to Q_A⁻ so as to account for the observed electrochromic shift. Previously, Chl_D1 was taken as the most likely candidate for the primary donor based on spectroscopy, sequence analysis and mechanistic arguments. Here, a more detailed comparison of the spectroscopic data with exciton modelling of the electrochromic pattern indicates that P_D2 is at least as likely as Chl_D1 to be responsible for the 726 nm absorption. The correspondence in sign and magnitude of the CD observed at 726 nm with that predicted from modelling favors P_D2 as the primary donor. The pros and cons of P_D2 vs Chl_D1 as the location of the FRL-primary donor are discussed.     

Cationic state distribution over the chlorophyll d-containing PD1/PD2 pair in photosystem II

Most of the chlorophyll (Chl) cofactors in photosystem II (PSII) from Acaryochloris marina are Chld, although a few Chla molecules are also present. To evaluate the possibility that Chla may participate in the P_D1/P_D2 Chl pair in PSII from A. marina, the P_D1^?+/P_D2^?+ charge ratio was investigated using the PSII crystal structure analyzed at 1.9-Å resolution, while considering all possibilities for the Chld-containing P_D1/P_D2 pair, i.e., Chld/Chld, Chla/Chld, and Chld/Chla pairs. Chld/Chld and Chla/Chld pairs resulted in a large P_D1^?+ population relative to P_D2^?+, as identified in Chla/Chla homodimer pairs in PSII from other species, e.g., Thermosynechococcus elongatus PSII. However, the Chld/Chla pair possessed a P_D1^?+/P_D2^?+ ratio of approximately 50/50, which is in contrast to previous spectroscopic studies on A. marina PSII. The present results strongly exclude the possibility that the Chld/Chla pair serves as P_D1/P_D2 in A. marina PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Formation and decay of P680 (PD1–PD2)PheoD1 radical ion pair in photosystem II core complexes

Under physiological conditions (278 K) femtosecond pump-probe laser spectroscopy with 20-fs time resolution was applied to study primary charge separation in spinach photosystem II (PSII) core complexes excited at 710 nm. It was shown that initial formation of anion radical band of pheophytin molecule (Pheo⁻) at 460 nm is observed with rise time of ~ 11 ps. The kinetics of the observed rise was ascribed to charge separation between Chl (chlorophyll a) dimer, primary electron donor in PSII (P680*) and Pheo located in D1 protein subunit (Pheo_D1) absorbing at 420 nm, 545 nm and 680 nm with formation of the ion-radical pair P680⁺Pheo_DI⁻. The subsequent electron transfer from Pheo_D1⁻ to primary plastoquinone electron acceptor (Q_A) was accompanied by relaxation of the 460-nm band and occurred within ~ 250 ps in good agreement with previous measurements in Photosystem II-enriched particles and bacterial reaction centers. The subtraction of the P680⁺ spectrum measured at 455 ps delay from the spectra at 23 ps or 44 ps delay reveals the spectrum of Pheo_DI⁻, which is very similar to that measured earlier by accumulation method. The spectrum of Pheo_DI⁻ formation includes a bleaching (or red shift) of the 670 nm band indicating that Chl-670 is close to Pheo_D1. According to previous measurements in the femtosecond–picosecond time range this Chl-670 was ascribed to Chl_D1 [Shelaev, Gostev, Vishnev, Shkuropatov, Ptushenko, Mamedov, Sarkisov, Nadtochenko, Semenov and Shuvalov, J. Photochemistry and Photobiology, B: Biology 104 (2011) 45–50]. Stimulated emission at 685 nm was found to have two decaying components with time constants of ~ 1 ps and ~ 14 ps. These components appear to reflect formation of P680⁺Chl_D1⁻ and P680⁺Pheo_D1⁻, respectively, as found earlier. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Function of two β-carotenes near the D1 and D2 proteins in photosystem II dimers

The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 Å resolution), in which 11 β-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (E_m) of one-electron oxidation for all Car (E_m(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (Car_D1) in van der Waals contact with the chlorin ring of Chl_Z(D1). Each Car_D1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between Car_D1 and plastoquinone/Q_A may imply a direct charge recombination of Car⁺Q_A⁻. The calculated E_m(Car_D1) and E_m(Chl_Z(D1)) are, respectively, 83 and 126 mV higher than E_m(Car_D2) and E_m(Chl_Z(D2)), which could explain why Car_D2⁺ and Chl_Z(D2)⁺ are observed rather than the corresponding Car_D1⁺ and Chl_Z(D1)⁺.     

Charge separation in photosystem II core complexes induced by 690-730 nm excitation at 1.7 K

The illumination of oxygen-evolving PSII core complexes at very low temperatures in spectral regions not expected to excite P680 leads to charge separation in a majority of centers. The fraction of centers photoconverted as a function of the number of absorbed photons per PSII core is determined by quantification of electrochromic shifts on Pheo_D1. These shifts arise from the formation of metastable plastoquinone anion (Q_A⁻) configurations. Spectra of concentrated samples identify absorption in the 700-730 nm range. This is well beyond absorption attributable to CP47. Spectra in the 690-730 nm region can be described by the ‘trap’ CP47 absorption at 689 nm, with dipole strength of ∼1 chlorophyll a (chl a), partially overlapping a broader feature near 705 nm with a dipole strength of ∼0.15 chl a. This absorption strength in the 700-730 nm region falls by 40% in the photoconverted configuration. Quantum efficiencies of photoconversion following illumination in the 690-700 nm region are similar to those obtained with green illumination but fall significantly in the 700-730 nm range. Two possible assignments of the long-wavelength absorption are considered. Firstly, as a low intensity component of strongly exciton-coupled reaction center chlorin excitations and secondly as a nominally ‘dark’ charge-transfer excitation of the ‘special pair’ P_D1-P_D2. The opportunities offered by these observations towards the understanding of the nature of P680 and PSII fluorescence are discussed.     

Role of pheophytin a in the primary charge separation of photosystem I from Acaryochloris marina: Femtosecond optical studies of excitation energy and electron transfer reactions

Photosystem I (PSI) of the cyanobacterium Acaryochloris marina is capable of performing an efficient photoelectrochemical conversion of far-red light due to its unique suite of cofactors. Chlorophyll d (Chl-d) has been long known as the major antenna pigment in the PSI from A. marina, while the exact cofactor composition of the reaction centre (RC) was established only recently by cryo-electron microscopy. The RC consists of four Chl-d molecules, and, surprisingly, two molecules of pheophytin a (Pheo-a), which provide a unique opportunity to resolve, spectrally and kinetically, the primary electron transfer reactions. Femtosecond transient absorption spectroscopy was here employed to observe absorption changes in the 400–860 nm spectral window occurring in the 0.1–500 ps timescale upon unselective antenna excitation and selective excitation of the Chl-d special pair P₇₄₀ in the RC. A numerical decomposition of the absorption changes, including principal component analysis, allowed the identification of P₇₄₀⁽⁺⁾Chl_d2⁽⁻⁾ as the primary charge separated state and P₇₄₀⁽⁺⁾Pheo_a3⁽⁻⁾ as the successive, secondary, radical pair. A remarkable feature of the electron transfer reaction between Chl_d2 and Pheo_a3 is the fast, kinetically unresolved, equilibrium with an estimated ratio of 1:3. The energy level of the stabilised ion-radical state P₇₄₀⁽⁺⁾Pheo_a3⁽⁻⁾ was determined to be ~60 meV below that of the RC excited state. In this regard, the energetics and the structural implications of the presence of Pheo-a in the electron transfer chain of PSI from A. marina are discussed, also in comparison with those of the most diffused Chl-a binding RC.     

Subunit composition of CP43-less photosystem II complexes of Synechocystis sp. PCC 6803: implications for the assembly and repair of photosystem II

Photosystem II (PSII) mutants are useful experimental tools to trap potential intermediates involved in the assembly of the oxygen-evolving PSII complex. Here, we focus on the subunit composition of the RC47 assembly complex that accumulates in a psbC null mutant of the cyanobacterium Synechocystis sp. PCC 6803 unable to make the CP43 apopolypeptide. By using native gel electrophoresis, we showed that RC47 is heterogeneous and mainly found as a monomer of 220 kDa. RC47 complexes co-purify with small Cab-like proteins (ScpC and/or ScpD) and with Psb28 and its homologue Psb28-2. Analysis of isolated His-tagged RC47 indicated the presence of D1, D2, the CP47 apopolypeptide, plus nine of the 13 low-molecular-mass (LMM) subunits found in the PSII holoenzyme, including PsbL, PsbM and PsbT, which lie at the interface between the two momomers in the dimeric holoenzyme. Not detected were the LMM subunits (PsbK, PsbZ, Psb30 and PsbJ) located in the vicinity of CP43 in the holoenzyme. The photochemical activity of isolated RC47-His complexes, including the rate of reduction of P680⁺, was similar to that of PSII complexes lacking the Mn₄CaO₅ cluster. The implications of our results for the assembly and repair of PSII in vivo are discussed.     

Evidence that chlorophyll f functions solely as an antenna pigment in far-red-light photosystem I from Fischerella thermalis PCC 7521

The Photosystem I (PSI) reaction center in cyanobacteria is comprised of ~96 chlorophyll (Chl) molecules, including six specialized Chl molecules denoted Chl1A/Chl1B (P₇₀₀), Chl2A/Chl2B, and Chl3A/Chl3B that are arranged in two branches and function in primary charge separation. It has recently been proposed that PSI from Chroococcidiopsis thermalis (Nürnberg et al. (2018) Science 360, 1210–1213) and Fischerella thermalis PCC 7521 (Hastings et al. (2019) Biochim. Biophys. Acta 1860, 452–460) contain Chl f in the positions Chl2A/Chl2B. We tested this proposal by exciting RCs from white-light grown (WL-PSI) and far-red light grown (FRL-PSI) F. thermalis PCC 7521 with femtosecond pulses and analyzing the optical dynamics. If Chl f were in the position Chl2A/Chl2B in FRL-PSI, excitation at 740 nm should have produced the charge-separated state P₇₀₀⁺A₀⁻ followed by electron transfer to A₁ with a τ of ≤25 ps. Instead, it takes ~230 ps for the charge-separated state to develop because the excitation migrates uphill from Chl f in the antenna to the trapping center. Further, we observe a strong electrochromic shift at 685 nm in the final P₇₀₀⁺A₁⁻ spectrum that can only be explained if Chl a is in the positions Chl2A/Chl2B. Similar arguments rule out the presence of Chl f in the positions Chl3A/Chl3B; hence, Chl f is likely to function solely as an antenna pigment in FRL-PSI. We additionally report the presence of an excitonically coupled homo- or heterodimer of Chl f absorbing around 790 nm that is kinetically independent of the Chl f population that absorbs around 740 nm.     

Primary radical ion pairs in photosystem II core complexes

Ultrafast absorption spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach photosystem II (PSII) reaction center (RC) and PSII core complex (RC complex with integral antenna) upon excitation at maximum wavelength 700–710 nm at 278 K. It was found that the initial charge separation between P680* and Chl_D1 (Chl-670) takes place with a time constant of ～1 ps with the formation of the primary charge-separated state P680* with an admixture of: P680^*(1?δ) (P680^δ+1Chl _D1 ^δ? ), where δ ～ 0.5. The subsequent electron transfer from P680^δ+Chl _D1 ^δ? to pheophytin (Pheo) occurs within 13 ps and is accompanied by a relaxation of the absorption band at 670 nm (Chl _D1 ^δ? ) and bleaching of the Pheo_D1 bands at 420, 545, and 680 nm with development of the Pheoband at 460 nm. Further electron transfer to Q_A occurs within 250 ps in accordance with earlier data. The spectra of P680⁺ and Pheo^? formation include a bleaching band at 670 nm; this indicates that Chl-670 is an intermediate between P680 and Pheo. Stimulated emission kinetics at 685 nm demonstrate the existence of two decaying components with time constants of ～1 and ～13 ps due to the formation of P680^δ+Chl _D1 ^δ? and P680⁺Pheo _D1 ^? , respectively.     

Spectroscopic properties of reaction center pigments in photosystem II core complexes: revision of the multimer model

Absorbance difference spectra associated with the light-induced formation of functional states in photosystem II core complexes from Thermosynechococcus elongatus and Synechocystis sp. PCC 6803 (e.g., ) are described quantitatively in the framework of exciton theory. In addition, effects are analyzed of site-directed mutations of D1-His¹⁹⁸, the axial ligand of the special-pair chlorophyll P_D1, and D1-Thr¹⁷⁹, an amino-acid residue nearest to the accessory chlorophyll Chl_D1, on the spectral properties of the reaction center pigments. Using pigment transition energies (site energies) determined previously from independent experiments on D1-D2-cytb559 complexes, good agreement between calculated and experimental spectra is obtained. The only difference in site energies of the reaction center pigments in D1-D2-cytb559 and photosystem II core complexes concerns Chl_D1. Compared to isolated reaction centers, the site energy of Chl_D1 is red-shifted by 4 nm and less inhomogeneously distributed in core complexes. The site energies cause primary electron transfer at cryogenic temperatures to be initiated by an excited state that is strongly localized on Chl_D1 rather than from a delocalized state as assumed in the previously described multimer model. This result is consistent with earlier experimental data on special-pair mutants and with our previous calculations on D1-D2-cytb559 complexes. The calculations show that at 5 K the lowest excited state of the reaction center is lower by ∼10 nm than the low-energy exciton state of the two special-pair chlorophylls P_D1 and P_D2 which form an excitonic dimer. The experimental temperature dependence of the wild-type difference spectra can only be understood in this model if temperature-dependent site energies are assumed for Chl_D1 and P_D1, reducing the above energy gap from 10 to 6 nm upon increasing the temperature from 5 to 300 K. At physiological temperature, there are considerable contributions from all pigments to the equilibrated excited state P*. The contribution of Chl_D1 is twice that of P_D1 at ambient temperature, making it likely that the primary charge separation will be initiated by Chl_D1 under these conditions. The calculations of absorbance difference spectra provide independent evidence that after primary electron transfer the hole stabilizes at P_D1, and that the physiologically dangerous charge recombination triplets, which may form under light stress, equilibrate between Chl_D1 and P_D1.     

What can we still learn from the electrochromic band-shifts in Photosystem II?

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 Q_X absorption of Phe_D1 differs, a band-shift in the Q_X region of Phe_D2 centered at ~ 544 nm has been identified upon the oxidation, at pH 8.6, of Tyr_D. In contrast, a band-shift due to the formation of either Q_A^•- or Tyr_Z^• is observed in PsbA3-PSII at ~ 546 nm, as expected with E130 H-bonded to Phe_D1 and at ~ 544 nm as expected with Q130 H-bonded to Phe_D1. 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 P_D1 is blue shifted and in the PsbA3/T179H mutant. Upon Tyr_Z^•Q_A^•- formation the Soret band of P_D1 is red shifted and the Soret band of Chl_D1 is blue shifted. In contrast, only P_D1 undergoes a detectable S-state dependent electrochromism. Thirdly, the time resolved S-state dependent electrochromism attributed to P_D1 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 S₂Tyr_Z^• is discussed in view of the models in the literature involving intermediate states in the S₂ to S₃ transition.     

D1 protein variants in Photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy

In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA₃ gene over the psbA₁ gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at < 10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Q_x band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the Pheo_D1 Q_x band induced by Q_A⁻ (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light. When green light was used there was little difference between the two variants. With far red light the stable (t_1/2 > 50 ms) Q_A⁻ yield was ∼ 95% in D1:3, and ∼ 60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Q_x band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 13¹-keto of Pheo_D1, which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of P_D1, in particular Ser153Ala. Photo-induced Q_A⁻ formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P⁺.     

Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b(559), with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and β-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q(Y) region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.     

Influence of Histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus

The influence of the histidine axial ligand to the P_D1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P_680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA₁ and psbA₂ genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT^?) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA₃ gene. The O₂-evolving activity of His-tagged PSII isolated from WT^? was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA₁ is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT^?. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P₆₈₀, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechocystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265-9281]. We conclude that the nature of the axial ligand to P_D1 is not an important determinant of the redox and spectroscopic properties of P₆₈₀ in T. elongatus.     

The Origin of the Low-Energy Form of Photosystem I Light-Harvesting Complex Lhca4: Mixing of the Lowest Exciton with a Charge-Transfer State

The peripheral light-harvesting complex of photosystem I contains red chlorophylls (Chls) that, unlike the typical antenna Chls, absorb at lower energy than the primary electron donor P700. It has been shown that the red-most absorption band arises from two excitonically coupled Chls, although this interaction alone cannot explain the extreme red-shifted emission (25 nm, ∼480 cm⁻¹ for Lhca4 at 4 K) that the red Chls present. Here, we report the electric field-induced absorption changes (Stark effect) on the Q_y region of the Lhca4 complex. Two spectral forms, centered around 690 nm and 710 nm, were necessary to describe the absorption and Stark spectra. The analysis of the lowest energy transition yields a high value for the change in dipole moment, Δμ_710nm ≈ 8 Df⁻¹, between the ground and excited states as compared with monomeric, Δμ = 1 D, or dimeric, Δμ = 5 D, Chl a in solution. The high value of the Δμ demonstrates that the origin of the red-shifted emission is the mixing of the lowest exciton state with a charge-transfer state of the dimer. This energetic configuration, an excited state with charge-transfer character, is very favorable for the trapping and dissipation of excitations and could be involved in the photoprotective mechanism(s) of the photosystem I complex.     

1D/2D coordination polymers of copper(II) having two superexchange pathways: syntheses, crystal structures and magnetic properties

Two new polynuclear complexes of Cu(II), [(μ-1,1,3-N₃)₂{Cu₂(me₂tn)₂(N₃)₂}]_n (1) (me₂tn=2,2-dimethylpropane-1,3-diamine) and [Cu₂(μ-C₂O₄)(μ-N₃)(ipr₂en)₂]_n(ClO₄)_n (2) (ipr₂en=N,N^′-di-isopropylethane-1,2-diamine) have been synthesized and structurally characterized by X-ray crystallography. The crystal structure of 1 displays a 2D network in which distorted octahedral copper(II) centers, chelated by a me₂tn ligand and bound to a terminal azide, are connected through μ-1,1,3 bridging azide anions. The structure of 2 shows 1D chains comprising alternating [(ipr₂en)Cu-Ox-Cu(ipr₂en)] units and end-to-end azide ligand. The chains on mutual H-bonding interaction through ClO₄⁻, give rise to a 2D supramolecular architecture. The magnetic data of complexes were recorded in the temperature range, 300-2 K. In case of complex 1, the magnetic data are consistent with a ferromagnetic interaction through the end-on azide bridge (J_FM=10 cm⁻¹) and a weak antiferromagnetic interaction (zj=−0.8 cm⁻¹) between the ferromagnetically coupled dimers and an average g-value of 2.05. The susceptibility data of 2 were fitted using an alternating AF-AF chain spin 1/2 law which leads to the following parameters J_oxalate=−180 cm⁻¹, J_azide=−43 cm⁻¹ and g=2.25 cm⁻¹.     

Evaluation of the association of mercury(II) with some dicysteinyl tripeptides

The present study was undertaken to gain insight into the associations of mercury(II) with dicysteinyl tripeptides in buffered media at pH 7.4. We investigated the effects of increasing the distance between cysteinyl residues on mercury(II) associations and complex formations. The peptide–mercury(II) formation constants and their associated thermodynamic parameters in 3-(N-morpholino)propanesulfonic acid (MOPS) buffered solutions were evaluated by isothermal titration calorimetry. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides in ammonium formate buffered solutions were characterized by LTQ Orbitrap mass spectrometry. The results from these studies show that n-alkyl dicysteinyl peptides (CP 1–4), and an aryl dicysteinyl peptide (CP 5) can serve as effective “double anchors” to accommodate the coordination sites of mercury(II) to form predominantly one-to-one Hg(peptide) complexes. The aryl dicysteinyl peptide (CP 5) also forms the two-to-two Hg₂(peptide)₂ complex. In the presence of excess peptide, Hg(peptide)₂ complexes are also detected. Notably, increasing the distance between the ligating groups or “anchor points” in CP 1–5 does not significantly affect their affinity for mercury(II). However, the enthalpy change (ΔH) values (ΔH₁ ∼ −91 kJ mol⁻¹ and ΔH₂ ∼ −66 kJ mol⁻¹) for complex formation between CP 4 and 5 with mercury(II) are about one and a half times larger than the related values for CP 1, 2 and 3 (ΔH₁ ∼ −66 kJ mol⁻¹ and ΔH₂ ∼ 46 kJ mol⁻¹). The corresponding entropy change (ΔS) values (ΔS₁ ∼ −129 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −116 J K⁻¹ mol⁻¹) of the structurally larger dicysteinyl peptides CP 4 and 5 are less entropically favorable than for CP 1, 2 and 3 (ΔS₁ ∼ −48 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −44 J K⁻¹ mol⁻¹). Generally, these associations result in a decrease in entropy, indicating that these peptide–mercury complexes potentially form highly ordered structures. The results from this study show that dicysteinyl tripeptides are effective in binding mercury(II) and they are promising motifs for the design of multi-cysteinyl peptides for binding more than one mercury(II) ion per peptide.     

Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene

The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA₃ gene compared to that deduced from the psbA₁ gene points a difference of 21 residues. In this work, PSII isolated from a wild type T. elongatus strain expressing PsbA1 or from a strain in which both the psbA₁ and psbA₂ genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of Pheo_D1 is increased by 17 mV from −522 mV in PsbA1-PSII to −505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S₂Q_A^−• charge recombination and the C ≡ N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the Q_B pocket. The temperature dependences of the S₂Q_A^−• charge recombination show that the strength of the H-bond to Pheo_D1 is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of Q_A (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P₆₈₀^+• and Y_Z is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P₆₈₀/P₆₈₀^+• couple (and hence that of ¹P₆₈₀^*/P₆₈₀^+•) is tuned as well when shifting from PsbA1 to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes.