Deactivation processes in PsbA1-Photosystem II and PsbA3-Photosystem II under photoinhibitory conditions in the cyanobacterium Thermosynechococcus elongatus

AbstractThe sensitivity to high light conditions of Photosystem II with either PsbA1 (WT*1) or PsbA3 (WT*3) as the D1 protein was studied in whole cells of the thermophilic cyanobacterium Thermosynechococcus elongatus. When the cells are cultivated under high light conditions the following results were found: (i) The O2 evolution activity decreases faster in WT*1 cells than in WT*3 cells both in the absence and in the presence of lincomycin, a protein synthesis inhibitor; (ii) In WT*1 cells, the rate constant for the decrease of the O2 evolution activity is comparable in the presence and in the absence of lincomycin; (iii) The D1 content revealed by western blot analysis decays similarly in both WT*1 and WT*3 cells and much slowly than O2 evolution; (iv) The faster decrease in O2 evolution in WT*1 than in WT*3 cells correlates with a much faster inhibition of the S2-state formation; (v) The shape of the WT*1 cells is altered. All these results are in agreement with a photo-inhibition process resulting in the loss of the O2 activity much faster than the D1 turnover in PsbA1-PSII and likely to a greater production of reactive oxygen species under high light conditions in WT*1 than in WT*3. This latter result is discussed in view of the known effects of the PsbA1 to PsbA3 substitution on the redox properties of the Photosystem II cofactors. The observation that under low light conditions WT*3 cells are able to express the psbA3 gene, whereas under similar conditions wild type cells are expressing mainly the psbA1 gene is also discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial

Evidence for different binding sites on the 33-kDa protein for DCMU, atrazine and QB

AbstractTwo DCMU-resistant strains of the cyanobacterium Synechocystis 6714 were used to analyse the binding sites of DCMU, atrazine and QB. DCMUr-IIA was DCMU and atrazine resistant; it presented an impaired electron flow and its 33-kDa protein was weakly attached to the membrane. DCMUr-IIB, derived from the former, simultaneously regained atrazine sensitivity, normal electron flow and a tight linkage of the 33-kDa protein to the membrane. This mutant shows that loss of DCMU binding does not necessarily affect the binding of either atrazine or QB. The role of the 33-kDa protein is discussed

EPR characterisation of the ferrous nitrosyl complex formed within the oxygenase domain of NO synthase.

International audienceNitric oxide is produced in mammals by a class of enzymes called NO synthases (NOSs). It plays a central role in cellular signalling but also has deleterious effects, as it leads to the production of reactive oxygen and nitrogen species. NO forms a relatively stable adduct with ferrous haem proteins, which, in the case of NOS, is also a key catalytic intermediate. Despite extensive studies on the ferrous nitrosyl complex of other haem proteins (in particular myoglobin), little characterisation has been performed in the case of NOS. We report here a temperature-dependent EPR study of the ferrous nitrosyl complex of the inducible mammalian NOS and the bacterial NOS-like protein from Bacillus subtilis. The results show that the overall behaviours are similar to those observed for other haem proteins, but with distinct ratios between axial and rhombic forms in the case of the two NOS proteins. The distal environment appears to control the existence of the axial form and the evolution of the rhombic form

Probing the role of chloride in Photosystem II from Thermosynechococcus elongatus by exchanging chloride for iodide

AbstractThe active site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5 cluster and TyrZ, a redox-active tyrosine residue. Chloride ions have been known for long time to be required for the function of the enzyme. However, X-ray data have shown that they are located about 7Å away from the Mn4CaO5 cluster, a distance that seems too large to be compatible with a direct involvement of chloride in the water splitting chemistry. We have investigated the role of this anion by substituting I− for Cl− in the cyanobacterium Thermosynechococcus elongatus with either Ca2+ or Sr2+ biosynthetically assembled into the Mn4 cluster. The electron transfer steps affected by the exchanges were investigated by time-resolved UV–visible absorption spectroscopy, time-resolved EPR at room temperature and low temperature cw-EPR spectroscopy. In both Ca-PSII and Sr-PSII, the Cl−/I− exchange considerably slowed down the two S3TyrZ•→(S3TyrZ•)′→S0 reactions in which the fast phase, S3TyrZ•→(S3TyrZ•)′, reflects the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge near/on TyrZ• and the slow phase corresponds to the S0 and O2 formations and to a second proton release. The t1/2 for S0 formation increased from 1.1ms in Ca/Cl-PSII to ≈6ms in Ca/I-PSII and from 4.8ms in Sr/Cl-PSII to ≈45ms in Sr/I-PSII. In all cases the TyrZ• reduction was the limiting step. The kinetic effects are interpreted by a model in which the Ca2+ binding site and the Cl− binding site, although spatially distant, interact. This interaction is likely mediated by the H-bond and/or water molecules network(s) connecting the Cl− and Ca2+ binding sites by which proton release may be channelled

Impact of energy limitations on function and resilience in long-wavelength Photosystem II

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 Acaryochloris marina has Chl-d replacing all but one of its 35 Chl-a, while PSII from Chroococcidiopsis thermalis, 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 PD1+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 QA 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

Ammonia binding to the oxygen-evolving complex of photosystem II identifies the solvent-exchangeable oxygen bridge (μ-oxo) of the manganese tetramer

The assignment of the two substrate water sites of the tetra-manganese penta-oxygen calcium (Mn4O5Ca) cluster of photosystem II is essential for the elucidation of the mechanism of biological O-O bond formation and the subsequent design of bio-inspired water-splitting catalysts. We recently demonstrated using pulsed EPR spectroscopy that one of the five oxygen bridges (μ-oxo) exchanges unusually rapidly with bulk water and is thus a likely candidate for one of the substrates. Ammonia, a water analog, was previously shown to bind to the Mn4O5Ca cluster, potentially displacing a water/substrate ligand [Britt RD, et al. (1989) J Am Chem Soc 111(10):3522–3532]. Here we show by a combination of EPR and time-resolved membrane inlet mass spectrometry that the binding of ammonia perturbs the exchangeable μ-oxo bridge without drastically altering the binding/exchange kinetics of the two substrates. In combination with broken-symmetry density functional theory, our results show that (i) the exchangable μ-oxo bridge is O5 {using the labeling of the current crystal structure [Umena Y, et al. (2011) Nature 473(7345):55–60]}; (ii) ammonia displaces a water ligand to the outer manganese (MnA4-W1); and (iii) as W1 is trans to O5, ammonia binding elongates the MnA4-O5 bond, leading to the perturbation of the μ-oxo bridge resonance and to a small change in the water exchange rates. These experimental results support O-O bond formation between O5 and possibly an oxyl radical as proposed by Siegbahn and exclude W1 as the second substrate water

Cytochrome c550 in the cyanobacterium Thermosynechococcus elongatus: Study of redox mutants

Cytochrome c550 is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c550 we constructed two mutants of Thermosynechococcus elongatus in which the residue His-92, the sixth ligand of the heme, was replaced by a Met or a Cys in order to modify the redox properties of the heme. The H92M and H92C mutations changed the midpoint redox potential of the heme in the isolated cytochrome by +125 mV and –30 mV, respectively, compared with the wild type. The binding-induced increase of the redox potential observed in the wild type and the H92C mutant was absent in the H92M mutant. Both modified cytochromes were more easily detachable from the Photosystem II compared with the wild type. The Photosystem II activity in cells was not modified by the mutations suggesting that the redox potential of the cytochrome c550 is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c550 was also constructed. It showed a lowered affinity for Cl– and Ca2+ as reported earlier for the cytochrome c550-less Synechocystis 6803 mutant, but it showed a shorter lived Formula state, rather than a stabilized S2 state and rapid deactivation of the enzyme in the dark, which were characteristic of the Synechocystis mutant. It is suggested that the latter effects may be caused by loss (or weaker binding) of the other extrinsic proteins rather than a direct effect of the absence of the cytochrome c55

Quantification of the number of spins in the S2- and S3-states of Ca2+-depleted photosystem II by pulsed-EPR spectroscopy

AbstractCa2+-depletion of the photosystem II enzyme by a NaCl-washing in the light inhibits oxygen evolution. In Ca2+-depleted photosystem II the S3 charge storage state exhibits a split EPR signal attributed to the magnetic interaction between a radical and the Mn-cluster. Further treatment of photosystem II by EGTA modifies the shape of the EPR signal of the Mn-cluster in the S2 charge storage state. The percentage of centers in which the S2 modified signal and the split S3 signal can be observed has been estimated by using pulsed-EPR spectroscopy. On the basis of one tyrosine D radical per reaction center, the field-swept spin echo spectrum of the modified S2 state in dark-adapted photosystem II was detected in a large majority of the reaction centers. The derivative of the S2 field-swept spectrum with respect to the magnetic field resulted in a spectrum similar to that observed by cw-EPR. The additional light-induced split S3 signal appeared on top of the envelope of the S2 signal and was detected in the same proportion of centers as that which exhibited the S2 signal prior to the illumination. In the formal S3 state, the hyperfine lines of the Mn field-swept echo spectrum were no longer detectable. The storage of PS-11 at 77 K after formation of the S3QA− state by freezing the membranes under continuous illumination resulted in a decrease of the S3 signal but the pulsed-EPR S2 manganese signal was conserved

Temperature dependence of the high-spin S2 to S3 transition in Photosystem II: Mechanistic consequences

International audienceThe 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 \textless 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