RESUMEN
Photosystem II is the water/plastoquinone photo-oxidoreductase of photosynthesis. The photochemistry and catalysis occur in a quasi-symmetrical heterodimer, D1D2, that evolved from a homodimeric ancestor. Here, we studied site-directed mutants in PSII from the thermophilic cyanobacterium Thermosynechoccocus elongatus, focusing on the primary electron donor chlorophyll a in D1, ChlD1, and on its symmetrical counterpart in D2, ChlD2, which does not play a direct photochemical role. The main conserved amino acid specific to ChlD1 is D1/T179, which H-bonds the water ligand to its Mg2+, while its counterpart near ChlD2 is the non-H-bonding D2/I178. The symmetrical-swapped mutants, D1/T179I and D2/I178T, and a second ChlD2 mutant, D2/I178H, were studied. The D1 mutations affected the 686 nm absorption attributed to ChlD1, while the D2 mutations affected a 663 nm feature, tentatively attributed to ChlD2. The mutations had little effect on enzyme activity and forward electron transfer, reflecting the robustness of the overall enzyme function. In contrast, the mutations significantly affected photodamage and protective mechanisms, reflecting the importance of redox tuning in these processes. In D1/T179I, the radical pair recombination triplet on ChlD1 was shared onto a pheophytin, presumably PheD1 and the detection of 3PheD1 supports the proposed mechanism for the anomalously short lifetime of 3ChlD1; e.g. electron transfer quenching by QA- of 3PheD1 after triplet transfer from 3ChlD1. In D2/I178T, a charge separation could occur between ChlD2 and PheD2, a reaction that is thought to occur in ancestral precursors of PSII. These mutants help understand the evolution of asymmetry in PSII.
Asunto(s)
Aminoácidos , Complejo de Proteína del Fotosistema II , Complejo de Proteína del Fotosistema II/metabolismo , Aminoácidos/genética , Clorofila A , Clorofila/metabolismo , Mutagénesis Sitio-Dirigida , AguaRESUMEN
Flash-induced absorption changes in the Soret region arising from the [PD1PD2]+ state, the chlorophyll cation radical formed upon light excitation of Photosystem II (PSII), were measured in Mn-depleted PSII cores at pH 8.6. Under these conditions, TyrD is i) reduced before the first flash, and ii) oxidized before subsequent flashes. In wild-type PSII, when TyrDâ is present, an additional signal in the [PD1PD2]+-minus-[PD1PD2] difference spectrum was observed when compared to the first flash when TyrD is not oxidized. The additional feature was "W-shaped" with troughs at 434 nm and 446 nm. This feature was absent when TyrD was reduced, but was present (i) when TyrD was physically absent (and replaced by phenylalanine) or (ii) when its H-bonding histidine (D2-His189) was physically absent (replaced by a Leucine). Thus, the simple difference spectrum without the double trough feature at 434 nm and 446 nm, seemed to require the native structural environment around the reduced TyrD and its H bonding partners to be present. We found no evidence of involvement of PD1, ChlD1, PheD1, PheD2, TyrZ, and the Cytb559 heme in the W-shaped difference spectrum. However, the use of a mutant of the PD2 axial His ligand, the D2-His197Ala, shows that the PD2 environment seems involved in the formation of "W-shaped" signal.
RESUMEN
In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for the Photosystem II (PSII) D1 subunit that interacts with most of the main cofactors involved in the electron transfers. Recently, the 3D crystal structures of both PsbA2-PSII and PsbA3-PSII have been solved [Nakajima et al., J. Biol. Chem. 298 (2022) 102668.]. It was proposed that the loss of one hydrogen bond of PheD1 due to the D1-Y147F exchange in PsbA2-PSII resulted in a more negative Em of PheD1 in PsbA2-PSII when compared to PsbA3-PSII. In addition, the loss of two water molecules in the Cl-1 channel was attributed to the D1-P173M substitution in PsbA2-PSII. This exchange, by narrowing the Cl-1 proton channel, could be at the origin of a slowing down of the proton release. Here, we have continued the characterization of PsbA2-PSII by measuring the thermoluminescence from the S2QA-/DCMU charge recombination and by measuring proton release kinetics using time-resolved absorption changes of the dye bromocresol purple. It was found that i) the Em of PheD1-/PheD1 was decreased by â¼30 mV in PsbA2-PSII when compared to PsbA3-PSII and ii) the kinetics of the proton release into the bulk was significantly slowed down in PsbA2-PSII in the S2TyrZ⢠to S3TyrZ and S3TyrZ⢠â (S3TyrZâ¢)' transitions. This slowing down was partially reversed by the PsbA2/M173P mutation and induced by the PsbA3/P173M mutation thus confirming a role of the D1-173 residue in the egress of protons trough the Cl-1 channel.
Asunto(s)
Cianobacterias , Complejo de Proteína del Fotosistema II , Complejo de Proteína del Fotosistema II/metabolismo , Protones , Cianobacterias/metabolismo , Transporte de ElectrónRESUMEN
Three psbA genes (psbA1, psbA2, and psbA3) encoding the D1 subunit of photosystem II (PSII) are present in the thermophilic cyanobacterium Thermosynechococcus elongatus and are expressed differently in response to changes in the growth environment. To clarify the functional differences of the D1 protein expressed from these psbA genes, PSII dimers from two strains, each expressing only one psbA gene (psbA2 or psbA3), were crystallized, and we analyzed their structures at resolutions comparable to previously studied PsbA1-PSII. Our results showed that the hydrogen bond between pheophytin/D1 (PheoD1) and D1-130 became stronger in PsbA2- and PsbA3-PSII due to change of Gln to Glu, which partially explains the increase in the redox potential of PheoD1 observed in PsbA3. In PsbA2, one hydrogen bond was lost in PheoD1 due to the change of D1-Y147F, which may explain the decrease in stability of PheoD1 in PsbA2. Two water molecules in the Cl-1 channel were lost in PsbA2 due to the change of D1-P173M, leading to the narrowing of the channel, which may explain the lower efficiency of the S-state transition beyond S2 in PsbA2-PSII. In PsbA3-PSII, a hydrogen bond between D1-Ser270 and a sulfoquinovosyl-diacylglycerol molecule near QB disappeared due to the change of D1-Ser270 in PsbA1 and PsbA2 to D1-Ala270. This may result in an easier exchange of bound QB with free plastoquinone, hence an enhancement of oxygen evolution in PsbA3-PSII due to its high QB exchange efficiency. These results provide a structural basis for further functional examination of the three PsbA variants.
Asunto(s)
Cianobacterias , Complejo de Proteína del Fotosistema II , Cianobacterias/metabolismo , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
The stoichiometry and kinetics of the proton release were investigated during each transition of the S-state cycle in Photosystem II (PSII) from Thermosynechococcus elongatus containing either a Mn4CaO5 (PSII/Ca) or a Mn4SrO5 (PSII/Sr) cluster. The measurements were done at pH 6.0 and pH 7.0 knowing that, in PSII/Ca at pH 6.0 and pH 7.0 and in PSII/Sr at pH 6.0, the flash-induced S2-state is in a low-spin configuration (S2LS) whereas in PSII/Sr at pH 7.0, the S2-state is in a high-spin configuration (S2HS) in half of the centers. Two measurements were done; the time-resolved flash dependent i) absorption of either bromocresol purple at pH 6.0 or neutral red at pH 7.0 and ii) electrochromism in the Soret band of PD1 at 440 nm. The fittings of the oscillations with a period of four indicate that one proton is released in the S1 to S2HS transition in PSII/Sr at pH 7.0. It has previously been suggested that the proton released in the S2LS to S3 transition would be released in a S2LSTyrZ⢠â S2HSTyrZ⢠transition before the electron transfer from the cluster to TyrZ⢠occurs. The release of a proton in the S1TyrZ⢠â S2HSTyrZ transition would logically imply that this proton release is missing in the S2HSTyrZ⢠to S3TyrZ transition. Instead, the proton release in the S1 to S2HS transition in PSII/Sr at pH 7.0 was mainly done at the expense of the proton release in the S3 to S0 and S0 to S1 transitions. However, at pH 7.0, the electrochromism of PD1 seems larger in PSII/Sr when compared to PSII/Ca in the S3 state. This points to the complex link between proton movements in and immediately around the Mn4 cluster and the mechanism leading to the release of protons into the bulk.
Asunto(s)
Cianobacterias , Complejo de Proteína del Fotosistema II , Cianobacterias/metabolismo , Transporte de Electrón , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/metabolismo , ProtonesRESUMEN
Photosystem II (PSII), the oxygen-evolving enzyme, consists of 17 trans-membrane and 3 extrinsic membrane proteins. Other subunits bind to PSII during assembly, like Psb27, Psb28, and Tsl0063. The presence of Psb27 has been proposed (Zabret et al. in Nat Plants 7:524-538, 2021; Huang et al. Proc Natl Acad Sci USA 118:e2018053118, 2021; Xiao et al. in Nat Plants 7:1132-1142, 2021) to prevent the binding of PsbJ, a single transmembrane α-helix close to the quinone QB binding site. Consequently, a PSII rid of Psb27, Psb28, and Tsl0034 prior to the binding of PsbJ would logically correspond to an assembly intermediate. The present work describes experiments aiming at further characterizing such a ∆PsbJ-PSII, purified from the thermophilic Thermosynechococcus elongatus, by means of MALDI-TOF spectroscopy, thermoluminescence, EPR spectroscopy, and UV-visible time-resolved spectroscopy. In the purified ∆PsbJ-PSII, an active Mn4CaO5 cluster is present in 60-70% of the centers. In these centers, although the forward electron transfer seems not affected, the Em of the QB/QB- couple increases by ≥ 120 mV , thus disfavoring the electron coming back on QA. The increase of the energy gap between QA/QA- and QB/QB- could contribute in a protection against the charge recombination between the donor side and QB-, identified at the origin of photoinhibition under low light (Keren et al. in Proc Natl Acad Sci USA 94:1579-1584, 1997), and possibly during the slow photoactivation process.
Asunto(s)
Cianobacterias , Complejo de Proteína del Fotosistema II , Cianobacterias/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Electrones , Complejo de Proteína del Fotosistema II/metabolismo , Subunidades de Proteína/metabolismoRESUMEN
The Mn4 CaO5 cluster of photosystem II (PSII) advances sequentially through five oxidation states (S0 to S4 ). Under the enzyme cycle, two water molecules are oxidized, O2 is generated and four protons are released into the lumen. Umena et al. (2011) have proposed that, with other charged amino acids, the R323 residue of the D1 protein could contribute to regulate a proton egress pathway from the Mn4 CaO5 cluster and TyrZ via a proton channel identified from the 3D structure. To test this suggestion, a PsbA3/R323E site-directed mutant has been constructed and the properties of its PSII have been compared to those of the PsbA3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV-visible absorption spectroscopy. Neither the oscillations with a period four nor the kinetics and S-state-dependent stoichiometry of the proton release were affected. However, several differences have been found: (1) the P680 + decay in the hundreds of ns time domain was much slower in the mutant, (2) the S2 QA - /DCMU and S3 QA - /DCMU radiative charge recombination occurred at higher temperatures and (3) the S0 TyrZ ⢠, S1 TyrZ ⢠, S2 TyrZ ⢠split EPR signals induced at 4.2 K by visible light from the S0 TyrZ , S1 TyrZ , S2 TyrZ , respectively, and the (S2 TyrZ ⢠)' induced by NIR illumination at 4.2 K of the S3 TyrZ state differed. It is proposed that the R323 residue of the D1 protein interacts with TyrZ likely via the H-bond network previously proposed to be a proton channel. Therefore, rather than participating in the egress of protons to the lumen, this channel could be involved in the relaxations of the H-bonds around TyrZ by interacting with the bulk, thus tuning the driving force required for TyrZ oxidation.
Asunto(s)
Arginina , Complejo de Proteína del Fotosistema II , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/metabolismo , ProtonesRESUMEN
Electrochromic band-shifts have been investigated in Photosystem II (PSII) from Thermosynechoccocus elongatus. Firstly, by using Mn-depleted PsbA1-PSII and PsbA3-PSII in which the QX absorption of PheD1 differs, a band-shift in the QX region of PheD2 centered at ~ 544 nm has been identified upon the oxidation, at pH 8.6, of TyrD. In contrast, a band-shift due to the formation of either QAâ¢- or TyrZ⢠is observed in PsbA3-PSII at ~ 546 nm, as expected with E130 H-bonded to PheD1 and at ~ 544 nm as expected with Q130 H-bonded to PheD1. Secondly, electrochromic band-shifts in the Chla Soret region have been measured in O2-evolving PSII in PsbA3-PSII, in the PsbA3/H198Q mutant in which the Soret band of PD1 is blue shifted and in the PsbA3/T179H mutant. Upon TyrZâ¢QAâ¢- formation the Soret band of PD1 is red shifted and the Soret band of ChlD1 is blue shifted. In contrast, only PD1 undergoes a detectable S-state dependent electrochromism. Thirdly, the time resolved S-state dependent electrochromism attributed to PD1 is biphasic for all the S-state transitions except for S1 to S2, and shows that: i) the proton release in S0 to S1 occurs after the electron transfer and ii) the proton release and the electron transfer kinetics in S2 to S3, in T. elongatus, are significantly faster than often considered. The nature of S2TyrZ⢠is discussed in view of the models in the literature involving intermediate states in the S2 to S3 transition.
Asunto(s)
Electrones , Complejo de Proteína del Fotosistema II/metabolismo , Clorofila/metabolismo , Luz , Modelos Moleculares , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/química , Synechococcus/metabolismo , Tirosina/metabolismoRESUMEN
Photosynthetic water oxidation takes place at the Mn4CaO5 cluster in photosystem II via a light-driven cycle of intermediates called S states (S0-S4). Clarifying how electron and proton transfer reactions are coupled with each other in the S2 â S3 transition, which occurs just before O-O bond formation, is crucial for understanding the water oxidation mechanism. Here, we investigated the pH dependence of the kinetics of the S2 â S3 transition using time-resolved infrared (TRIR) spectroscopy to identify the proton release phase in this transition. TRIR measurements of YD-less PSII core complexes from the D2-Y160F mutant of Thermosynechococcus elongatus showed that the last phase in this transition (τ â¼ 350 µs at pH 6) was strongly dependent on pH, and its time constant at pH 5 was larger than that at pH 8 by a factor of >3. In contrast, the earlier phase with a time constant of â¼100 µs was virtually independent of pH. These results strongly support the view that proton release is a rate-limiting step of the proton-coupled electron transfer in the last phase of the S2 â S3 transition. This proton release enables electron transfer by removing an excessive positive charge from the catalytic center and hence decreasing its redox potential.
Asunto(s)
Cianobacterias/química , Complejo de Proteína del Fotosistema II/química , Protones , Espectrofotometría Infrarroja/métodos , Agua/química , Dominio Catalítico , Transporte de Electrón , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción/efectos de la radiación , Complejo de Proteína del Fotosistema II/aislamiento & purificación , ThermosynechococcusRESUMEN
Photosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus, homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Emâ¯=â¯-440â¯mV, is lower than that of Fd1, Emâ¯=â¯-372â¯mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP+ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38â¯Å resolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV-Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys53ValAsnCys56 of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Ferredoxinas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cianobacterias/genética , Ferredoxinas/química , Ferredoxinas/genética , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Filogenia , Alineación de Secuencia , ThermosynechococcusRESUMEN
The monomeric chlorophyll, ChlD1, which is located between the PD1PD2 chlorophyll pair and the pheophytin, PheoD1, 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 *ChlD1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of PD1PD2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the ChlD1+PheoD1- 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 ChlD1 upon QA- formation, indicates that in the T179H-PSII and in the WT*3-PSII, the ChlD1 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, ChlD1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of ChlD1 very likely contribute to the well-known electrochromic shifts observed at ~430â¯nm during the S-state cycle.
Asunto(s)
Proteínas Bacterianas/química , Clorofila/química , Cianobacterias/enzimología , Luz , Complejo de Proteína del Fotosistema II/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Clorofila/genética , Clorofila/metabolismo , Cianobacterias/genética , Transporte de Electrón/fisiología , Mutagénesis Sitio-Dirigida , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
Cytb559 in Photosystem II is a heterodimeric b-type cytochrome. The subunits, PsbE and PsbF, consist each in a membrane α-helix. Mutants were previously designed and studied in Thermosynechococcus elongatus (Sugiura et al., Biochim Biophys Acta 1847:276-285, 2015) either in which an axial histidine ligand of the haem-iron was substituted for a methionine, the PsbE/H23M mutant in which the haem was lacking, or in which the haem environment was modified, the PsbE/Y19F and PsbE/T26P mutants. All these mutants remained active showing that the haem has no structural role provided that PsbE and PsbF subunits are present. Here, we have carried on the characterization of these mutants. The following results were obtained: (i) the Y19F mutation hardly affect the Em of Cytb559, whereas the T26P mutation converts the haem into a form with a Em much below 0 mV (so low that it is likely not reducible by QB-) even in an active enzyme; (ii) in the PsbE/H23M mutant, and to a less extent in PsbE/T26P mutant, the electron transfer efficiency from QA- to QB is decreased; (iii) the lower Em of the QA/QA- couple in the PsbE/H23M mutant correlates with a higher production of singlet oxygen; (iv) the superoxide and/or hydroperoxide formation was not increased in the PsbE/H23M mutant lacking the haem, whereas it was significantly larger in the PsbE/T26P. These data are discussed in view of the literature to discriminate between structural and redox roles for the haem of Cytb559 in the production of reactive oxygen species.
Asunto(s)
Cianobacterias/metabolismo , Grupo Citocromo b/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Cianobacterias/genética , Grupo Citocromo b/química , Grupo Citocromo b/genética , Mutación/genética , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824-6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV-visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking TyrZ and the halide. The V185 contributes to the stabilization of S2 into the low spin (LS), Sâ¯=â¯1/2, configuration. Indeed, in the V185T mutant a high proportion of S2 exhibits a high spin (HS), Sâ¯=â¯5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S1TyrZâ¯ââ¯S2HSTyrZ transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S2LSTyrZâ¯ââ¯S3TyrZ transition was observed and we propose that it occurs in the S2LSTyrZâ¯ââ¯S2HSTyrZ intermediate step before the S2HSTyrZâ¯ââ¯S3TyrZ transition occurs. The dramatic slowdown of the S3TyrZâ¯ââ¯S0TyrZ transition in the V185T mutant does not originate from a structural modification of the Mn4CaO5 cluster since the spin Sâ¯=â¯3â¯S3 EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.
Asunto(s)
Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Valina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Mediciones Luminiscentes , Modelos Moleculares , Synechococcus/metabolismo , Factores de TiempoRESUMEN
Photosynthetic water oxidation takes place at the Mn4CaO5 cluster in photosystem II through a light-driven cycle of intermediates called S states (S0-S4). To unravel the mechanism of water oxidation, it is essential to understand the coupling of electron- and proton-transfer reactions during the S-state transitions. Here, we monitored the reaction process in the S0 â S1 transition using time-resolved infrared (TRIR) spectroscopy. The TRIR signals of the pure contribution of the S0 â S1 transition was obtained by measurement upon a flash after dark adaptation following three flashes. The S0 â S1 traces at the vibrational frequencies of carboxylate groups and hydrogen bond networks around the Mn4CaO5 cluster showed a single phase with a time constant of â¼45 µs. A relatively small H/D kinetic isotope effect of â¼1.2 together with the absence of a slower phase suggests that proton release is coupled with electron transfer, which is a rate-limiting step. The high rate of proton-coupled electron transfer, which is even higher than pure electron transfer in the S1 â S2 transition, is consistent with the previous theoretical prediction that a hydroxo bridge of the Mn4CaO5 cluster gives rise to barrierless deprotonation upon S1 formation through a strongly hydrogen-bonded water molecule.
RESUMEN
Small heat shock protein 27 (HSP27) is an essential element of the proteostasis network in human cells. The HSP27 monomer coexists with the dimer, which can bind unfolded client proteins. Here, we evaluated the in-cell dimer-monomer equilibrium and its relevance to the binding of client proteins in a normal human vascular endothelial cell line. When cells were treated with a membrane-permeable crosslinker, the protein existed primarily as a free monomer (27â¯kDa) with a markedly smaller percentage of dimer (54â¯kDa), hetero-conjugates, and minor smear-like bands. When the protein was crosslinked in a cell-free lysate, two of the hetero-conjugates that were crosslinked in live cells were also detected, but the dimer and other complexes were absent. However, when cells were pretreated with fatty acid (FA) and/or heat (42.5⯰C), dissociation of the dimer was selectively prevented and two types of covalently linked dimers were increased. These changes occurred most prominently in cells treated with docosahexaenoic acid (DHA) and heat, which appeared to intensify the heat resistance of the cell. Both the formation of covalently linked dimers and heat resistance were prevented by N-acetylcysteine. By contrast, nearly all of the free monomers in the lysate converted to disulfide bond-linked dimers by a simple, long incubation at 4⯰C. These results strongly suggest that the monomer-dimer equilibrium of HSP27 was inversed between the in-cell and cell-free systems. Temperature- and amphiphile-regulated dimerization was restricted probably due to the low hydration of the in-cell crowding environment.
Asunto(s)
Ácidos Docosahexaenoicos/farmacología , Proteínas de Choque Térmico HSP27/metabolismo , Respuesta al Choque Térmico/efectos de los fármacos , Calor , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Acetilcisteína/farmacología , Ácido Araquidónico/farmacología , Células Cultivadas , Ácido Eicosapentaenoico/farmacología , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Chaperonas Moleculares , Peso Molecular , Conformación Proteica , Multimerización de Proteína , Relación Estructura-Actividad , Factores de TiempoRESUMEN
In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S2 using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S2LS and S2HS is pH dependent, with a pKaâ¯≈â¯8.3 (nâ¯≈â¯4) for the native Mn4CaO5 and pKaâ¯≈â¯7.5 (nâ¯≈â¯1) for Mn4SrO5. The DFT results suggest that exchanging Ca with Sr modifies the electronic structure of several titratable groups within the active site, including groups that are not direct ligands to Ca/Sr, e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of the pKa upon the Ca/Sr exchange. EPR also showed that NH3 addition reversed the effect of high pH, NH3-S2LS being present at all pH values studied. Absorption spectroscopy indicates that NH3 is no longer bound in the S3TyrZ state, consistent with EPR data showing minor or no NH3-induced modification of S3 and S0. In both Ca-PSII and Sr-PSII, S2HS was capable of advancing to S3 at low temperature (198â¯K). This is an experimental demonstration that the S2LS is formed first and advances to S3via the S2HS state without detectable intermediates. We discuss the nature of the changes occurring in the S2LS to S2HS transition which allow the S2HS to S3 transition to occur below 200â¯K. This work also provides a protocol for generating S3 in concentrated samples without the need for saturating flashes.
Asunto(s)
Cianobacterias/enzimología , Complejo de Proteína del Fotosistema II/química , Agua/química , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/metabolismo , Agua/metabolismoRESUMEN
Photosystem II catalyzes light-induced water oxidation leading to the generation of dioxygen indispensable for sustaining aerobic life on Earth. The Photosystem II reaction center is composed of D1 and D2 proteins encoded by psbA and psbD genes, respectively. In cyanobacteria, different psbA genes are present in the genome. The thermophilic cyanobacterium Thermosynechococcus elongatus contains three psbA genes: psbA1, psbA2, and psbA3, and a new c-type heme protein, Tll0287, was found to be expressed in a strain expressing the psbA2 gene only, but the structure and function of Tll0287 are unknown. Here we solved the crystal structure of Tll0287 at a 2.0 Å resolution. The overall structure of Tll0287 was found to be similar to some kinases and sensor proteins with a Per-Arnt-Sim-like domain rather than to other c-type cytochromes. The fifth and sixth axial ligands for the heme were Cys and His, instead of the His/Met or His/His ligand pairs observed for most of the c-type hemes. The redox potential, E½, of Tll0287 was -255 ± 20 mV versus normal hydrogen electrode at pH values above 7.5. Below this pH value, the E½ increased by ≈57 mV/pH unit at 15 °C, suggesting the involvement of a protonatable group with a pKred = 7.2 ± 0.3. Possible functions of Tll0287 as a redox sensor under microaerobic conditions or a cytochrome subunit of an H2S-oxidizing system are discussed in view of the environmental conditions in which psbA2 is expressed, as well as phylogenetic analysis, structural, and sequence homologies.
Asunto(s)
Proteínas Bacterianas/química , Cianobacterias/química , Hemoproteínas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Cianobacterias/genética , Cianobacterias/metabolismo , Proteínas de Unión al Hemo , Hemoproteínas/genética , Hemoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Dominios ProteicosRESUMEN
Two mutants, D1-H198Q and D1-H198A, have been previously constructed in Thermosynechococcus elongatus with the aim at modifying the redox potential of the P680â¢+/P680 couple by changing the axial ligand of PD1, one the two chlorophylls of the Photosystem II primary electron donor [Sugiura et al., Biochim. Biophys. Acta 1777 (2008) 331-342]. However, after the publication of this work it was pointed out to us by Dr. Eberhard Schlodder (Technische Universität Berlin) that in both mutants the pheophytin band shift which is observed upon the reduction of QA was centered at 544nm instead of 547nm, clearly showing that the D1 protein corresponded to PsbA1 whereas the mutants were supposedly constructed in the psbA3 gene so that the conclusions in our previous paper were wrong. O2 evolving mutants have been therefore reconstructed and their analyze shows that they are now correct mutants which are suitable for further studies. Indeed, the D1-H198Q mutation downshifted by ≈3nm the P680â¢+/P680 difference absorption spectrum in the Soret region and increased the redox potential of the P680â¢+/P680 couple and the D1-H198A mutation decreased the redox potential of the P680â¢+/P680 couple all these effects being comparable to those which were observed in Synechocystis sp. PCC 6803 [Diner et al., Biochemistry 40 (2001) 9265-9281 and Merry et al. Biochemistry 37 (1998) 17,439-17,447]. We apologize for having presented wrong data and wrong conclusions in our earlier publication.
RESUMEN
The 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 close to a redox-active tyrosine residue (YZ). Cl- is also required for enzyme activity. By using EPR spectroscopy it has been shown that both Ca2+/Sr2+ exchange and Cl-/I- exchange perturb the proportions of centers showing high (S=5/2) and low spin (S=1/2) forms of the S2-state. The S3-state was also found to be heterogeneous with: i) a S=3 form that is detectable by EPR and not sensitive to near-infrared light; and ii) a form that is not EPR visible but in which Mn photochemistry occurs resulting in the formation of a (S2YZ)' split EPR signal upon near-infrared illumination. In Sr/Cl-PSII, the high spin (S=5/2) form of S2 shows a marked heterogeneity with a g=4.3 form generated at low temperature that converts to a relaxed form at g=4.9 at higher temperatures. The high spin g=4.9 form can then progress to the EPR detectable form of S3 at temperatures as low as 180K whereas the low spin (S=1/2) S2-state can only advance to the S3 state at temperatures≥235 K. Both of the two S2 configurations and the two S3 configurations are each shown to be in equilibrium at ≥235 K but not at 198 K. Since both S2 configurations are formed at 198 K, they likely arise from two specific populations of S1. The existence of heterogeneous populations in S1, S2 and S3 states may be related to the structural flexibility associated with the positioning of the oxygen O5 within the cluster highlighted in computational approaches and which has been linked to substrate exchange. These data are discussed in the context of recent in silico studies of the electron transfer pathways between the S2-state(s) and the S3-state(s).