Qc Site Research Articles

Structural insights into the action mechanisms of artificial electron acceptors in photosystem II

Photosystem II (PSII) utilizes light energy to split water, and the electrons extracted from water are transferred to QB, a plastoquinone molecule bound to the D1 subunit of PSII. Many artificial electron acceptors (AEAs) with molecular structures similar to that of plastoquinone can accept electrons from PSII. However, the molecular mechanism by which AEAs act on PSII is unclear. Here, we solved the crystal structure of PSII treated with three different AEAs, 2,5-dibromo-1,4-benzoquinone, 2,6-dichloro-1,4-benzoquinone, and 2-phenyl-1,4-benzoquinone, at 1.95 to 2.10Å resolution. Our results show that all AEAs substitute for QB and are bound to the QB-binding site (QB site) to receive electrons, but their binding strengths are different, resulting in differences in their efficiencies to accept electrons. The acceptor 2-phenyl-1,4-benzoquinone binds most weakly to the QB site and showed the highest oxygen-evolving activity, implying a reverse relationship between the binding strength and oxygen-evolving activity. In addition, a novel quinone-binding site, designated the QD site, was discovered, which is located in the vicinity of QB site and close to QC site, a binding site reported previously. This QD site is expected to play a role as a channel or a storage site for quinones to be transported to the QB site. These results provide the structural basis for elucidating the actions of AEAs and exchange mechanism of QB in PSII and also provide information for the design of more efficient electron acceptors.

Regulation of light energy conversion between linear and cyclic electron flow within photosystem II controlled by the plastoquinone/quinol redox poise.

Ultrapurified Photosystem II complexes crystalize as uniform microcrystals (PSIIX) of unprecedented homogeneity that allow observation of details previously unachievable, including the longest sustained oscillations of flash-induced O2 yield over > 200 flashes and a novel period-4.7 water oxidation cycle. We provide new evidence for a molecular-based mechanism for PSII-cyclic electron flow that accounts for switching from linear to cyclic electron flow within PSII as the downstream PQ/PQH2 pool reduces in response to metabolic needs and environmental input. The model is supported by flash oximetry of PSIIX as the LEF/CEF switch occurs, Fourier analysis of O2 flash yields, and Joliot-Kok modeling. The LEF/CEF switch rebalances the ratio of reductant energy (PQH2) to proton gradient energy (H+o/H+i) created by PSII photochemistry. Central to this model is the requirement for a regulatory site (QC) with two redox states in equilibrium with the dissociable secondary electron carrier site QB. Both sites are controlled by electrons and protons. Our evidence fits historical LEF models wherein light-driven water oxidation delivers electrons (from QA-) and stromal protons through QB to generate plastoquinol, the terminal product of PSII-LEF in vivo. The new insight is the essential regulatory role of QC. This site senses both the proton gradient (H+o/H+i) and the PQ pool redox poise via e-/H+ equilibration with QB. This information directs switching to CEF upon population of the protonated semiquinone in the Qc site (Q-H+)C, while the WOC is in the reducible S2 or S3 states. Subsequent photochemical primary charge separation (P+QA-) forms no (QH2)B, but instead undergoes two-electron backward transition in which the QC protons are pumped into the lumen, while the electrons return to the WOC forming (S1/S2). PSII-CEF enables production of additional ATP needed to power cellular processes including the terminal carboxylation reaction and in some cases PSI-dependent CEF.

Structural basis for safe and efficient energy conversion in a respiratory supercomplex

Proton-translocating respiratory complexes assemble into supercomplexes that are proposed to increase the efficiency of energy conversion and limit the production of harmful reactive oxygen species during aerobic cellular respiration. Cytochrome bc complexes and cytochrome aa3 oxidases are major drivers of the proton motive force that fuels ATP generation via respiration, but how wasteful electron- and proton transfer is controlled to enhance safety and efficiency in the context of supercomplexes is not known. Here, we address this question with the 2.8 Å resolution cryo-EM structure of the cytochrome bcc-aa3 (III2-IV2) supercomplex from the actinobacterium Corynebacterium glutamicum. Menaquinone, substrate mimics, lycopene, an unexpected Qc site, dioxygen, proton transfer routes, and conformational states of key protonable residues are resolved. Our results show how safe and efficient energy conversion is achieved in a respiratory supercomplex through controlled electron and proton transfer. The structure may guide the rational design of drugs against actinobacteria that cause diphtheria and tuberculosis.

Optimizing combining green manures and pelletized manure for organic spring wheat production

Well-planned crop rotations and targeted use of organic amendments are critical to success in organic wheat production. The impact of green manure (GMr) type, GMr termination timing, and “Acti-Sol” [pelletized dehydrated poultry manure (DPM); 5-2-3] on organic wheat productivity and quality was evaluated from 2014 to 2016 in Truro, NS, and Saint-Mathieu-de-Beloeil, QC. Crops prior to wheat were soybean or GMr of hairy vetch/oat (HVO), common vetch/oat (CVO), and red clover (RC) (NS site), and HVO, red clover/oat (RCO), and oat (QC site). Trials were split-split-plot designs with treatments of precrops, GMr termination (fall vs. spring), and DPM at 0, 40, 80, and 120 kg total N ha−1. Wheat yields ranged from 1500 to 1800 kg ha−1 if unfertilized with DPM and following soybean or oat precrops. All legume GMrs (HVO, CVO, and RC/RCO) and DPM applications increased grain yield (2000–4200 kg ha−1) and protein content (13%–16%), wheat total N uptake [49–60 kg N ha−1 (QC); 87–125 kg N ha−1 (NS)] and soil mineral N content mid-season and postharvest, and responses were consistently greatest following HVO. Timing of GMr incorporation largely had no impact. Applying DPM at 80 kg N ha−1 was an effective substitute for a GMr precrop.

Mutations of Cytochrome b559 and PsbJ on and near the QC Site in Photosystem II Influence the Regulation of Short-Term Light Response and Photosynthetic Growth of the Cyanobacterium Synechocystis sp. PCC 6803.

The characteristic features of two types of short-term light adaptations of the photosynthetic apparatus of the cyanobacterium Synechocystis sp. PCC 6803, state transition and blue-green light-induced fluorescence quenching, were compared in wild-type and cytochrome b559 and PsbJ mutant cells with mutations on and near the QC site in photosystem II (PSII). All mutant cells grew photoautotrophically and assembled stable PSII. Thermoluminescence emission experiments showed a decrease in the stability of the S3QB(-)/S2QB(-) charge pairs in the A16FJ, S28Aβ, and V32Fβ mutant cells. When dark-adapted wild-type and mutant cells were illuminated by medium-intensity blue light, the increase in the PSII fluorescence yield (indicating a transition to state 1) was more prominent in mutant than wild-type cells. Strong blue-light conditions induced a quenching of fluorescence corresponding to nonphotochemical fluorescence quenching (NPQ). The extension of NPQ decreased significantly in the mutants, and the kinetics appeared to be affected. When similar measures were repeated on an orange carotenoid protein (OCP)-deficient background, little or no quenching was observed, which confirms that the decrease in fluorescence under strong blue light corresponded to the OCP-dependent NPQ. Immunoblot results showed that the attenuated effect of blue light-induced NPQ in mutant cells was not due to a lack of OCP. Photosynthetic growth and biomass production were greater for A16FJ, S28Aβ, and V32Fβ mutant cells than for wild-type cells under normal growth conditions. Our results suggest that mutations of cytochrome b559 and PsbJ on and near the QC site of PSII may modulate the short-term light response in cyanobacteria.

Oxidation of plastohydroquinone by photosystem II and by dioxygen in leaves

In sunflower leaves linear electron flow LEF=4O2 evolution rate was measured at 20ppm O2 in N2. PSII charge separation rate CSRII=aII∙PAD∙(Fm−F)/Fm, where aII is excitation partitioning to PSII, PAD is photon absorption density, Fm and F are maximum and actual fluorescence yields. Under 630nm LED+720nm far-red light (FRL), LEF was equal to CSRII with aII=0.51 to 0.58. After FRL was turned off, plastoquinol (PQH2) accumulated, but LEF decreased more than accountable by F increase, indicating PQH2-oxidizing cyclic electron flow in PSII (CEFII). CEFII was faster under conditions requiring more ATP, consistent with CEFII being coupled with proton translocation. We propose that PQH2 bound to the QC site is oxidized, one e− moving to P680+, the other e− to Cyt b559. From Cyt b559 the e− reduces QB− at the QB site, forming PQH2. About 10–15% electrons may cycle, causing misses in the period-4 flash O2 evolution and lower quantum yield of photosynthesis under stress. We also measured concentration dependence of PQH2 oxidation by dioxygen, as indicated by post-illumination decrease of Chl fluorescence yield. After light was turned off, F rapidly decreased from Fm to 0.2 Fv, but further decrease to F0 was slow and O2 concentration dependent. The rate constant of PQH2 oxidation, determined from this slow phase, was 0.054s−1 at 270μM (21%) O2, decreasing with Km(O2) of 60μM (4.6%) O2. This eliminates the interference of O2 in the measurements of CEFII.

Evidence for the involvement of loosely bound plastosemiquinones in superoxide anion radical production in photosystem II.

Recent evidence has indicated the presence of novel plastoquinone-binding sites, QC and QD, in photosystem II (PSII). Here, we investigated the potential involvement of loosely bound plastosemiquinones in superoxide anion radical (O2 •−) formation in spinach PSII membranes using electron paramagnetic resonance (EPR) spin-trapping spectroscopy. Illumination of PSII membranes in the presence of the spin trap EMPO (5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide) resulted in the formation of O2 •−, which was monitored by the appearance of EMPO-OOH adduct EPR signal. Addition of exogenous short-chain plastoquinone to PSII membranes markedly enhanced the EMPO-OOH adduct EPR signal. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, the EMPO-OOH adduct EPR signal was suppressed by 50% when the urea-type herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) was bound at the QB site. However, the EMPO-OOH adduct EPR signal was enhanced by binding of the phenolic-type herbicide dinoseb (2,4-dinitro-6-sec-butylphenol) at the QD site. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, DCMU and dinoseb inhibited photoreduction of the high-potential form of cytochrome b 559 (cyt b 559). Based on these results, we propose that O2 •− is formed via the reduction of molecular oxygen by plastosemiquinones formed through one-electron reduction of plastoquinone at the QB site and one-electron oxidation of plastoquinol by cyt b 559 at the QC site. On the contrary, the involvement of a plastosemiquinone formed via the one-electron oxidation of plastoquinol by cyt b 559 at the QD site seems to be ambiguous. In spite of the fact that the existence of QC and QD sites is not generally accepted yet, the present study provided more spectroscopic data on the potential functional role of these new plastoquinone-binding sites.

Differences in the Interactions between the Subunits of Photosystem II Dependent on D1 Protein Variants in the Thermophilic Cyanobacterium Thermosynechococcus elongatus

The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA(1) or the psbA(3) gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491-1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b(559) (Cyt b(559)) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/ΔPsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b(559) properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/ΔPsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b(559) likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

The functional role of cytochrome (cyt) b(559) in photosystem II (PSII) was investigated in H22K alpha and Y18S alpha cyt b(559) mutants of the cyanobacterium Synechocystis sp. PCC6803. H22K alpha and Y18S alpha cyt b(559) mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b(559) in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b(559) in H22K alpha mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b(559) in Y18S alpha mutant PSII core complexes was shifted to 561 nm. Y18S alpha and H22K alpha mutant PSII core complexes contained predominately the low potential form of cyt b(559). The findings lend support to the concept that the redox properties of cyt b(559) are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b(559) mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b(559) in protection of PSII under photoinhibition conditions in vivo.

Electrogenic events in chromatophores fromRhodobacter sphaeroides lacking high-potential cytochrome b of thebc1-complex

The ubiquinol:cytochrome c oxidoreductase (bc tcomplex), contains a cytochrome (cyt) b subunit, which contributes to both quinol oxidizing (Qz) and quinone reductase (Qc) sites. As shown by electrochromic measurements [1,2], the antimycin-insensitive electrogenic reaction linked to electron transfer between cyt bj and b h contributes about 50% of the total electrogenesis of the bcrcomplex. The nature of the remaining antimycin-sensitive electrogenic step is controversial. The antimycin sensitivity of both the forward and the reverse (in the presence of myxothiazol) reaction shows that the phase is linked to reduction of Q at the Qc site by cyt b h [1-5]. Direct electrometry [3] suggests that the electron transfer between b h and Qc is not electrogenic. Probably, the antimycin-sensitive electrogenesis is at least partially due to the protonation that accompanies the reduction of QcAnother possible electrogenic step could be the release of protons, trapped at the Qz site on oxidation of QH 2, as b, becomes oxidized [6]. The goal of the present work is to clarify these points by measurement of the flash-induced electrical potential difference (A~0) generated by chromatophores from Rhodobacter sphaeroides mutants which have lost the heme of cyt b,. Replacement of the axial ligands of cyt b, (His-lll by either Asp (Hl l lD) or Asn (Hll lN), or His-212 by Asp (H212D)) resulted in selective loss of cyt b, but retention of cyt b~ [7]. Flash kinetic studies of the H l l l N mutant showed that the

On an Inhibitor of Ferredoxin Catalyzed Cyclic Photophosphorylation in Thylakoid Membranes*

AbstractDibromo‐ and diiodo‐naphthoquinones are shown to be inhibitors of the cytochrome b6/f complex in isolated thylakoid membranes from spinach chloroplasts. Dibromo‐naphthoquinone inhibits ferredoxin catalyzed cyclic photophosphorylation at 0.1 μM concentrations, but non cyclic e‐flow only at 10 μM. It does not inhibit cyclic systems with artifical cofactors, nor non‐cyclic electron flow from duroquinol through photosystem I via the cytochrome b6/f complex. Dibromo‐naphthoquinone does however, lower the stoichiometry for ATP formation in the duroquinol donor system. This inhibitory pattern is quite different from that of DBMIB, but very similar to that of antimycin. This antimycin‐like behaviour of these inhibitors is interpreted to indicate a) the existence of a Qc site in the cytochrome b6/f complex and its obligate function in ferredoxin catalyzed cyclic electron flow and b) a non‐essential role of the Qc site in non‐cyclic electron flow, but which — when operative — pumps an extra proton across the thylakoid membrane increasing the ATP yield.