High Potential Form Of Cytochrome B-559 Research Articles

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.

Differential mechanism of light-induced and oxygen-dependent restoration of the high-potential form of cytochrome b559 in Tris-treated Photosystem II membranes

The effect of illumination and molecular oxygen on the redox and the redox potential changes of cytochrome b 559 (cyt b 559) has been studied in Tris-treated spinach photosystem II (PSII) membranes. It has been demonstrated that the illumination of Tris-treated PSII membranes induced the conversion of the intermediate-potential (IP) to the reduced high-potential (HP Fe2+) form of cyt b 559, whereas the removal of molecular oxygen resulted in the conversion of the IP form to the oxidized high-potential (HP Fe3+) form of cyt b 559. Light-induced conversion of cyt b 559 from the IP to the HP form was completely inhibited above pH 8 or by the modification of histidine ligand that prevents its protonation. Interestingly, no effect of high pH or histidine modification was observed during the conversion of the IP to the HP form of cyt b 559 after the removal of molecular oxygen. These results indicate that conversion from the IP to the HP form of cyt b 559 proceeds via different mechanisms. Under illumination, conversion of the IP to the HP form of cyt b 559 depends primarily on the protonation of the histidine residue, whereas under anaerobic conditions, the conversion of the IP to the HP form of cyt b 559 is driven by higher hydrophobicity of the environment around the heme iron resulting from the absence of molecular oxygen.

Interaction of α-tocopherol quinone, α-tocopherol and other prenyllipids with photosystem II

We have found that plastoquinone-A (PQ-A) and α-tocopherol (α-Toc) increased the reduction level of the high-potential form of cytochrome b-559 (cyt. b-559 HP) and α-tocopherol quinone (α-TQ) decreased the level of this cytochrome form in Scenedesmus obliquus wild-type, while the investigated prenyllipids were not active in the restoration of the cyt. b-559 HP form in Scenedesmus PS28 mutant and Synechococcus 6301 ( Anacystis nidulans) where the cyt. b-559 HP form is naturally not present. Among the tested prenyllipids, α-TQ quenched fluorescence in thylakoids of S. obliquus wild-type, the PS28 mutant and tobacco to the highest extent, while PQ-A was less effective in this respect. α-Tocopherol showed the opposite effect to α-TQ and it was rather small. The fluorescence quenching measurements of thylakoids in the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) showed that the α-Toc and FCCP (carbonylcyanide-p-trifluoromethoxy-phenyl-hydrazone) did not quench non-photochemically chlorophyll fluorescence while PQ-9 and α-TQ were effective fluorescence quenchers at higher concentrations (> 15 μM). However, at the lower α-TQ concentrations where its effective fluorescence quenching was found in DCMU-free samples, there was nearly no quenching effect by α-TQ observed in DCMU-treated thylakoids. This suggested a specific, not non-photochemical, DCMU sensitive, fluorescence quenching of photosystem II (PSII) at low α-TQ concentrations which is probably connected with the cyclic electron transport around PSII and might have a function of excess light energy dissipation. The effects of α-TQ on PSII resembled those of FCCP under many respects which might suggest similar mechanism of action of these compounds on PSII, i.e. the catalytic deprotonation and/or redox changes of some components of PSII such as the water splitting system, tyrosine D, Chl z or cytochrome b-559.

Restoration of the high-potential form of cytochrome b559 of photosystem II occurs via a two-step mechanism under illumination in the presence of manganese ions

Spinach photosystem II membranes that had been depleted of the Mn cluster contained four forms of cytochrome (Cyt) b559, namely, high-potential (HP), HP′, intermediate-potential (IP) and low-potential (LP) forms that exhibited the redox potentials of +400, +310, +170 and +35 mV, respectively, in potentiometric titration. When the membranes were illuminated with flashing light in the presence of 0.1 mM Mn 2+, the IP form was converted to the HP′ form by two flashes and then the HP′ form was converted to the HP form by an additional flash. The quantum efficiency of the first conversion appeared to be quite high since the conversion was almost complete after two flashes. By contrast, the second conversion proceeded with low quantum efficiency and 40 flashes were required for completion. The effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) suggested that the first conversion did not require electron transfer from Q A to Q B while the second conversion had an absolute requirement for it. It was also suggested that the first conversion involved the reduction of the heme of Cyt b559, probably by Q A −, and we propose that direct reduction by Q A − induces a shift in the redox potential of the heme. The second conversion was also accompanied by the reduction of heme but it appeared that this conversion did not necessarily involve the reduction. The effects of DCMU on the reduction of heme suggested that the heme became reducible by Q B − after the first conversion had been completed. This observation implies that the efficiency of electron transfer from Q A to Q B increased upon the conversion of the IP form to the HP′ form, and we propose that restoration of the high-potential forms of Cyt b559 itself acts to make the acceptor side of photosystem II functional.

Restoration of the high-potential form of cytochrome b-559 by electron transport reactions through Photosystem II in Tris-treated Photosystem II membranes

The conversion of cytochrome (Cyt) b-559 from the low-potential (LP) to the high-potential (HP) form under conditions for photoactivation of O 2 evolution (reconstitution of the Mn cluster) was investigated using Photosystem II (PS II) membranes that had been depleted of the Mn cluster by treatment with Tris. Illumination of the PS II membranes with continuous or flashing light in the presence of 0.1 mM Mn 2+ reactivated O 2 evolution and increased the level of the HP form of Cyt b-559 with a concomitant decrease in the level of the LP form. When illumination was achieved with flashing light, the restoration of the HP form occurred after two flashes, while reactivation of O 2 evolution required more than six flashes. It was also found that the HP form could be restored when the PS II membranes were illuminated in the presence of artificial electron donors instead of Mn 2+. NH 2OH (10–100 μM), 1,5-diphenylcarbazide (50–100 μM) and semicarbazide (0.5–1 mM) were effective in restoring the HP form. These observations suggest that, under photoactivation conditions, not the reconstitution of the Mn cluster but electron donation by Mn 2+ to PS II is responsible for the restoration of the HP form. The restoration of the HP form by illumination in the presence of Mn 2+ was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) but it was completely suppressed by artificial electron acceptors which bind to the Q B site and reoxidize Q A −, namely, 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB) and 2,6-dichloro-1,4-benzoquinone. These results suggest that some redox reaction(s) at the acceptor side of PS II, which probably involves Q A −, occurs during the course of the restoration of the HP form.

Acridones: a chemically new group of protonophores.

Although the interaction of proton-conducting ionophores (protonophores) with photosynthetic electron transport has been extensively studied during the past decade, the mode of action of protonophores remained uncertain. For a better understanding of the molecular mechanism of the action of protonophores, the introduction of chemically new types of molecules will be required. In this work, we demonstrate that acridones (9-azaanthracene-10-ones) completely fulfill this requirement. At low concentrations of acridones, the thermoluminescence bands at +20 degrees C and +10 degrees C were strongly inhibited, while normal electron transport activity was retained. This indicates that the concentrations of S2 and S3 states involved in the generation of these bands are reduced. At higher concentrations, an increased activity of electron transport was observed, which is attributed to the typical uncoupler effect of protonophores. Indeed, acridones accelerate the decay of the electrochromic absorbance change at 515 nm and also inhibit the generation of the transmembrane proton gradient, measured as an absorbance transient of neutral red. Variable fluorescence induction was quenched even at low concentrations of acridones but was restored by either a long-term illumination or high light intensity. Acridones, similarly to other protonophores, promoted the autooxidation of the high-potential form of cytochrome b559 and partially converted it to lower potential forms. These results suggest that acridones, acting as typical protonophores, uncouple electron transport, accelerate the deactivation of the S2 and S3 states on the donor side, and facilitate the oxidation of cytochrome b559 on the acceptor side of photosystem II.

Two populations of the high-potential form of cytochrome b-559 in chloroplasts treated with 2-(3-chloro-4-trifluoromethyl)amino-3,5-dinitrothiophene (Ant 2p)

Two populations of the high-potential form of cytochrome b-559 (cyt b-559HP) can be distinguished in chloroplasts. In the presence of ≥ 1 μM Ant 2p [2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene] about one-half of the cyt b-559 is autooxidized in the dark. This fraction of cyt b-559HP undergoes rapid (t12≈2 ms) photoreduction followed by reoxidation in the dark with t12 100–200 ms. Photoreduction can be inhibited by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and UHDBT [5-(n-undecyl)-6-hydroxy-4,7-dioxobezothiazole] but not by DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinine), which suggests the involvement of QB or the plastoquinone pool. Another fraction of cyt b-559HP undergoes slow (t12 ≥ 1 s), irreversible photooxidation in the presence of ≥ 0.1 μM Ant 2p. The presently available data suggest that the heterogeneity of cyt b-559HP originates from PS IIα centers.

The structure and function of the thylakoid membrane

Thylakoid membranes are laterally differentiated into appressed and non-appressed regions called grana and stroma lamellae. Pure stroma lamellae isolated from wild type maize and barley leaves contain photosystem I and its light-harvesting antennae, the cytochromeb6/f complex and coupling factor. Maize stroma lamellae contained only 2% of the total photosystem II polypeptides found in whole thylakoids, and most of the small amount of the photosystem II light-harvesting complex (LHCII) was associated with photosystem I. These results were consistent with the low rates of photosystem II electron transport and low levels of the high potential form of cytochromeb-559. Immune blot assays indicated that about half of the low potential form of cytochromeb-559 in stroma lamellae was antigenically distinct from that derived from the high potential form. The amount of LHCII in stroma lamellae could be increased by exposing leaves to bright white light (state 2) prior to the isolation of stromal lamellae. This LHCII caused a 15% increase in photosystem I antenna size and was different from the LHCII found in grana lamellae, since it lacked a 26 kD polypeptide possibly involved in thylakoid appression. These results demonstrate that the migration of LHCII from appressed to non-appressed lamellae as a result of changes in the relative amounts of energy absorbed by the 2 different photosystems, also occurs in vivo.

Differential inhibition by plastoquinone analogues of photoreduction of cytochrome b-559 in chloroplasts

The high-potential form of cytochrome b-559 ( b-559 HP) is closely linked to the oxygenic photosystem (photosystem II) but its relation to other redox components of the photosynthetic apparatus, including plastoquinone, is still obscure. We investigated the photoreduction of cytochrome b-559 HP by isolated chloroplasts in the presence of 3 antagonists of plastoquinone, of which, DBMIB (dibromothymoquinone) and DNP-INT (dinitrophenyl ether of iodonitrothymol) are known to inhibit the oxidation of the plastoquinone pool (PQ) by the FeS-cytochrome ƒ/ b 6 complex and one, UHDBT (5- n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) is known to inhibit the reduction of PQ by Q B.Q B is a protein-bound plastoquinone that serves as a two-electron gate for the reduction of PQ. We found that DBMIB and DNP-INT did not inhibit but low concentrations of UHDBT severely inhibited the photoreduction of cytochrome b-559 HP. These results suggest that the electron donor for the reduction of cytochrome b-559 HP was either Q B or a portion of the PQ pool that was oxidized by a new pathway free of binding sites for DBMIB and DNP-INT.

Quantitative analysis of membrane components in a highly active O 2-evolving photosystem II preparation from spinach chloroplasts

Stoichiometry of membrane components associated with Photosystem II was determined in a highly active O 2-evolving Photosystem II preparation isolated from spinach chloroplasts by the treatment with digitonin and Triton X-100. From the analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Triton X-114 phase partitioning, the preparation was shown to contain the reaction center protein (43 kDa), the light-harvesting chlorophyll-protein complex (the main band, 27 kDa), the herbicide-binding protein (32 kDa) and cytochrome b-559 (10 kDa) as hydrophobic proteins, and three proteins (33, 24 and 18 kDa) which probably constitute the O 2-evolution enzyme complex as hydrophilic proteins. These proteins were associated stoichiometrically with the Photosystem II reaction center: one Photosystem II reaction center, approx. 200 chlorophyll, one high-potential form of cytochrome b-559, one low-potential form of cytochrome b-559, one 33 kDa protein, one (to two) 24 kDa protein and one (to two) 18 kDa protein. Measurement of fluorescence induction showed the presence of three electron equivalents in the electron acceptor pool on the reducing side of Photosystem II in our preparation. Three molecules of plastoquinone A were detected per 200 chlorophyll molecules with high-performance liquid chromatography. The Photosystem II preparation contained four managanese atoms per 200 chlorophyll molecules.

La photooxydation du cytochrome b-559, en presence de [formula omitted] et de 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone, ou de p-benzoquinone, chez trois mutants non-photosynthétiques de Chlamydomonas reinhardti

Cytochrome b-559 photooxidation in the presence of carbonyl cyanide p-trifluorometh-oxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone or p-benzoquinone in three non-photosynthetic mutants of Chlamydomonas reinhardti Studies of absorbance changes related to the cytochrome b-559 photooxidation induced by FCCP, with and without addition of 3- p-chlorophenyl-1, 1-dimethylurea (CMU), DBMIB or p-benzoquinone, in whole cells and in chloroplast fragments of Chlamydomonas reinhardti, were carried out. In addition to the wild type, three strains of non-photosynthetic mutants were used: Fl 5, which lacks P 700; Fl 9 and Fl 15, which are deficient in bound cytochrome c-553 and in cytochrome b-563. In the presence of FCCP, whole cells and chloroplast fragments of the four strains showed a System II-dependent photooxidation of cytochrome b-559. This photooxidation was inhibited by CMU but it occurred again in presence of FCCP, CMU and DBMIB. In chloroplast fragments, cytochrome b-559 photooxidation was also inhibited by an excess of FCCP; it was recovered, likewise, by addition of DBMIB. In whole cells, the highest measured redox changes were: 1 μmol oxidized cytochrome b-559 per 1 mmol chlorophyll, corresponding approximately to about one seventh (wild type, Fl 5) or one fifth ( Fl 9, Fl 15) of the total amount of this cytochrome. Another kind of cytochrome b-559 photooxidation, CMU-insensitive, also occurred in the mutants Fl 9 and Fl 15 and in the wild type, but not in the mutant Fl 5. This latter kind of photooxidation was observed with chloroplast fragments in the presence of FCCP and CMU and also with whole cells in the presence of FCCP, CMU and p-benzoquinone. These reactions can be attributed to the Photosystem I; they do not require the intervention of the cytochrome c-553. A high-potential form of cytochrome b-559, hydroquinone-reducible, was involved in these two kinds of photooxidation. In addition, a lower potential form, reducible only by ascorbate, appeared to be able to interfere also. An interpretation is attempted, taking into consideration the various effects of FCCP and DBMIB, at different concentrations, on photosynthetic electron transport.