Cytochrome B559 In Photosystem Research Articles

New Structural and Mechanistic Insights Into Functional Roles of Cytochrome b 559 in Photosystem II.

Cytochrome (Cyt) b559 is a key component of the photosystem II (PSII) complex for its assembly and proper function. Previous studies have suggested that Cytb559 has functional roles in early assembly of PSII and in secondary electron transfer pathways that protect PSII against photoinhibition. In addition, the Cytb559 in various PSII preparations exhibited multiple different redox potential forms. However, the precise functional roles of Cytb559 in PSII remain unclear. Recent site-directed mutagenesis studies combined with functional genomics and biochemical analysis, as well as high-resolution x-ray crystallography and cryo-electron microscopy studies on native, inactive, and assembly intermediates of PSII have provided important new structural and mechanistic insights into the functional roles of Cytb559. This mini-review gives an overview of new exciting results and their significance for understanding the structural and functional roles of Cytb559 in PSII.

Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b559 in Photosystem II

Photosystem II (PSII), a large pigment protein complex, undergoes rapid turnover under natural conditions. During assembly of PSII, oxidative damage to vulnerable assembly intermediate complexes must be prevented. Psb28, the only cytoplasmic extrinsic protein in PSII, protects the RC47 assembly intermediate of PSII and assists its efficient conversion into functional PSII. Its role is particularly important under stress conditions when PSII damage occurs frequently. Psb28 is not found, however, in any PSII crystal structure, and its structural location has remained unknown. In this study, we used chemical cross-linking combined with mass spectrometry to capture the transient interaction of Psb28 with PSII. We detected three cross-links between Psb28 and the α- and β-subunits of cytochrome b559, an essential component of the PSII reaction-center complex. These distance restraints enable us to position Psb28 on the cytosolic surface of PSII directly above cytochrome b559, in close proximity to the QB site. Protein-protein docking results also support Psb28 binding in this region. Determination of the Psb28 binding site and other biochemical evidence allow us to propose a mechanism by which Psb28 exerts its protective effect on the RC47 intermediate. This study also shows that isotope-encoded cross-linking with the "mass tags" selection criteria allows confident identification of more cross-linked peptides in PSII than has been previously reported. This approach thus holds promise to identify other transient protein-protein interactions in membrane protein complexes.

New interpretation of the redox properties of cytochrome b559 in photosystem II.

A model of heme-quinone redox interaction has been developed for cytochrome b559 in photosystem II. The quinone QC in the singly protonated form may function as an interacting quinone. The electrostatic effect between the charges on the heme iron of the cytochrome and QCH leads to appearance of three forms of the cytochrome with different redox potentials. A simple and effective mechanism of redox regulation of the electron transfer pathways in photosystem II is proposed.

Investigating the photoprotective role of cytochrome b-559 in photosystem II in a mutant with altered ligation of the haem.

Despite many years of study, the physiological role of cytochrome b-559 (Cyt b-559) within the photosystem II (PSII) complex still remains unclear. Here we describe the analysis of a mutant of the green alga Chlamydomonas reinhardtii in which the His ligand to the haem, provided by the alpha subunit, has been replaced by a Cys residue. The mutant is unable to grow photoautotrophically but can assemble oxygen-evolving PSII supercomplexes to 15-20% of the levels found in the wild-type control. Haem is still detected in the isolated PSII supercomplexes but at sub-stoichiometric levels consistent with weaker binding to the mutated cytochrome. Analysis of PSII activity in cells indicates slowed electron transfer in the mutant between plastoquinones QA and QB. We show that PSII activity in the mutant is more sensitive to chronic photoinhibition than the WT control because of two effects: a faster rate of damage and an impaired PSII repair cycle at the level of synthesis and/or incorporation of D1 into PSII. We also demonstrate that Cyt b-559 plays a role during the critical stage of assembling the Mn4CaO5 cluster. Overall we conclude that Cyt b-559 optimises electron transfer on the acceptor side of PSII and plays physiologically important roles in the assembly, repair and maintenance of the complex.

Towards an understanding of the nature of the redox forms of cytochrome b559 in photosystem II

Towards an understanding of the nature of the redox forms of cytochrome b559 in photosystem II

Spectroscopic and functional characterization of cyanobacterium Synechocystis PCC 6803 mutants on the cytoplasmic-side of cytochrome b559 in photosystem II

We performed spectroscopic and functional characterization on cyanobacterium Synechocystis PCC6803 with mutations of charged residues of the cytoplasmic side of cytochrome (Cyt) b559 in photosystem II (PSII). All of the mutant cells grew photoautotrophically and assembled stable PSII. However, R7Eα, R17Eα and R17Lβ mutant cells grew significantly slower and were more susceptible to photoinhibition than wild-type cells. The adverse effects of the arginine mutations on the activity and the stability of PSII were in the following order (R17Lβ>R7Eα>R17Eα and R17Aα). All these arginine mutants exhibited normal period-four oscillation in oxygen yield. Thermoluminescence characteristics indicated a slight decrease in the stability of the S3QB−/S2QB− charge pairs in the R7Eα and R17Lβ mutant cells. R7Eα and R17Lβ PSII core complexes contained predominantly the low potential form of Cyt b559. EPR results indicated the displacement of one of the two axial ligands to the heme of Cyt b559 in R7Eα and R17Lβ mutant reaction centers. Our results demonstrate that the electrostatic interactions between these arginine residues and the heme propionates of Cyt b559 are important to the structure and redox properties of Cyt b559. In addition, the blue light-induced nonphotochemical quenching was significantly attenuated and its recovery was accelerated in the R7Lα and R17Lβ mutant cells. Furthermore, ultra performance liquid chromatography–mass spectrometry results showed that the PQ pool was more reduced in the R7Eα and R17Lβ mutant cells than wild-type cells in the dark. Our data support a functional role of Cyt b559 in protection of PSII under photoinhibition conditions in vivo.

A unique regulation of the expression of the psbA, psbD, and psbE genes, encoding the D1, D2 and cytochrome b559 subunits of the Photosystem II complex in the chlorophyll d containing cyanobacterium Acaryochloris marina

Photosynthetic electron transport, chromatic photoacclimation and expression of the genes encoding the D1, D2, and cytochrome b559 subunits of the Photosystem II complex were studied in the chlorophyll d containing cyanobacterium Acaryochloris marina MBIC11017 under various environmental conditions. During oxygen deprivation and inhibition of photosynthetic electron transport by dibromothymoquinone the psbA1 gene encoding a D1′ isoform was induced. All of the three psbA and one of the three psbD (psbD2) genes, encoding two different isoforms of the D1 and the abundant isoform of the D2 proteins, respectively were induced under exposure to UV-B radiation and high intensity visible light. Under far red light the amount of Photosystem II complexes increased, and expression of the psbE2 gene encoding the alpha-subunit of cytochrome b559 was enhanced. However, the psbF and psbE1 genes encoding the beta- and another isoform of alpha-cytochrome b559, respectively remained lowly expressed under all conditions. Far red light also induced the psbD3 gene encoding a D2′ isoform whose primary structure is different from the abundant D2 isoform. psbD3 was also induced under low intensity visible light, when chromatic photoacclimation was indicated by a red-shifted absorption of chlorophyll d. Our results show that differential expression of multigene families encoding different isoforms of D1 and D2 plays an important role in the acclimation of A. marina to contrasting environmental conditions. Moreover, the disproportionate quantity of transcripts of the alpha and beta subunits of cytochrome b559 implies the existence of an alpha–alpha homodimer organization of cytochrome b559 in Photosystem II complexes.

Properties of photoreduction reaction of cytochrome b559 in photosystem II membrane fragments

Properties of photoreduction reaction of cytochrome b559 in photosystem II membrane fragments

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.

Superoxide oxidase and reductase activity of cytochrome b559 in photosystem II

This study provides evidence for the superoxide oxidase and the superoxide reductase activity of cytochrome b 559 (cyt b 559) in PSII. It is reported that in Tris-treated PSII membranes upon illumination, both the intermediate potential (IP) and the reduced high potential (HP red) forms of cyt b 559 exhibit superoxide scavenging activity and interconversion between IP and HP red form. When Tris-treated PSII membranes were illuminated in the presence of spin trap EMPO, the formation of superoxide anion radical (O 2 − ) was observed, as confirmed by EPR spin-trapping spectroscopy. The observations that the addition of enzymatic (superoxide dismutase) and non-enzymatic (cytochrome c, α-tocopherol and Trolox) O 2 − scavengers prevented the light-induced conversion of IP ↔ HP red cyt b 559 confirmed that IP and HP red cyt b 559 are reduced and oxidized by O 2 − , respectively. Redox changes in cyt b 559 by an exogenous source of O 2 − reconfirmed the superoxide oxidase and reductase activity of cyt b 559. Furthermore, the light-induced conversion of IP to HP red form of cyt b 559 was completely inhibited at pH > 8 and by chemical modification of the imidazole ring of histidine residues using diethyl pyrocarbonate. We proposed that a change in the environment around the heme iron, induced by the protonation and deprotonation of His 22 residue generates a favorable condition for the oxidation and reduction of O 2 − , respectively.

Site-directed mutagenesis on the heme axial-ligands of cytochrome b559 in photosystem II by using cyanobacteria Synechocystis PCC 6803

Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the α or the β subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kα mutant grew photoautotrophically, and accumulated stable PSII reaction centers (∼ 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Yβ mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers (∼ 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559α and β polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559β polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YβPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YβPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YβPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kα mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kα mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kα and H22YβPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII.

Construction and Characterization of Genetically Modified Synechocystis sp. PCC 6803 Photosystem II Core Complexes Containing Carotenoids with Shorter π-Conjugation than β-Carotene

Beta-carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes Chl(Z) and cytochrome b(559) in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the zeta-carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated pi-electron systems and higher reduction potentials than beta-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced beta-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and beta-zeacarotene (9 conjugated double bonds and 1 beta-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the beta-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl(+)/Car(+) is higher in the mutant core complexes, consistent with the higher reduction potential for Car(+). As the temperature increases, other carotenoids become accessible to oxidation by P(680)(+).

Spin conversion of cytochrome b559 in photosystem II induced by exogenous high potential quinone

The spin-state of cytochrome b 559 (Cyt b 559 ) was studied in photosystem II (PSII) membrane fragments by low-temperature EPR spectroscopy. Treatment of the membranes with 2,3-dichloro-5,6-dicyano- p -benzoquinone (DDQ) converts the native low-spin (LS) form of Cyt b 559 to the high-spin (HS) form characterized with the g= 6.19 and g= 5.95 split signal. The HS Cyt b 559 was pH dependent with the amplitude increasing toward more acidic pH values (pH 5.5–8.5). The HS state was not photochemically active upon 77 and 200 K continuous illumination under our conditions and was characterized by a low reduction potential (⩽0 V). It was also demonstrated that DDQ treatment damages the oxygen evolving complex, leading to inhibition of oxygen evolution, decrease of the S 2 -state EPR multiline signal and release of Mn 2+ . In parallel, studies of model systems containing iron(III) protoporphyrin IX chloride (Fe III Por), which is a good model compound for the Cyt b 559 prosthetic group, were performed by using optical and EPR spectroscopy. The interaction of Fe III Por with imidazole (Im) in weakly polar solvent results in formation of bis -imidazole coordinated heme iron (Fe III Por Im 2 ) which mimic the bis -histidine axial ligation of Cyt b 559 . The reaction of DDQ with the LS Fe III Por Im 2 complex leads to its transformation into the HS state ( g ⊥ =5.95, g ∥ =2.00). It was shown that the spin conversion occurs due to the donor–acceptor interaction of coordinated imidazole with this high-potential quinone causing the displacement of imidazole from the axial position. The similar mechanism of DDQ-induced spin change is assumed to be valid for the native membrane Cyt b 559 in PSII centers.

Structural model of cytochrome b559 in photosystem II based on a mutant with genetically fused subunits.

Photosystem II is a reaction center protein complex located in photosynthetic membranes of plants, algae, and cyanobacteria. Using light energy, photosystem II catalyzes the oxidation of water and the reduction of plastoquinone, resulting in the release of molecular oxygen. A key component of photosystem II is cytochrome b559, a membrane-embedded heme protein with an unknown function. The cytochrome is unusual in that a heme links two separate polypeptide subunits, alpha and beta, either as a heterodimer (alphabeta) or as two homodimers (alpha2 and beta2). To determine the structural organization of cytochrome b559 in the membrane, we used site-directed mutagenesis to fuse the coding regions of the two respective genes in the cyanobacterium Synechocystis sp. PCC 6803. In this construction, the C terminus of the alpha subunit (9 kDa) is attached to the N terminus of the beta subunit (5 kDa) to form a 14-kDa alphabeta fusion protein that is predicted to have two membrane-spanning alpha-helices with antiparallel orientations. Cells containing the alphabeta fusion protein grow photoautotrophically and assemble functional photosystem II complexes. Optical spectroscopy shows that the alphabeta fusion protein binds heme and is incorporated into photosystem II. These data support a structural model of cytochrome b559 in which one heme is coordinated to an alpha2 homodimer and a second heme is coordinated to a beta2 homodimer. In this model, each photosystem II complex contains two cytochrome b559 hemes, with the alpha2 heme located near the stromal side of the membrane and the beta2 heme located near the lumenal side.

Reevaluation of the stoichiometry of cytochrome b559 in photosystem II and thylakoid membranes.

The stoichiometry of cytochrome b559 (one or two copies) per reaction center of photosystem II (PSII) has been the subject of considerable debate. The molar ratio of cytochrome b559 has a number of significant implications on our understanding of the functional role of cytochrome b559, the mechanism of electron donation in PSII, and the stoichiometry of the other redox-active, reaction center components. We have reinvestigated the stoichiometry of cytochrome b559 in PSII-enriched and thylakoid membranes, using differential absorbance and electron paramagnetic resonance spectroscopies. The data from both quantitation procedures strongly indicate only one copy of cytochrome b559 per reaction center in PSII-enriched membranes and also suggest one copy of cytochrome b559 per reaction center in thylakoid membranes.

Simultaneous photoreduction and photooxidation of cytochrome b-559 in Photosystem II treated with carbonylcyanide-m-chlorophenylhydrazone

The possibility of a Photosystem II (PS II) cyclic electron flow via Cyt b-559 catalyzed by carbonylcyanide m-chlorophenylhydrazone (CCCP) was further examined by studying the effects of the PS II electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ) on the light-induced changes of the redox states of Cyt b-559. Addition to barley thylakoids of micromolar concentrations of DCBQ completely inhibited the changes of the absorbance difference corresponding to the photoreduction of Cyt b-559 observed either in the presence of 10 μM ferricyanide or after Cyt b-559 photooxidation in the presence of 2 μM CCCP. In CCCP-treated thylakoids, the concentration of photooxidized Cyt b-559 decreased as the irradiance of actinic light increased from 2 to 80 W m(-2) but remained close to the maximal concentration (0.53 photooxidized Cyt b-559 per photoactive Photosystem II) in the presence of 50 μM DCBQ. The stimulation of Cyt b-559 photooxidation in parallel with the inhibition of its photoreduction caused by DCBQ demonstrate that the extent of the light-induced changes of the redox state of Cyt b-559 in the presence of CCCP is determined by the difference between the rates of photooxidation and photoreduction of Cyt b-559 occuring simultaneously in a cyclic electron flow around PS II.We also observed that the Photosystem I electron acceptor methyl viologen (MV) at a concentration of 1 mM barely affected the rate and extent of the light-induced redox changes of Cyt b-559 in the presence of either FeCN or CCCP. Under similar experimental conditions, MV strongly quenched Chl-a fluorescence, suggesting that Cyt b-559 is reduced directly on the reducing side of Photosystem II.

Distinctive stability of the reduced and oxidized forms of high-potential cytochrome b-559 in photosystem II particles

Heating, aging or Triton X-100 treatment of oxygen-evolving spinach photosystem II (PS II) particles promotes the progressive conversion of the high-potential (HP) couple of cytochrome b-559 to its low-potential (LP) couple. Any of these treatments affects, however, the oxidized form of the HP couple more intensely than the reduced form counterpart of the same HP couple, the resulting form being in both cases the oxidized form of the LP couple. This finding indicates that the HP oxidized form is more labile towards these treatments than the HP reduced form. Moreover, incubation of PS II particles at pH 8.0 promotes also the gradual conversion of the HP couple of cytochrome b-559 to its LP couple. The treatment affects, again, more intensely the oxidized form of the HP couple than the correponding reduced HP form and in both cases results in the formation of the oxidized form of the LP couple. The results support the suggestion that the oxidized form of the HP couple, which is apparently protonated, is energized and labile with respect to the stable oxidized form of the LP couple.

Characterization of the multiple forms of cytochrome b559 in photosystem II

Cytochrome b559 is an essential component of the photosystem II (PSII) protein complex. Its function, which has long been an unsolved puzzle, is likely to be related to the unique ability of PSII to oxidize water. We have used EPR spectroscopy and spectrophotometric redox titrations to probe the structure of cytochrome b559 in PSII samples that have been treated to remove specific components of the complex. The results of these experiments indicate that the low-temperature photooxidation of cytochrome b559 does not require the presence of the 17-, 23-, or 33-kDa extrinsic polypeptides or the Mn complex (the active site in water oxidation). We observe a shift in the g value of the EPR signal of cytochrome b559 upon warming a low-temperature photooxidized sample, which presumably reflects a change in conformation to accommodate the oxidized state. At least three redox forms of cytochrome b559 are observed. Untreated PSII membranes contain one high-potential (375 mV) and one intermediate-potential (230 mV) cytochrome b559 per PSII. Thylakoid membranes also appear to contain one high-potential and one intermediate-potential cytochrome b559 per PSII, although this measurement is more difficult due to interference from other cytochromes. Removal of the 17- and 23-kDa extrinsic polypeptides from PSII membranes shifts the composition to one intermediate-potential (170 mV) and one low-potential (5 mV) cytochrome b559. This large decrease in potential is accompanied by a very small g-value change (0.04 at gz), indicating that it is the environment and not the ligand field of the heme which changes significantly upon the removal of the 17- and 23-kDa polypeptides.

Stoichiometry of cytochrome b-559 in Photosystem II

A careful determination of the stoichiometry of cytochrome b -559 per Photosystem II reaction centre has been made for different Photosystem II preparations. Using an extinction coefficient determined from the α-band of reduced pyridine haemochrome and chemical analyses of pigment levels it is concluded that there is one cytochrome b -559 haem per Photosystem II reaction centre. This conclusion assumes that by analogy with the purple bacterial reaction centre there are two pheophytin molecules per Photosystem II reaction centre.

Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

The genes encoding the two subunits (alpha and beta) of the cytochrome b559 (cyt b559) protein, psbE and psbF, were cloned from the unicellular, transformable cyanobacterium, Synechocystis 6803. Cyt b559, an intrinsic membrane protein, is a component of photosystem II, a membrane-protein complex that catalyzes photosynthetic oxygen evolution. However, the role of cyt b559 in photosynthetic electron transport is yet to be determined. A high degree of homology was found between the cyanobacterial and green plant chloroplastidic psbE and psbE genes and in the amino acid sequences of their corresponding protein products. Cartridge mutagenesis techniques were used to generate a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes were replaced by a kanamycin-resistance gene cartridge. Physiological analyses indicated that the PSII complexes of the mutant were inactivated. We conclude that cyt b559 is an essential component of PSII.