Photooxidation of cytochrome b559 in oxygen-evolving photosystem II.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron-transfer events leading to reconstitution of oxygen-evolution activity in manganese-depleted photosystem II membranes

O2-evolution activity and the Mn complex can be reconstituted in photosystem II by a process called photoactivation. We have studied the elementary steps in photoactivation by using electron paramagnetic resonance spectroscopy to probe electron transport in Mn-depleted photosystem II membranes. The electron donation reactions in Mn-depleted photosystem II were found to be identical with those in untreated photosystem II, except that electron donation from the Mn complex was absent and could be replaced by slower electron donation from exogenous Mn2+. Mn2+ photooxidation by Mn-depleted photosystem II membranes correlates with reconstitution of O2-evolution activity. However, photooxidation of Mn2+ occurs in competition with photooxidation of the tyrosine residue YD, and cytochrome b-559. Thus, these two species are excluded from direct participation in the initial steps in the assembly of the Mn complex. Because photooxidation of Mn2+ is slower than photooxidation of the competing electron donors, cytochrome b-559 and chlorophyll, as well as recombination of the charge-separated states chlorophyll+QA- or YZ+QA-, these other reactions dominate in a single photochemical turnover reaction. This provides a molecular basis for both the low yield and low quantum yield of photoactivation. The first photochemical step in the assembly of the Mn complex results in photooxidation of one Mn2+ ion. Therefore, the first intermediate in assembly of the Mn complex contains Mn3+. On the basis of these results and previous kinetic studies [Miller, A.-F., & Brudvig, G. W. (1989) Biochemistry 28, 8181], we conclude that the second intermediate of Mn complex assembly contains Mn2+Mn3+, which is photooxidized to Mn3+2.     

Photoinhibition of hydroxylamine-extracted photosystem II membranes: identification of the sites of photodamage.

Electron paramagnetic resonance (EPR) analyses (g = 2 region) and optical spectrophotometric analyses of P680+ were made of NH2OH-extracted photosystem II (PSII) membranes after various durations of weak-light photoinhibition, in order to identify the sites of damage responsible for the observed kinetic components of the loss of electron transport [Blubaugh, D.J., & Cheniae, G.M. (1990) Biochemistry 29, 5109-5118]. The EPR spectra, recorded in the presence of K3Fe(CN)6, gave evidence for rapid (t1/2 = 2-3 min) and slow (t1/2 = 3-4) losses of formation of the tyrosyl radicals YZ+ and YD+, respectively, and the rapid appearance (t1/2 = 0.8 min) of a 12-G-wide signal, centered at g = 2.004, which persisted at 4 degrees C in subsequent darkness in rather constant abundance (approximately 1/2 spin per PSII). This latter EPR signal is correlated with quenching of the variable chlorophyll a fluorescence yield and is tentatively attributed to a carotenoid (Car) cation. Exogenous reductants (NH2OH greater than or equal to NH2NH2 greater than DPC much greater than Mn2+) were observed to reduce the quencher, but did not reverse other photoinhibition effects. An additional 10-G-wide signal, tentatively attributed to a chlorophyll (Chl) cation, is observed during illumination of photoinhibited membranes and rapidly decays following illumination. The amplitude of formation of the oxidized primary electron donor, P680+, was unaffected throughout 120 min of photoinhibition, indicating no impairment of charge separation from P680, via pheophytin (Pheo), to the first stable electron acceptor, QA. However, a 4-microsecond decay of P680+, reflecting YZ----P680+, was rapidly (t1/2 = 0.8 min) replaced by an 80-140 microsecond decay, presumably reflecting QA-/P680+ back-reaction. Photoinhibition caused no discernible decoupling of the antenna chlorophyll from the reaction center complex. We conclude that the order of susceptibility of PSII components to photodamage when O2 evolution is impaired is Chl/Car greater than YZ greater than YD much greater than P680, Pheo, QA.     

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.     

Carotenoid oxidation in photosystem II.

The oxidation of carotenoid upon illumination at low temperature has been studied in Mn-depleted photosystem II (PSII) using EPR and electronic absorption spectroscopy. Illumination of PSII at 20 K results in carotenoid cation radical (Car+*) formation in essentially all of the centers. When a sample which was preilluminated at 20 K was warmed in darkness to 120 K, Car+* was replaced by a chlorophyll cation radical. This suggests that carotenoid functions as an electron carrier between P680, the photooxidizable chlorophyll in PSII, and ChlZ, the monomeric chlorophyll which acts as a secondary electron donor under some conditions. By correlating with the absorption spectra at different temperatures, specific EPR signals from Car+* and ChlZ+* are distinguished in terms of their g-values and widths. When cytochrome b559 (Cyt b559) is prereduced, illumination at 20 K results in the oxidation of Cyt b559 without the prior formation of a stable Car+*. Although these results can be reconciled with a linear pathway, they are more straightforwardly explained in terms of a branched electron-transfer pathway, where Car is a direct electron donor to P680(+), while Cyt b559 and ChlZ are both capable of donating electrons to Car+*, and where the ChlZ donates electrons when Cyt b559 is oxidized prior to illumination. These results have significant repercussions on the current thinking concerning the protective role of the Cyt b559/ChlZ electron-transfer pathways and on structural models of PSII.     

Beta-carotene redox reactions in photosystem II: electron transfer pathway

A carotenoid (Car), a chlorophyll (Chl(Z)), and cytochrome b(559) (Cyt b(559)) are able to donate electrons with a low quantum yield to the photooxidized chlorophyll, P680(+), when photosystem II (PSII) is illuminated at low temperatures. Three pathways for electron transfer from Cyt b(559) to P680(+) are considered: (a) the "linear pathway" in which Cyt b(559) donates via Chl(Z) to Car, (b) the "branched pathway" in which Cyt b(559) donates via Car and where Chl(Z) is also able to donate to Car, and (c) the "parallel pathway" where Cyt b(559) donates to P680 without intermediate electron carriers and electron donation from Chl(Z) and Car occurs by a competing pathway. Experiments were performed using EPR and spectrophotometry in an attempt to distinguish among these pathways, and the following observations were made. (1) Using PSII with an intact Mn cluster in which Cyt b(559) was preoxidized, Car oxidation was dominant upon illumination at < or =20 K, while electron donation from Chl dominated at >120 K. (2) When Cyt b(559) was prereduced, its light-induced oxidation occurred at < or =20 K in what appeared to be all of the centers and without the formation of a detectable Car(+) intermediate. The small and variable quantity of Car(+) photoinduced in these experiments can be attributed to the residual centers in which Cyt b(559) remained oxidized prior to illumination. (3) The relative rates for irreversible electron donation from Cyt b(559) and Car were determined indirectly at 20 K by monitoring the flash-induced loss of charge separation (i.e., the accumulation of Cyt b(559)(+)Q(A)(-) or Car(+)Q(A)(-)). Similar yields per flash were observed (13% for Cyt b(559) and 8% for Car), indicating similar donation rates. The slightly lower yield with Car as a donor is attributed at least in part to slow charge recombination occurring from the Car(+)Q(A)(-) radical pair in a fraction of centers. (4) Light-induced oxidation of Cyt b(559) and Car at 20 K was monitored directly by EPR, and the rates were found to be indistinguishable. The parallel pathway predicts that when both Cyt b(559) and Car are prereduced, the relative amounts of Cyt b(559)(+) and Car(+) produced upon illumination at 20 K should depend directly on their relative electron donation rates. The measured similarity in the donation rates thus predicts comparable yields of oxidation for both donors. However, what is observed experimentally is that Cyt b(559) oxidation occurs almost exclusively, and this argues strongly against the parallel pathway. The lack of Car(+) as a detectable intermediate is attributed to rapid electron transfer from Cyt b(559) to Car(+). The trapping of Car(+) at low temperature when Cyt b(559) is preoxidized but its absence when Cyt b(559) is prereduced is taken as an argument against the simple linear pathway. Overall, the data reported here and previously favor the branched pathway over the linear pathway, while the parallel pathway is thought to be unlikely. Structural considerations provide further arguments in favor of the branched model.     

Water-splitting manganese complex controls light-induced redox changes of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>559</Subscript> in Photosystem II

The effect of water-splitting Mn complex on light-induced redox changes of cytochrome b ₅₅₉ (cyt b ₅₅₉) was studied in spinach photosystem II (PSII) membranes. Photoreduction of the heme iron in the intact PSII membranes was completely suppressed by DCMU, whereas photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were unaffected by DCMU. Interesingly, photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were completely diminished by exogenous superoxide dismutase (SOD), whereas no effect of SOD on photoreduction of the heme iron was observed in the intact PSII membranes. The current work shows that the light-induced redox changes of cyt b ₅₅₉ proceed via a different mechanism in the both types of PSII membranes. In the intact PSII membranes, photoreduction of the heme iron is mediated by plastoquinol. However, in the Mn-depleted PSII membranes, photoreduction and photooxidation of the heme iron are mediated by superoxide anion radical formed in PSII.     

Electron transfer in photosystem II at cryogenic temperatures

The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH dependent competition between Y(Z) and Y(D) in photosystem II probed by illumination at 5 K

The photosystem II (PSII) reaction center contains two redox active tyrosines, YZ and YD, situated on the D1 and D2 proteins, respectively. By illumination at 5 K, oxidation of YZ in oxygen-evolving PSII can be observed as induction of the Split S1 EPR signal from YZ* in magnetic interaction with the CaMn4 cluster, whereas oxidation of YD can be observed as the formation of the free radical EPR signal from YD*. We have followed the light induced induction at 5 K of the Split S1 signal between pH 4-8.5. The formation of the signal, that is, the oxidation of YZ, is pH independent and efficient between pH 5.5 and 8.5. At low pH, the split signal formation decreases with pKa approximately 4.7-4.9. In samples with chemically pre-reduced YD, the pH dependent competition between YZ and YD was studied. Only YZ was oxidized below pH 7.2, but at pH above 7.2, the oxidation of YD became possible, and the formation of the Split S1 signal diminished. The onset of YD oxidation occurred with pKa approximately 8.0, while the Split S1 signal decreased with pKa approximately 7.9 demonstrating that the two tyrosines compete in this pH interval. The results reflect the formation and breaking of hydrogen bonds between YZ and D1-His190 (HisZ) and YD and D2-His190 (HisD), respectively. The oxidation of respective tyrosine at 5 K demands that the hydrogen bond is well-defined; otherwise, the low-temperature oxidation is not possible. The results are discussed in the framework of recent literature data and with respect to the different oxidation kinetics of YZ and YD.     

Photoconsumption of molecular oxygen on both donor and acceptor sides of photosystem II in Mn-depleted subchloroplast membrane fragments

Oxygen consumption in Mn-depleted photosystem II (PSII) preparations under continuous and pulsed illumination is investigated. It is shown that removal of manganese from the water-oxidizing complex (WOC) by high pH treatment leads to a 6-fold increase in the rate of O₂ photoconsumption. The use of exogenous electron acceptors and donors to PSII shows that in Mn-depleted PSII preparations along with the well-known effect of O₂ photoreduction on the acceptor side of PSII, there is light-induced O₂ consumption on the donor side of PSII (nearly 30% and 70%, respectively). It is suggested that the light-induced O₂ uptake on the donor side of PSII is related to interaction of O₂ with radicals produced by photooxidation of organic molecules. The study of flash-induced O₂ uptake finds that removal of Mn from the WOC leads to O₂ photoconsumption with maximum in the first flash, and its yield is comparable with the yield of O₂ evolution on the third flash measured in the PSII samples before Mn removal. The flash-induced O₂ uptake is drastically (by a factor of 1.8) activated by catalytic concentration (5-10 μM, corresponding to 2-4 Mn per RC) of Mn²⁺, while at higher concentrations (> 100 μM) Mn²⁺ inhibits the O₂ photoconsumption (like other electron donors: ferrocyanide and diphenylcarbazide). Inhibitory pre-illumination of the Mn-depleted PSII preparations (resulting in the loss of electron donation from Mn²⁺) leads to both suppression of flash-induced O₂ uptake and disappearance of the Mn-induced activation of the O₂ photoconsumption. We assume that the light-induced O₂ uptake in Mn-depleted PSII preparations may reflect not only the negative processes leading to photoinhibition but also possible participation of O₂ or its reactive forms in the formation of the inorganic core of the WOC.     

A theoretical study on effect of the initial redox state of cytochrome b559 on maximal chlorophyll fluorescence level (F(M)): implications for photoinhibition of photosystem II

In this work, we extended the reversible radical pair model which describes energy utilization and electron transfer up to the first quinone electron acceptor (Q(A)) in photosystem II (PSII), by redox reactions involving cytochrome (cyt) b559. In the model, cyt b559 accepts electrons from the reduced primary electron acceptor in PSII, pheophytin, and donates electrons to the oxidized primary electron donor in PSII (P680+). Theoretical simulations of chlorophyll fluorescence rise based on the model show that the maximal fluorescence, F(M), increases with an increasing amount of initially reduced cyt b559. In this work we applied, the first to our knowledge, metabolic control analysis (MCA) to a model of reactions in PSII. The MCA was used to determine to what extent the reactions occurring in the model control the F(M) level and how this control depends on the initial redox state of cyt b559. The simulations also revealed that increasing the amount of initially reduced cyt b559 could protect PSII against photoinhibition. Also experimental data, which might be used to validate our theory, are presented and discussed.     

Unique binding site for Mn2+ ion responsible for reducing an oxidized YZ tyrosine in manganese-depleted photosystem II membranes.

Binding of Mn2+ to manganese-depleted photosystem II and electron donation from the bound Mn2+ to an oxidized YZ tyrosine were studied under the same equilibrium conditions. Mn2+ associated with the depleted membranes in a nonsaturating manner when added alone, but only one Mn2+ ion per photosystem II (PS II) was bound to the membranes in the presence of other divalent cations including Ca2+ and Mg2+. Mn2+-dependent electron donation to photosystem II studied by monitoring the decay kinetics of chlorophyll fluorescence and the electron paramagnetic resonance (EPR) signal of an oxidized YZ tyrosine (YZ+) after a single-turnover flash indicated that the binding of only one Mn2+ ion to the manganese-depleted PS II is sufficient for the complete reduction of YZ+ induced by flash excitation. The results indicate that the manganese-depleted membranes have only one unique binding site, which has higher affinity and higher specificity for Mn2+ compared with Mg2+ and Ca2+, and that Mn2+ bound to this unique site can deliver an electron to YZ+ with high efficiency. The dissociation constant for Mn2+ of this site largely depended on pH, suggesting that a single amino acid residue with a pKa value around neutral pH is implicated in the binding of Mn2+. The results are discussed in relation to the photoactivation mechanism that forms the active manganese cluster.     

Cytochrome b-559 may function to protect photosystem II from photoinhibition

Although cytochrome b-559 is an integral component of the photosystem II complex (PSII), its function is unknown. Because cytochrome b-559 has been shown to be both photooxidized and photoreduced in PSII, one of several proposals is that it mediates cyclic electron transfer around PSII, possibly as a protective mechanism. We have used electron paramagnetic resonance spectroscopy to investigate the pathway of photooxidation of cytochrome b-559 in PSII and have shown that it proceeds via photooxidation of chlorophyll. We propose that this photooxidation of chlorophyll is the first step in the photoinhibition of PSII. The unique susceptibility of PSII to photoinhibition is probably due to the fact that it is the only reaction center in photosynthesis which generates an oxidant with a reduction potential high enough to oxidize chlorophyll. We propose that the function of cytochrome b-559 is to mediate cyclic electron transfer to rereduce photooxidized chlorophyll and protect PSII from photoinhibition. We also suggest that the chlorophyll(s) which are susceptible to photooxidation are analogous to the monomer chlorophylls found in the bacterial photosynthetic reaction center complex.     

Role of D1-His190 in the proton-coupled oxidation of tyrosine YZ in manganese-depleted photosystem II.

To further characterize the role of D1-His190 in the oxidation of tyrosine Y(Z) in photosystem II, the pH dependence of P(680)(*)()(+) reduction was measured in H190A and Mn-depleted wild-type PSII particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803. Measurements were conducted in the presence and absence of imidazole and other small organic bases. In H190A PSII particles, rapid reduction of P(680)(*)()(+) attributed to electron transfer from Y(Z) increased dramatically above pH 9, with an apparent pK(A) of approximately 10.3. In the presence of ethanolamine and imidazole, this dramatic increase occurred at lower pH values, with the efficiency of Y(Z) oxidation correlating with the solution pK(A) value of the added base. We conclude that the pK(A) of Y(Z) is approximately 10.3 in D1-H190A PSII particles. In Mn-depleted wild-type PSII particles, P(680)(*)()(+) reduction was accelerated by all exogenous bases examined (substituted imidazoles, histidine, Tris, and 1,4-diazabicyclo[2.2.2]octane). We conclude that Y(Z) is solvent accessible in Mn-depleted wild-type PSII particles and that its pK(A) is near that of tyrosine in solution. In Mn-depleted wild-type PSII particles, over 80% of the kinetics of P(680)(*)()(+) reduction after a flash could be described by three kinetic components. The individual rate constants of these components varied slightly with pH, but their relative proportions varied dramatically with pH, showing apparent pK(A) values of 7.5 and 6.25 (6.9 and 5.8 in the presence of Ca(2+) and Mg(2+) ions). An additional pK(A) value (pK(A) < 4.5) may also be present. To describe these data, we propose (1) the pK(A) of His190 is 6.9-7.5, depending on buffer ions, (2) the deprotonation of Y(Z) is facilitated by the transient formation of a either a hydrogen bond or a hydrogen-bonded water bridge between Y(Z) and D1-His190, and (3) when protonated, D1-His190 interacts with nearby residues having pK(A) values near 6 and 4. Because Y(Z) and D1-His190 are located near the Mn cluster, these residues may interact with the Mn cluster in the intact system.     

The effect of temperature on the primary reaction of chloroplast Photosystem II Evidence for a temperature-dependent back reaction

The primary reaction of Photosystem II has been studied over the temperature range from −196 to −20 °C. The photooxidation of the reaction-center chlorophyll (P680) was followed by the free-radical electron paramagnetic resonance signal of P680⁺, and the photoreduction of the Photosystem II primary electron acceptor was monitored by the C-550 absorbance change.

At temperatures below −100 °C, the primary reaction of Photosystem II is irreversible. However, at temperatures between −100 and −20 °C a back reaction that is insensitive to 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) occurs between P680⁺ and the reduced acceptor.

The amount of reduced acceptor and P680⁺ present under steady-state illumination at temperatures between −100 and −20 °C is small unless high light intensity is used to overcome the competing back reaction. The amount of reduced acceptor present at low light intensity can be increased by adjusting the oxidation-reduction potential so that P680⁺ is reduced by a secondary electron donor (cytochrome b₅₅₉) before P680⁺ can reoxidize the reduced primary acceptor. The photooxidation of cytochrome b₅₅₉ and the accompanying photoreduction of C-550 are inhibited by DCMU. The inhibition of C-550 photoreduction by DCMU, the dependence of P680 photooxidation and C-550 photoreduction on light intensity, and the effect of the availability of reduced cytochrome b₅₅₉ on C-550 photoreduction are unique to the temperature range where the Photosystem II primary reaction is reversible and are not observed at lower temperatures.     

Directed mutagenesis indicates that the donor to P+680 in photosystem II is tyrosine-161 of the D1 polypeptide

Photosystem II contains two redox-active tyrosines. One of these, YZ, reduces the reaction center chlorophyll, P680, and transfers the oxidizing equivalent to the oxygen-evolving complex. The second, YD, has a long-lived free radical state of unknown function. We recently established that YD is Tyr-160 of the D2 polypeptide by site-directed mutagenesis of a psbD gene in the unicellular cyanobacterium Synechocystis 6803 [Debus, R. J., Barry, B. A., Babcock, G. T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430]. YZ is most likely the symmetry-related Tyr-161 of the D1 polypeptide. To test this hypothesis, we have changed Tyr-161 to phenylalanine by site-directed mutagenesis of a psbA gene in Synechocystis. The resulting mutant assembles PSII, as judged by its ability to produce the stable Y+D radical, but is unable to grow photosynthetically and exhibits altered fluorescence properties. The nature of the fluorescence change indicates that forward electron transfer to P+680 is disrupted in the mutant. These results provide strong support for our identification of Tyr-161 in the D1 polypeptide with YZ.     

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.     

Composition and function of cytochrome b559 in reaction centers of photosystem II of green plants

A review of a recent study of the spectral and thermodynamic properties of cytochrome b559 as well as of the electron transfer between b559 and photosystem II reaction center cofactors in isolated D1/D2/cytochrome b559 complex RC-2 is presented. Attention is paid to the existence of intermediary-potential (IP, +150 mV) and extra-low-potential (XLP, –45 mV) hemes located close to the acceptor (quinone) and donor (P680) sides of the reaction center cofactors, respectively. These hemes found in isolated RC-2 probably correspond to the high-potential and low-potential hemes in chloroplasts, respectively. The above location of the hemes is believed to allow the photoreduction of the XLP heme and photooxidation of the IP heme. The electron transfer between the two hemes is discussed in terms of the cyclic electron flow and possible involvement in water splitting.     

Multiple redox-active chlorophylls in the secondary electron-transfer pathways of oxygen-evolving photosystem II

Photosystem II (PS II) is unique among photosynthetic reaction centers in having secondary electron donors that compete with the primary electron donors for reduction of P680(+). We have characterized the photooxidation and dark decay of the redox-active accessory chlorophylls (Chl) and beta-carotenes (Car) in oxygen-evolving PS II core complexes by near-IR absorbance and EPR spectroscopies at cryogenic temperatures. In contrast to previous results for Mn-depleted PS II, multiple near-IR absorption bands are resolved in the light-minus-dark difference spectra of oxygen-evolving PS II core complexes including two fast-decaying bands at 793 and 814 nm and three slow-decaying bands at 810, 825, and 840 nm. We assign these bands to chlorophyll cation radicals (Chl(+)). The fast-decaying bands observed after illumination at 20 K could be generated again by reilluminating the sample. Quantization by EPR gives a yield of 0.85 radicals per PS II, and the yield of oxidized cytochrome b 559 by optical difference spectroscopy is 0.15 per PS II. Potential locations of Chl(+) and Car(+) species, and the pathways of secondary electron transfer based on the rates of their formation and decay, are discussed. This is the first evidence that Chls in the light-harvesting proteins CP43 and CP47 are oxidized by P680(+) and may have a role in Chl fluorescence quenching. We also suggest that a possible role for negatively charged lipids (phosphatidyldiacylglycerol and sulfoquinovosyldiacylglycerol identified in the PS II structure) could be to decrease the redox potential of specific Chl and Car cofactors. These results provide new insight into the alternate electron-donation pathways to P680(+).     

P680(+)* reduction kinetics and redox transition probability of the water oxidizing complex as a function of pH and H/D isotope exchange in spinach thylakoids.

The rise of fluorescence as an indicator for P680(+)* reduction by YZ and the period-four oscillation of oxygen yield induced by a train of saturating flashes were measured in dark-adapted thylakoids as a function of pH in the absence of exogenous electron acceptors. The results reveal that: (i) the average amplitude of the nanosecond kinetics and the average of the maximum fluorescence attained at 100 micros after the flash in the acidic range decrease with decreasing pH; (ii) the oxygen yield exhibits a pronounced period-four oscillation at pH 6.5 and higher damping at both pH 5.0 and pH 8.0; (iii) the probability of misses in the Si-state transitions of the water oxidizing complex is affected characteristically when exchangeable protons are replaced by deuterons [at pH <6.5, the ratio alpha(D)/alpha(H) is larger than 1 whereas at pH >7.0 values of <1 are observed]. The results are discussed within the framework of a combined mechanism for P680(+)* reduction where the nanosecond kinetics reflect an electron transfer coupled with a "rocket-type" proton shift within a hydrogen bridge from YZ to a nearby basic group, X [Eckert, H.-J., and Renger, G. (1988) FEBS Lett. 236, 425-431], and subsequent relaxations within a network of hydrogen bonds. It is concluded that in the acidic region the hydrogen bond between YZ and X (most likely His 190 of polypeptide D1) is interrupted either by direct protonation of X or by conformational changes due to acid-induced Ca2+ release. This gives rise to a decreased P680(+)* reduction by nanosecond kinetics and an increase of dissipative P680(+)* recombination at low pH. A different mechanism is responsible for the almost invariant amplitude of nanosecond kinetics and increase of alpha in the alkaline region.