D1-D2-cytochrome b559 complex from the aquatic plant Spirodela oligorrhiza: correlation between complex integrity, spectroscopic properties, photochemical activity, and pigment composition

A D1-D2-cyt b559 complex with about four attached chlorophylls and two pheophytins has been isolated from photosystem II of the aquatic plant Spirodela oligorrhiza and used for studying the detergent-induced changes in spectroscopic properties and photochemical activity. Spectral analyses (absorption, CD, and fluorescence) of D1-D2-cyt b559 preparations that were incubated with different concentrations of the detergent Triton X-100 indicate two forms of the D1-D2-cyt b559 complexes. One of them is photochemically active and has an absorption maximum at 676 nm, weak fluorescence at 685 nm, and a strong CD signal. The other is photochemically inactive, with an absorption maximum at 670 nm, strong fluorescence at 679 nm, and much weaker CD. The relative concentrations of the two forms determine the overall spectra of the D1-D2-cyt b559 preparation and can be deduced from the wavelength of the lowest energy absorption band: preparations having maximum absorption at 674, 672, or 670.5 nm have approximately 20, 60, or 85% inactive complexes. The active form contains two chlorophylls with maximum absorption at 679 nm and CD signals at 679 (+) and 669 nm (-). These chlorophylls make a special pair that is identified as the primary electron donor P-680. The calculated separation between the centers of these two pigments (using an extended version of the exciton theory) is about 10 A, the pigments' molecular planes are tilted by about 20 degrees, and their N1-N3 axes are rotated by 150 degrees relative to each other. The other two chlorophylls and one of the two pheophytins in the D1-D2-cyt b559 complex have their maximum absorption at 672 nm, while the maximum absorption of the photochemically active pheophytin is probably at 672-676 nm. During incubation with Triton X-100, the photochemically active complex is transformed into an inactive form with first-order kinetics. In the inactive form the maximum absorption of the 679 nm absorbing Chls is blue-shifted to 669 nm. The first-order decay of the photochemical activity suggests that the isolated D1-D2-cyt b559 complex is stable as an aggregate but becomes unstable on dissociation into individual D1-D2-cyt b559 units.     

Excitation energy transfer and charge separation in photosystem II membranes revisited

We have performed time-resolved fluorescence measurements on photosystem II (PSII) containing membranes (BBY particles) from spinach with open reaction centers. The decay kinetics can be fitted with two main decay components with an average decay time of 150 ps. Comparison with recent kinetic exciton annihilation data on the major light-harvesting complex of PSII (LHCII) suggests that excitation diffusion within the antenna contributes significantly to the overall charge separation time in PSII, which disagrees with previously proposed trap-limited models. To establish to which extent excitation diffusion contributes to the overall charge separation time, we propose a simple coarse-grained method, based on the supramolecular organization of PSII and LHCII in grana membranes, to model the energy migration and charge separation processes in PSII simultaneously in a transparent way. All simulations have in common that the charge separation is fast and nearly irreversible, corresponding to a significant drop in free energy upon primary charge separation, and that in PSII membranes energy migration imposes a larger kinetic barrier for the overall process than primary charge separation.     

Determination of the excitation migration time in Photosystem II consequences for the membrane organization and charge separation parameters

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3+/-1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes approximately 23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5+/-0.4 ps)(-1), the rate of secondary charge separation is (137+/-5 ps)(-1) and the drop in free energy upon primary charge separation is 826+/-30 cm(-1). These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. R?gner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].     

Functional heterogeneity of photosystem II in domain specific regions of the thylakoid membrane of spinach (Spinacia oleracea L.)

A mild sonication and phase fractionation method has been used to isolate five regions of the thylakoid membrane in order to characterize the functional lateral heterogeneity of photosynthetic reaction centers and light harvesting complexes. Low-temperature fluorescence and absorbance spectra, absorbance cross-section measurements, and picosecond time-resolved fluorescence decay kinetics were used to determine the relative amounts of photosystem II (PSII) and photosystem I (PSI), to determine the relative PSII antenna size, and to characterize the excited-state dynamics of PSI and PSII in each fraction. Marked progressive increases in the proportion of PSI complexes were observed in the following sequence: grana core (BS), whole grana (B3), margins (MA), stroma lamellae (T3), and purified stromal fraction (Y100). PSII antenna size was drastically reduced in the margins of the grana stack and stroma lamellae fractions as compared to the grana. Picosecond time-resolved fluorescence decay kinetics of PSII were characterized by three exponential decay components in the grana fractions, and were found to have only two decay components with slower lifetimes in the stroma. Results are discussed in the framework of existing models of chloroplast thylakoid membrane lateral heterogeneity and the PSII repair cycle. Kinetic modeling of the PSII fluorescence decay kinetics revealed that PSII populations in the stroma and grana margin fractions possess much slower primary charge separation rates and decreased photosynthetic efficiency when compared to PSII populations in the grana stack.     

Charge Separation, Stabilization, and Protein Relaxation in Photosystem II Core Particles with Closed Reaction Center

The fluorescence kinetics of cyanobacterial photosystem II (PSII) core particles with closed reaction centers (RCs) were studied with picosecond resolution. The data are modeled in terms of electron transfer (ET) and associated protein conformational relaxation processes, resolving four different radical pair (RP) states. The target analyses reveal the importance of protein relaxation steps in the ET chain for the functioning of PSII. We also tested previously published data on cyanobacterial PSII with open RCs using models that involved protein relaxation steps as suggested by our data on closed RCs. The rationale for this reanalysis is that at least one short-lived component could not be described in the previous simpler models. This new analysis supports the involvement of a protein relaxation step for open RCs as well. In this model the rate of ET from reduced pheophytin to the primary quinone Q_A is determined to be 4.1 ns⁻¹. The rate of initial charge separation is slowed down substantially from ∼170 ns⁻¹ in PSII with open RCs to 56 ns⁻¹ upon reduction of Q_A. However, the free-energy drop of the first RP is not changed substantially between the two RC redox states. The currently assumed mechanistic model, assuming the same early RP intermediates in both states of RC, is inconsistent with the presented energetics of the RPs. Additionally, a comparison between PSII with closed RCs in isolated cores and in intact cells reveals slightly different relaxation kinetics, with a ∼3.7 ns component present only in isolated cores.     

Absence of the psbH gene product destabilizes photosystem II complex and bicarbonate binding on its acceptor side in Synechocystis PCC 6803.

The PsbH protein, a small subunit of the photosystem II complex (PSII), was identified as a 6-kDa protein band in the PSII core and subcore (CP47-D1-D2-cyt b-559) from the wild-type strain of the cyanobacterium Synechocystis PCC 6803. The protein was missing in the D1-D2-cytochrome b-559 complex and also in all PSII complexes isolated from IC7, a mutant lacking the psbH gene. The following properties of PSII in the mutant contrasted with those in wild-type: (a) CP47 was released during nondenaturing electrophoresis of the PSII core isolated from IC7; (b) depletion of CO2 resulted in a reversible decrease of the QA- reoxidation rate in the IC7 cells; (c) light-induced decrease in PSII activity, measured as 2,5-dimethyl-benzoquinone-supported Hill reaction, was strongly dependent on the HCO3- concentration in the IC7 cells; and (d) illumination of the IC7 cells lead to an extensive oxidation, fragmentation and cross-linking of the D1 protein. We did not find any evidence for phosphorylation of the PsbH protein in the wild-type strain. The results showed that in the PSII complex of Synechocystis attachment of CP47 to the D1-D2 heterodimer appears weakened and binding of bicarbonate on the PSII acceptor side is destabilized in the absence of the PsbH protein.     

Excitation energy transfer and trapping in higher plant Photosystem II complexes with different antenna sizes

We performed picosecond fluorescence measurements on well-defined Photosystem II (PSII) supercomplexes from Arabidopsis with largely varying antenna sizes. The average excited-state lifetime ranged from 109 ps for PSII core to 158 ps for the largest C₂S₂M₂ complex in 0.01% α-DM. Excitation energy transfer and trapping were investigated by coarse-grained modeling of the fluorescence kinetics. The results reveal a large drop in free energy upon charge separation (>700 cm⁻¹) and a slow relaxation of the radical pair to an irreversible state (∼150 ps). Somewhat unexpectedly, we had to reduce the energy-transfer and charge-separation rates in complexes with decreasing size to obtain optimal fits. This strongly suggests that the antenna system is important for plant PSII integrity and functionality, which is supported by biochemical results. Furthermore, we used the coarse-grained model to investigate several aspects of PSII functioning. The excitation trapping time appears to be independent of the presence/absence of most of the individual contacts between light-harvesting complexes in PSII supercomplexes, demonstrating the robustness of the light-harvesting process. We conclude that the efficiency of the nonphotochemical quenching process is hardly dependent on the exact location of a quencher within the supercomplexes.     

Dimeric and monomeric organization of photosystem II. Distribution of five distinct complexes in the different domains of the thylakoid membrane

The supramolecular organization of photosystem II (PSII) was characterized in distinct domains of the thylakoid membrane, the grana core, the grana margins, the stroma lamellae, and the so-called Y100 fraction. PSII supercomplexes, PSII core dimers, PSII core monomers, PSII core monomers lacking the CP43 subunit, and PSII reaction centers were resolved and quantified by blue native PAGE, SDS-PAGE for the second dimension, and immunoanalysis of the D1 protein. Dimeric PSII (PSII supercomplexes and PSII core dimers) dominate in the core part of the thylakoid granum, whereas the monomeric PSII prevails in the stroma lamellae. Considerable amounts of PSII monomers lacking the CP43 protein and PSII reaction centers (D1-D2-cytochrome b559 complex) were found in the stroma lamellae. Our quantitative picture of the supramolecular composition of PSII, which is totally different between different domains of the thylakoid membrane, is discussed with respect to the function of PSII in each fraction. Steady state electron transfer, flash-induced fluorescence decay, and EPR analysis revealed that nearly all of the dimeric forms represent oxygen-evolving PSII centers. PSII core monomers were heterogeneous, and a large fraction did not evolve oxygen. PSII monomers without the CP43 protein and PSII reaction centers showed no oxygen-evolving activity.     

Model for the fluorescence induction curve of photoinhibited thylakoids.

The fluorescence induction curve of photoinhibited thylakoids measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea was modeled using an extension of the model of Lavergne and Trissl (Biophys. J. 68:2474-2492), which takes into account the reversible exciton trapping by photosystem II (PSII) reaction centers and exciton exchange between PSII units. The model of Trissl and Lavergne was modified by assuming that PSII consists of photosynthetically active and photoinhibited (inactive in oxygen evolution) units and that the inactive PSII units can efficiently dissipate energy even if they still retain the capacity for the charge separation reaction. Comparison of theoretical and experimental fluorescence induction curves of thylakoids, which had been subjected to strong light in the presence of the uncoupler nigericin, suggests connectivity between the photoinhibited and active PSII units. The model predicts that photoinhibition lowers the yield of radical pair formation in the remaining active PSII centers. However, the kinetics of PSII inactivation in nigericin-treated thylakoids upon exposure to photoinhibitory light ranging from 185 to 2650 micromol photons m-2 s-1 was strictly exponential. This may suggest that photoinhibition occurs independently of the primary electron transfer reactions of PSII or that increased production of harmful substances by photoinhibited PSII units compensates for the protection afforded by the quenching of excitation energy in photoinhibited centers.     

Charge separation and energy transfer in the photosystem II core complex studied by femtosecond midinfrared spectroscopy

The core of photosystem II (PSII) of green plants contains the reaction center (RC) proteins D1D2-cytb559 and two core antennas CP43 and CP47. We have used time-resolved visible pump/midinfrared probe spectroscopy in the region between 1600 and 1800 cm(-1) to study the energy transfer and charge separation events within PSII cores. The absorption difference spectra in the region of the keto and ester chlorophyll modes show spectral evolution with time constants of 3 ps, 27 ps, 200 ps, and 2 ns. Comparison of infrared (IR) difference spectra obtained for the isolated antennas CP43 and CP47 and the D1D2-RC with those measured for the PSII core allowed us to identify the features specific for each of the PSII core components. From the presence of the CP43 and CP47 specific features in the spectra up to time delays of 20-30 ps, we conclude that the main part of the energy transfer from the antennas to the RC occurs on this timescale. Direct excitation of the pigments in the RC evolution associated difference spectra to radical pair formation of PD1+PheoD1- on the same timescale as multi-excitation annihilation and excited state equilibration within the antennas CP43 and CP47, which occur within approximately 1-3 ps. The formation of the earlier radical pair ChlD1+PheoD1-, as identified in isolated D1D2 complexes with time-resolved mid-IR spectroscopy is not observed in the current data, probably because of its relatively low concentration. Relaxation of the state PD1+PheoD1-, caused by a drop in free energy, occurs in 200 ps in closed cores. We conclude that the kinetic model proposed earlier for the energy and electron transfer dynamics within the D1D2-RC, plus two slowly energy-transferring antennas C43 and CP47 explain the complex excited state and charge separation dynamics in the PSII core very well. We further show that the time-resolved IR-difference spectrum of PD1+PheoD1- as observed in PSII cores is virtually identical to that observed in the isolated D1D2-RC complex of PSII, demonstrating that the local structure of the primary reactants has remained intact in the isolated D1D2 complex.     

Determination of the excitation migration time in Photosystem II: Consequences for the membrane organization and charge separation parameters

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3 ± 1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes ~ 23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5 ± 0.4 ps)^− 1, the rate of secondary charge separation is (137 ± 5 ps)^− 1 and the drop in free energy upon primary charge separation is 826 ± 30 cm^− 1. These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. Rögner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].     

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.     

Perturbation of the structure of P680 and the charge distribution on its radical cation in isolated reaction center complexes of photosystem II as revealed by fourier transform infrared spectroscopy

The structure and the electronic properties of P680 and its radical cation in photosystem II (PSII) were studied by means of Fourier transform infrared spectroscopy (FTIR). Light-induced P680+/P680 FTIR difference spectra in the mid- and near-IR regions were measured using PSII membranes from spinach, core complexes from Thermosynechococcus elongatus, and reaction center (RC) complexes (D1-D2-Cytb559) from spinach. The spectral features of the former two preparations were very similar, indicating that the structures of P680 and its radical cation are virtually identical between membranes and cores and between plants and cyanobacteria. In sharp contrast, the spectrum of the RC complexes exhibited significantly different features. A positive doublet at approximately 1724 and approximately 1710 cm-1 due to the 131-keto C=O stretches of P680+ in the membrane and core preparations were changed to a prominent single peak at 1712 cm-1 in the RC complexes. This observation was interpreted to indicate that a positive charge on P680+ was extensively delocalized over the chlorophyll dimer in RC, whereas it was mostly localized on one chlorophyll molecule (70-80%) in intact P680. The significant change in the electronic structure of P680+ in RC was supported by a dramatic change in the characteristics of a broad intervalence band in the near-IR region and relatively large shifts of chlorin ring bands. It is proposed that the extensive charge delocalization in P680+ mainly causes the decrease in the redox potential of P680+/P680 in isolated RC complexes. This potential decrease explains the well-known phenomenon that YZ is not oxidized by P680+ in RC complexes.     

Picosecond time-resolved fluorescence studies on excitation energy transfer in a histidine 117 mutant of the D2 protein of photosystem II in Synechocystis 6803

The role of the peripheral reaction center chlorophyll a molecule associated with His117 of the D2 polypeptide in photosystem II was investigated in Synechocystis sp. PCC 6803 using a combination of steady state, pump-probe, and picosecond time-resolved fluorescence spectroscopy. Data were obtained from intact cells and isolated thylakoid membranes of a control mutant and a D2-H117T mutant, both of which lacked photosystem I. Excitation energy transfer and trapping were investigated by analyzing the data with a kinetic model that used an exact numerical solution of the Pauli master equation, taking into account available photosystem II spectral and structural information. The results of our kinetic analysis revealed the observed difference in excited-state dynamics between the H117T mutant and the control to be consistent with a retardation of the rate of excitation energy transfer from the peripheral chlorophyll of D2 (Chl at His117) to the electron-transfer pigments and an increase of the rate constant for charge recombination in the H117T mutant. The kinetic model was able to account for the experimentally observed changes in absorption cross section and fluorescence decay kinetics between the control and mutant by invoking changes in only these two rate constants. The results rule out quenching of excitation by a chlorophyll cation radical as a mechanism responsible for the lower efficiency of excitation energy utilization in the H117T mutant. Our work also demonstrates the importance of the chlorophyll associated with His117 of the D2 protein for excitation energy transfer to the PSII electron-transfer pigments and for the effective stabilization of the primary radical pair.     

Electron-transfer reactions in manganese-depleted photosystem II

We have used flash-detection optical and electron paramagnetic resonance spectroscopy to measure the kinetics and yield per flash of the photooxidation of cytochrome b559 and the yield per flash of the photooxidation of the tyrosine residue YD in Mn-depleted photosystem II (PSII) membranes at room temperature. The initial charge separation forms YZ+ QA-. Following this, cytochrome b559 is oxidized on a time scale of the same order and with the same pH dependence as is observed for the decay of YZ+; under the conditions of our experiments, the decay of YZ+ is determined by the lifetime of YZ+ QA-. In order to explain this observation, we have constructed a model for electron donation in which YZ+ and P680+ are in redox equilibrium and cytochrome b559 and YD are oxidized via P680+. Using our results, together with data from earlier investigations of the kinetics of electron transfer from YZ to P680+ and charge recombination of YZ+ QA-, we have obtained the first global fit for electron donation in Mn-depleted PSII that accounts for the data over the pH range from 5 to 7.5. From these calculations, we have obtained the intrinsic rate constants of all the electron-donation reactions in Mn-depleted PSII. These rate constants allow us to calculate the free energy difference between YZ+ P680 and YZ P680+, which is found to increase by 47 +/- 4 mV/pH from pH 5 to 6 and is observed to increase more slowly per pH unit for pH greater than 6. An important conclusion of our experimental work is that the rates of photooxidation of cytochrome b559 and YD are determined by the lifetime of the oxidizing equivalent on YZ/P680. Extension of our model to oxygen-evolving PSII samples leads to the prediction that the kinetics and yields of electron donation from cytochrome b559 and YD to P680+ will depend on the S2- or S3-state lifetime.     

Oxygen-evolving Photosystem II core complexes: a new paradigm based on the spectral identification of the charge-separating state, the primary acceptor and assignment of low-temperature fluorescence.

We review our recent low-temperature absorption, circular dichroism (CD), magnetic CD (MCD), fluorescence and laser-selective measurements of oxygen-evolving Photosystem II (PSII) core complexes and their constituent CP 4 3, CP 47 and D1/D2/cytb(559) sub-assemblies. Quantitative comparisons reveal that neither absorption nor fluorescence spectra of core complexes are simple additive combinations of the spectra of the sub-assemblies. The absorption spectrum of the D1/D2/cytb(559) component embedded within the core complex appears significantly better structured and red-shifted compared to that of the isolated sub-assembly. A characteristic MCD reduction or 'deficit' is a useful signature for the central chlorins in the reaction centre. We note a congruence of the MCD deficit spectra of the isolated D1/D2/cytb(559) sub-assemblies to their laser-induced transient bleaches associated with P 680. A comparison of spectra of core complexes prepared from different organisms helps distinguish features due to inner light-harvesting assemblies and the central reaction-centre chlorins. Electrochromic spectral shifts in core complexes that occur following low-temperature illumination of active core complexes arise from efficient charge separation and subsequent plastoquinone anion (Q(A)(-)) formation. Such measurements allow determinations of both charge-separation efficiencies and spectral characteristics of the primary acceptor, Pheo(D1). Efficient charge separation occurs with excitation wavelengths as long as 700 nm despite the illuminations being performed at 1.7 K and with an extremely low level of incident power density. A weak, homogeneously broadened, charge-separating state of PSII lies obscured beneath the CP 47 state centered at 690 nm. We present new data in the 690-760 nm region, clearly identifying a band extending to 730 nm. Active core complexes show remarkably strong persistent spectral hole-burning activity in spectral regions attributable to CP 43 and CP 47. Measurements of homogeneous hole-widths have established that, at low temperatures, excitation transfer from these inner light-harvesting assemblies to the reaction centre occurs with approximately 70-270 ps(-1) rates, when the quinone acceptor is reduced. The rate is slower for lower-energy sub-populations of an inhomogeneously broadened antenna (trap) pigment. The complex low-temperature fluorescence behaviour seen in PSII is explicable in terms of slow excitation transfer from traps to the weak low-energy charge-separating state and transfer to the more intense reaction-centre excitations near 685 nm. The nature and origin of the charge-separating state in oxygen-evolving PSII preparations is briefly discussed.     

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) b₅₅₉ in photosystem II (PSII) was investigated in H22Kα and Y18Sα cyt b₅₅₉ mutants of the cyanobacterium Synechocystis sp. PCC6803. H22Kα and Y18Sα cyt b₅₅₉ mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b₅₅₉ 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 b₅₅₉ in H22Kα mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b₅₅₉ in Y18Sα mutant PSII core complexes was shifted to 561 nm. Y18Sα and H22Kα mutant PSII core complexes contained predominately the low potential form of cyt b₅₅₉. The findings lend support to the concept that the redox properties of cyt b₅₅₉ are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b₅₅₉ 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 b₅₅₉ in protection of PSII under photoinhibition conditions in vivo.     

A three-state model for energy trapping and chlorophyll fluorescence in photosystem II incorporating radical pair recombination

The multiphasic fluorescence induction kinetics upon a high intensity light pulse have been measured and analyzed at a time resolution of 10 micros in intact leaves of Peperomia metallica and Chenopodium album and in chloroplasts isolated from the latter. Current theories and models on the relation between chlorophyll fluorescence yield and primary photochemistry in photosystem II (PSII) are inadequate to describe changes in the initial phase of fluorescence induction and in the dark fluorescence level F(0) caused by pre-energization of the system with single turnover excitation(s). A novel model is presented, which gives a quantitative relation between the efficiencies of primary photochemistry, energy trapping, and radical pair recombination in PSII. The model takes into account that at least two turnovers are required for stationary closure of a reaction center. An open reaction center is transferred with high efficiency into its semiclosed (-open) state. This state is characterized by Q(A) and P680 in the fully reduced state and a lifetime equal to the inverse of the rate constant of Q(A)(-) oxidation (approx. 250 micros). The fluorescence yield of the system with 100% of the centers in the semiclosed state is 50% of the maximal yield with all centers in the closed state at fluorescence level F(m). A situation with approximately 100% of the centers in the semiclosed state is reached after a single turnover excitation in the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). The lifetime of this state under these conditions is approximately 10 s. Closure of a semiclosed (-open) center occurs with low efficiency in a second turnover. The low(er) efficiency is caused by the rate of P(+) reduction by the secondary donor Y(Z) being competitive with the rate of radical pair recombination in second and following turnovers. The single-turnover-induced alterations in the initial kinetics of the fluorescence concomitantly with a 15-25% increase in F(o) can be simulated with the present so called three-state model of energy trapping. The experimental data suggest evidence for an electrostatic effect of local charges in the vicinity of the reaction center affecting the rate of radical pair recombination in the reaction center.