Flash-induced Fluorescence Yield Research Articles

D1-Tyr246 and D2-Tyr244 in photosystem II: Insights into bicarbonate binding and electron transfer from QA•− to QB

In photosystem II (PSII), D1-Tyr246 and D2-Tyr244 are symmetrically located at the binding site of the bicarbonate ligand of the non-heme Fe complex. Here, we investigated the role of the symmetrically arranged tyrosine pair, D1-Tyr246 and D2-Tyr244, in the function of PSII, by generating four chloroplast mutants of PSII from Chlamydomonas reinhardtii: D1-Y246F, D1-Y246T, D2-Y244F, and D2-Y244T. The mutants exhibited altered photoautotrophic growth, reduced PSII protein accumulation, and impaired O2-evolving activity. Flash-induced fluorescence yield decay kinetics indicated a significant slowdown in electron transfer from QA•− to QB in all mutants. Bicarbonate reconstitution resulted in enhanced O2-evolving activity, suggesting destabilization of bicarbonate binding in the mutants. Structural analyses based on a quantum mechanical/molecular mechanical approach identified the existence of a water channel that leads to incorporation of bulk water molecules and destabilization of the bicarbonate binding site. The water intake channels, crucial for bicarbonate stability, exhibited distinct paths in the mutants. These findings shed light on the essential role of the tyrosine pair in maintaining bicarbonate stability and facilitating efficient electron transfer in native PSII.

Simulation of chlorophyll fluorescence rise and decay kinetics, and P700-related absorbance changes by using a rule-based kinetic Monte-Carlo method.

A model of primary photosynthetic reactions in the thylakoid membrane was developed and its validity was tested by simulating three types of experimental kinetic curves: (1) the light-induced chlorophyll a fluorescence rise (OJIP transients) reflecting the stepwise transition of the photosynthetic electron transport chain from the oxidized to the fully reduced state; (2) the dark relaxation of the flash-induced fluorescence yield attributed to the QA- oxidation kinetics in PSII; and (3) the light-induced absorbance changes near 820 or 705nm assigned to the redox transitions of P700 in PSI. A model was implemented by using a rule-based kinetic Monte-Carlo method and verified by simulating experimental curves under different treatments including photosynthetic inhibitors, heat stress, anaerobic conditions, and very high light intensity.

Chlorophyll fluorescence induction and relaxation system for the continuous monitoring of photosynthetic capacity in photobioreactors.

The development of high-performance photobioreactors equipped with automatic systems for non-invasive real-time monitoring of cultivation conditions and photosynthetic parameters is a challenge in algae biotechnology. Therefore, we developed a chlorophyll (Chl) fluorescence measuring system for the online recording of the light-induced fluorescence rise and the dark relaxation of the flash-induced fluorescence yield (Qa- - re-oxidation kinetics) in photobioreactors. This system provides automatic measurements in a broad range of Chl concentrations at high frequency of gas-tight sampling, and advanced data analysis. The performance of this new technique was tested on the green microalgae Chlamydomonas reinhardtii subjected to a sulfur deficiency stress and to long-term dark anaerobic conditions. More than thousand fluorescence kinetic curves were recorded and analyzed during aerobic and anaerobic stages of incubation. Lifetime and amplitude values of kinetic components were determined, and their dynamics plotted on heatmaps. Out of these data, stress-sensitive kinetic parameters were specified. This implemented apparatus can therefore be useful for the continuous real-time monitoring of algal photosynthesis in photobioreactors.

Effects of heat stress on PSII photochemistry in a cyanobacterium Spirulina platensis

The effects of heat stress (30–50 °C) on photosystem II (PSII) photochemistry in a cyanobacterium Spirulina platensis grown at 30 °C were studied by measuring chlorophyll fluorescence induction kinetics and thermoluminescence. Heat stress inhibited significantly the maximum efficiency of PSII photochemistry. An investigation of the kinetics of flash-induced fluorescence yield decay revealed that heat stress caused a shift of the equilibrium towards Q A but showed no effect on the kinetics of flash-induced fluorescence yield decay in the presence of DCMU. An analysis of polyphasic rise of fluorescence transients indicated that heat stress induced a decrease in the total number of PSII active reaction centers. Thermoluminescence measurements demonstrated that the B band in Spirulina cells corresponding to the S 2Q B − state consisted of two different components, the B 32 and the B 24 bands with the peak temperatures at 32 and 24 °C, respectively. The intensity of the B 32 band and the B 24 band decreased significantly with increasing temperature but the decrease of the intensity of the B 24 band was much greater than that of the B 32 band. There were no significant changes in the peak temperatures of the B 24 and B 32 bands. The intensity of the Q band corresponding to the recombination of the S 2Q A − charge pair decreased significantly with increasing temperature. The period-four oscillation of the B band was significantly damped at temperatures higher than 45 °C. The contents of D1 and PsbO proteins decreased with increasing temperature. The results in this study suggest that heat stress shows no effects on the stability of the S 2Q A − and S 2Q B − states and that the different populations of the active PSII reaction centers show different sensitivity to heat stress in S. platensis cells.

Investigations on the Reaction Pattern of Photosystem II in Leaves fromArabidopsis thalianaby Time-Resolved Fluorometric Analysis

The transients of normalized fluorescence yield induced by an actinic laser flash in dark adapted leaves of Arabidopsis thaliana plants were measured with new equipment, that was developed as part of this work and permits the covarage of a wide time domain of 8 decades from 100 ns to 10 s. The raw data obtained were processed and analyzed within the framework of the "3-quencher" model with Q(A) as photochemical and P680(+)(*) and (3)Car as nonphotochemical quenchers. Comparative measurements with hydroxylamine treated PS II membrane fragments from spinach revealed that the widely used "dogma"of virtually identical efficiency of photochemical (Q(A)) and nonphotochemical (P680(+)(*)) quenching has to be revised: the constant of the latter exceeds that of the former by a factor of about 2. As a consequence, the probability of recombination between P680(+)(*) and Q(A)(-) and its kinetics have to be explicitly taken into account for the interpretation of flash induced fluorescence yield transients. The analysis of the experimental data within this extended "3-quencher" model reveals that a fully consistent description is achieved for the data gathered from measurements with intact leaves from wild type plants excited with actinic laser flashes of different energies (number of photons per flash and unit area). On the basis of these results it is shown that, in dark adapted leaves excited with a single laser flash, P680(+)(*) is predominantly (about 80% of the total reaction) reduced by Y(Z) via nanosecond kinetics and Q(A)(-) reoxidation is dominated by a kinetics of about 150 mus that are ascribed to PS II complexes with the Q(B) site occupied by PQ. The excess of excited chlorophyll singlet states decays to a significant extent via the carotenoid "triplet valve"with transient population of (3)Car. The present data provide the basis for analyses of A. thaliana mutants with modified lipid content and composition. The results of these investigations are described in an accompanying report (Steffen, R., Kelly, A. A., Huyer, J., Dormann, P., and Renger, G. (2005) Investigations on the reaction pattern of photosystem II in leaves from Arabidopsis thaliana wild type plants and mutants with genetically modified lipid content, Biochemistry 44, 3134-3142).

The rate of charge recombination in Photosystem II

Loss by recombination of the charge separated state P 680 +Q A − limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P 680 + by the secondary electron donor Tyrosine Z cannot occur because Y Z is already oxidized. The 1.4 ms recombination is seen in Y Z-less mutants of the cyanobacterium Synechocystis. However, the rate of P 680 +Q A − recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P 680 + reduction by Y Z were obtained, and the rate of the competing P 680 +Q A − recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of Y Z ox slightly decreased the efficiency of excitation trapping but did not seem to accelerate P 680 +Q A − recombination. The two P 680 +Q A − lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.

Two functionally distinct manganese clusters formed by introducing a mutation in the carboxyl terminus of a photosystem II reaction center polypeptide, D1, of the green alga Chlamydomonas reinhardtii

To study the function of the carboxyl-terminal domain of a photosystem II (PSII) reaction center polypeptide, D1, chloroplast mutants of the green alga Chlamydomonas reinhardtii have been generated in which Leu-343 and Ala-344 have been simultaneously or individually replaced by Phe and Ser, respectively. The mutants carrying these replacements individually, L343F and A344S, showed a wild-type phenotype. In contrast, the double mutant, L343FA344S, evolved O 2 at only 20–30% of the wild-type rate and was unable to grow photosynthetically. In this mutant, PSII accumulated to 60% of the wild-type level, indicating that the O 2-evolving activity per PSII was reduced to approximately half that of the wild-type. However, the amount of Mn atom detected in the thylakoids suggested that a normal amount of Mn cluster was assembled. An investigation of the kinetics of flash-induced fluorescence yield decay revealed that the electron transfer from Q − A to Q B was not affected. When a back electron transfer from Q − A to a donor component was measured in the presence of 3-(3,4-dichlorophenol)-1,1-dimethylurea, a significantly slower component of the Q − A oxidation was detected in addition to the normal component that corresponds to the back electron transfer from the Q − A to the S 2-state of the Mn cluster. Thermoluminescence measurements revealed that L343FA344S cells contained two functionally distinct Mn clusters. One was equivalent to that of the wild-type, while the other was incapable of water oxidation and was able to advance the transition from the S 1-state to the S 2-state. These results suggested that a fraction of the Mn cluster had been impaired by the L343FA344S mutation, leading to decreased O 2 evolution. We concluded that the structure of the C-terminus of D1 is critical for the formation of the Mn cluster that is capable of water oxidation, in particular, transition to higher S-states.

Fluorometric equipment for monitoring P680+• reduction in PS II preparations and green leaves

Newly developed equipment is described that permits the monitoring of laser flash induced transients of the normalised chlorophyll-a fluorescence quantum yield in isolated PS II preparations and whole leaves with a high time resolution. The essential operational unit of the set-up is a rapidly gated photomultiplier. In this way, the fluorescence artefact, due to the high intensity excitation laser flash, is sufficiently suppressed and the dead time of the signal response is reduced to about 500 ns. It is shown that the fluorescence rise kinetics in the μs time-domain, after flash excitation is strongly dependent on the redox state of the primary electron donor of PS II (P680). At high excitation energies, the decay of carotenoid triplets, which are very efficient quenchers of chlorophyll singlet states, dominates the rise kinetics of the flash induced fluorescence yield in the μs time domain.

A two-electron reaction at the catalytic site of water oxidation in manganese-depleted Photosystem II vesicles in the presence of an electron donor. Coupled electron and proton transfer processes

We have studied the electron and proton transfer at the manganese-depleted catalytic site of water oxidation in the presence of the electron donor 1,5-diphenylcarbazide (DPC). On the basis of measurements of flash-induced fluorescence yield, electrochromic bandshifts and pH transients into the aqueous medium, we have been led to the following conclusions: (1) The electron donor DPC was involved in a two sequential photostep process just like Mn 2+ in photoactivation. After a first flash, the product of the reaction of DPC with the first Photosystem II charge separation was unstable (half-decay around 1 s at pH 6.5) until converted into a stable one by a second flash. The formation of the unstable intermediate was rather favored at alkaline pH. Its concentration increased progressively with pH to reach a maximum at pH 7.2–7.4. Nevertheless, consumption of this intermediate required a proton binding, since its regeneration after the second flash needed a latency dark time inversely proportional to proton concentration. (2) Each step of the process was accompanied by proton exchange with the medium. At acidic pH, the odd flashes in a series induced acidification, whereas on even flashes occurred alkalinization. The pattern of proton release/uptake varied extremely as a function of pH, between 4 H + and −2 H +/e −, with opposite changes at acidic and basic pH. At neutral pH, 1 H +/e − was assumed to be released at each step. On the basis of these results and data already in the literature, the molecular details of the pH-dependent part of the deprotonation are discussed in terms of electrostatic interactions between the polarized catalytic center and bound H + and OH − ions. The described two-photostep process is proposed to reflect the two-electron water oxidation to an hydrogen peroxide intermediate.

Events near the reaction center in O2 evolving PS II enriched thylakoid membranes: The presence of an electric field during the S2 state in a population of centers

Flash-induced absorption changes at 515 nm observed as a function of flash number are examined in relation to the flash-induced fluorescence yields in inside-out thylakoids. After partial dissipation of the delocalized transmembrane electric field by adding gramicidin, the analysis of period 4 oscillations and of the kinetics in the 10 ms-1 s range suggest that the variation of the absorption changes at 515 nm as a function of flash number is the result of at least two processes:1) an electric field increase related to the S2 state and 2) the fact that the field generated by the water protons inside the membrane decreases when these protons are released outside the membrane. The former field correlates with the flash-induced fluorescence yield increase induced by the donor side of Photosystem II. Both measurements show similar oscillations as a function of flash number, with maxima on the 1st, 5th and 9th flash. These oscillations, after a shift of two flashes, appear to be different from those of the O2 yield observed under similar conditions. It is proposed that, in a population of centers the electric field during the S2 state reflects the presence of a stabilized positive equivalent in the protein close to the Mn complex.

Interaction of erogenous quinones with photosystem II in inside-out thylakoids

The presence of erogenous quinones PPBQ, DCBQ and DMQ in inside-out thylakoids alters the oscillation pattern with period 4 of the flash-induced fluorescence yield. The results can be interpreted by assuming that at ambient temperature PPBQ replaces PQ in 100% of the PS II centers detected by fluorescence, DMQ and DCBQ being bound to 40–60% of these centers only. The evaluation of the percentage of double hits in a 3 μs flash allows estimation of the electron-transfer rate from Q - A to Fe 3+, which varies from 9 μs (PPBQ) to 20μs (DMQ).

Characterization of two types of oxygen-evolving Photosystem II reaction center by the flash-induced oxygen and fluorescence yield

In freshly prepared inside-out thylakoids, the periodicity of four of the oscillation patterns of fluorescence yield differs fundamentally from that of the oxygen yield (Delrieu, M.J. and Rosengard, F. (1987) Biochim. Biophys. Acta 892, 163–171). In this paper, further investigation of the oxygen and fluorescence patterns provides information on the origin of this discrepancy, i.e., the heterogeneity of the oxygen-evolving centers. The following results are reported. (1) The oscillation pattern of fluorescence yield was characteristic of an S 2 state, having maxima after one and five flashes, showing that the decay of the flash-induced fluorescence yield matches the deactivation of S 2 (half-time, 40 s), after a stability time of 2 s. (2) Under the conditions to be described, large changes in the oscillation pattern of the oxygen yield were observed when the fluorescence pattern remained the same. In freshly prepared inside-out thylakoids, a large miss (17.5%) was detected in the oxygen-yield pattern, even after addition of ferricyanide which could relieve the acceptor side of a large number of centers. In contrast, when the sample was stored with 30% ethylene glycol in liquid nitrogen and then thawed, the oscillation pattern of the oxygen yield was characterized by a progressive decrease in the mean value of the oxygen yield as a function of flash number, even in the presence of ferricyanide, but the true miss value did not exceed 7%. In this stored sample the oscillation patterns of the oxygen and fluorescence yield look very similar. Consequently, a functionally homogeneous population of oxygen-evolving centers was isolated, i.e., those which induce period-4 fluorescence oscillations. These centers, related to the plastoquinone pool, could be identified with the α centers. During flash illumination the reducing equivalents accumulated in the plastoquinone pool, through the secondary acceptor Q B 2−, prevent the centers from being reactivated between the flashes, and thus are responsible for the disappearance of a small number of centers after each flash of a series. The other types of center observed in freshly prepared samples, characterized by large misses, are not related to the plastoquinone pool and could thus be the β centers.