Gábor Laczkó

Quinone-dependent delayed fluorescence from the reaction center of photosynthetic bacteria.

Millisecond delayed fluorescence from the isolated reaction center of photosynthetic bacteria Rhodobacter sphaeroides was measured after single saturating flash excitation and was explained by thermal repopulation of the excited bacteriochlorophyll dimer from lower lying charge separated states. Three exponential components (fastest, fast, and slow) were found with lifetimes of 1.5, 102, and 865 ms and quantum yields of 6.4 x 10(-9), 2.2 x 10(-9), and 2.6 x 10(-9) (pH 8.0), respectively. While the two latter phases could be related to transient absorption changes, the fastest one could not. The fastest component, dominating when the primary quinone was prereduced, might be due to a small fraction of long-lived triplet states of the radical pair and/or the dimer. The fast phase observed in the absence of the secondary quinone, was sensitive to pH, temperature, and the chemical nature of the primary quinone. The standard free energy of the primary stable charge pair relative to that of the excited dimer was -910 +/- 20 meV at pH 8 and with native ubiquinone, and it showed characteristic changes upon pH and quinone replacement. The interaction energy ( approximately 50 meV) between the cluster of the protonatable groups around GluL212 and the primary semiquinone provides evidence for functional linkage between the two quinone binding pockets. An empirical relationship was found between the in situ free energy of the primary quinone and the rate of charge recombination, with practical importance in the estimation of the free energy levels from the easily available lifetime of the charge recombination. The ratio of the slow and fast components could be used to determine the pH dependence of the free energy level of the secondary stable charge pair relative to that of the excited dimer.

Delayed fluorescence study on P*QA→ P+QA− charge separation energetics linked to protons and salt in reaction centers from Rhodobacter sphaeroides

The energetics of the first stable charge separated state, P+QA− relative to that of P−QA was examined in isolated RC from Rhodobacter sphaeroides by delayed fluorescence. The temperature dependence of the delayed fluorescence indicates that the charge separation is a highly enthalpy-driven process (ΔH = – 818 ± 20 meV at pH 8) and the free energy gap between P−QA and P+QA− drops with increasing pH (40 ± 4 meV between pH 6 and 10). The pH-dependence of the free energy change of the P+QA− state runs parallel to the (integrated) net proton uptake due to the PQA/P+QA− redox change in a wide pH range and under different ionic conditions. Elevation of the ionic strength increases the delayed fluorescence intensity and decreases the (dark and light) pKa values as well as the light-induced ΔpKa changes of the protonatable groups of the protein. The observed dependence of the energetics of P+QA− on the concentration and composition of mobile ions is discussed in terms of binding and screening of protonatable groups and surface charges as dominant modes of electrostatic interaction between RC and salt.

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.

Pulsed polarographic studies of photosynthetic oxygen evolution

Synchronously grown cultures of dark-adapted Chlorella fusca cells were illuminated with single flashes and series of flashes from a xenon discharge tube and a nitrogen laser, and the photosynthetic oxygen evolution was measured polarographically under the condition of photosynthetic saturation at ambient temperature. The mechanical and electrical construction of the Joliot-type oxygen polarograph is described. Kinetic analysis of the amperometric signal obtained after single flashes revealed that the diffusion of the oxygen molecules through the thylakoid membrane is the controlling process of the response of the electrode and leads to a lag phase at the beginning of the rise of the signal. The oscillation of the oxygen evolution produced by series of flashes is damped much more with laser flashes. This is explained in terms of the side carrier model of the losses in the oxygen production, assuming that photoactivation of the side carrier by visible light leads to less damped oscillations with xenon flashes. These experiments indirectly corroborate the validity of the side carrier model.

CCD for speeding up multichannel analysers

The design of a low cost and fast analogue buffer memory based on a charged-coupled device analogue shift register is described for extending the time resolution of multichannel analysers down to 20 ns.

Comparison of Energetics of P*Q A → P + Q A - and P*Q B → P + Q B - Charge Separation by Detection of Delayed Fluorescence of the Bacteriochlorophyll Dimer in Reaction Centers of Rb. sphaeroides

Flash absorbed by the reaction center (RC) initiates electron transfer from the excited bacteriochlorophyll dimer (P*) to the adjacent cofactors (bacteriopheophytin I, primary quinone QA and secondary quinone Q8) (Fig. 1). The free energy levels of the charge separated states P+Q A - and P+Q B - relative to that of the P*Q excited state can be directly determined from delayed luminescence (DL) of P* (1–3) and related to results obtained from proton binding measurements (2–4). Open image in new window Figure 1 Origin of the leakage-type delayed fluorescence and the free energy levels of the redox states P+QH- and PQAQ, relative to that of the excited (P*Q) state of the RC with approximate numerical values of the rate constants. Note that there is no direct recombination from charge separated state P+Q B - .

Photochemical and thermal phases in the short-term chlorophyll fluorescence induction kinetics of Chlorella fusca

Abstract The yield of chlorophyll fluorescence from photosystem II of Chlorella fusca was measured during a short xenon flash (half-width, 2 μs) and rectangular laser pulse (duration, 100 μs) of variable intensity. At the highest light intensity (above 105Wm−2), the photochemical rise and the decay attributed to the carotenoid triplet quenching could be well separated and a non-photochemical phase was observed which completed within 60 μs. If the algae are pre-excited with a saturating nitrogen laser pulse, the yield of fluorescence can exceed the maximum value measured routinely (stationary conditions and longer time range). It is argued that the unusually high fluorescence state can arise from a model of the reaction centre of two photoreactions separated by a thermal step. A numerical simulation of the model, based on the freely moving exciton approximation and competition between the carotenoid triplet traps and the different states of the reaction centre for exciton capture, gave reasonably good agreement with the experimental results.

Application of Polarized Luminescence in Biology and Medicine

Our purpose is to provide a brief report on the recent developments and trends in the field and to illustrate their manysided usefulness in diverse topics by many examples, mainly from the literature of the past two years. More details can be found in the books of Steiner (1983), Bayley and Dale (1985) and Lakowicz (1983).

USE OF LASERS IN PHOTOPHYSICAL RESEARCH OF PHOTOSYNTHESIS

During the past decade, extensive research has been carried out to utilize the unique characteristics of laser light. Mainly the high intensity, short pulse and small divergence of the laser beam have been made use of, and some of the results are briefly summarized here. The effect of the coherence of actinic light on the primary photochemical charge separation is discussed in some detail.

Determination of the distance law of the transfer of electronic excitation energy

Abstract In a frequently applied method the exponent, n , of the distance R , of interacting molecules, appearing in the expression of the efficiency, f , of transfer, is determined from the slope of a straight line obtained by plotting In ( f −1 −1) vs. In c c 0 ( c and c 0 are the concentration of the solution and a constant critical concentration characteristic of the interacting molecules and their environment, as defined in the Forster theory of transfer). The dependence of f on c is usually determined experimentally from the concentration quenching of fluorescence. From the theoretical quenching curve and the analytical expression for the slope, it is concluded that In ( f −1 −1) vs. In c c 0 is not strictly linear, but a curve with slopes yielding exponents from n = −6− n = −3, in contrast to the fact that the interaction theoretically remains very weak (with n = −6). A correct exponent is obtained experimentally from the high-concentration part of the quenching curve or by using the theoretical dependence of the slope on the concentration in the case of very weak interactions. For strong interactions, In ( f −1 −1) vs. In c c 0 is linear, and n = −3 in the whole concentration range. However, f is slightly volume-dependent.