HETEROGENEITY OF THE PHOTOCHEMICAL CENTERS IN SYSTEM II OF CHLOROPLASTS*

Abstract— In 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts of algae and‘ higher plants the area over the fluorescence induction curve increases with biphasic first order kinetics (Melis and Homann, 1975). Two possibilities are considered to explain the biphasic nature of the area growth. The first is a sequential double reduction of the primary electron acceptor in system II while the second envisages a heterogeneity of its photochemical centers. The kinetic properties of the area growth after firing a single saturating flash proved to be incompatible with the predictions of the “sequential double reduction” model. This conclusion was corroborated by results obtained from a kinetic analysis of the area restoration process in the dark, and an analysis of the partially restored areas. Assuming an existence of a heterogeneous pool of photochemical centers, the growth of the area over the fluorescence curve could be further analyzed to yield two components, a fast a-component, and a relatively slow β-component. The kinetic characteristics of these components, and the effect of a short saturating flash on their respective size, led to the conclusion that one type of photochemical center had a faster recombination rate of the photochemically separated charges and was less efficient in trapping excitation energy.     

ON THE SPATIAL CORRELATION BETWEEN THE PROTEIN SURFACE AND THE ELECTRON TRANSFER CYCLE IN BACTERIAL PHOTOSYNTHETIC REACTION CENTERS

Abstract— n -Doxylstearic acids are shown to be electron acceptors in reaction centers from R. spheroides R-26 mutant illuminated with 860 nm light. The electron accepting efficiency varies with n , thus with the location of the nitroxide moiety on the stearic acid chain, and is maximum for 12-doxylstearic acid. In this molecule the nitroxide moiety is 1.4 nm from the polar carboxyl carbon based on molecular models. If the nitroxide moiety is located in or near the polar reaction center protein surface, the results suggest that the quinone-iron complex, which is the ultimate electron acceptor from a bacterio-chlorophyll dimer in the reaction center, is located ∼ 1.4 nm from the protein surface. The protein itself is estimated to have a diameter of 5.4 nm assuming spherical symmetry, so it is postulated that the quinone-iron complex is located on one side of the protein.     

LIGHT-INDUCED FOURIER TRANSFORM INFRARED SPECTROSCOPIC INVESTIGATIONS OF THE INTERMEDIARY ELECTRON ACCEPTOR REDUTION IN BACTERIAL PHOTOSYNTHESIS

Abstract— Molecular changes associated with the light-induced reduction of the intermediary electron acceptor I (bacteriopheophytin, BPh) in bacterial photosynthesis were studied by means of Fourier transform infrared (FTIR) difference spectroscopy. Chromatophore membranes and reconstituted reaction centers (RCs) of Rhodopseudomonas viridis were prereduced with sodium dithionite and illuminated in order to trap photochemically the state I⁻. Fourier transform infrared spectra of these samples were recorded before, during and after illumination, with an accuracy better than 10⁻³ absorbance units. Difference spectra of I⁻ in chromatophores and in RCs closely correspond to each other. In the carbonyl stretching frequency region between 1640 and 1750 cm⁻¹, bands are tentatively attributed to a shift (from 1713 to 1683 cm⁻¹) of a keto carbonyl group, a change of an acetyl carbonyl grou at 1656 cm⁻¹ and a decrease in absorbance strength of ester carbonyl groups (at 1746 and 1732 cm⁻P) after reduction of I. These groups likely belong to the BPh molecule, although at least one of the ester carbonyls could be assigned to an amino acid side chain. The absence of strong bands in the amide I and amide II region excludes large protein conformational changes associated with I reduction.     

THE ELECTRON SPIN POLARIZATION OF THE DONOR-TRIPLET STATE IN NATIVE and MODIFIED PHOTOSYNTHETIC REACTION CENTERS FROM

The electron spin polarization (ESP) pattern of the donor-triplet state (P^R) of reaction centers (RC's) of the purple bacterium Rhodobacter (formerly Rhodopseudomonas) sphaeroides R-26 was investigated. δm =±1 triplet EPR spectra were recorded of unmodified RC's as well as of RC's from which Fe²⁺ or ubiquinone was removed, or ubiquinone was substituted by menaquinone.
The relative amplitude of the Y peaks in the triplet EPR powder spectrum of P^R decreases when the temperature is increased from 8 K to 100 K in RC's with an intact quinone-iron complex. This decrease is more pronounced when the primary ubiquinone is substituted by menaquinone. These observations provide further support for the hypothesis that the observed lineshape of the P^R triplet state EPR spectrum reflects the presence of a third electron spin, magnetically coupled to I^- in the P⁺I^- radical pair, as suggested by Van Wijk et al. (1986) (Photobiochem. Photobiophys . 11, 95–100). Our observations suggest that this phenomenon may be general in purple bacteria.     

PHOTOCHEMISTRY OF THE REACTION CENTERS OF SYSTEM II UNDER REPETITIVE FLASH GROUP EXCITATION IN ISOLATED CHLOROPLASTS

Abstract— Absorption changes induced in isolated chloroplasts by excitation with repetitive flash groups have been measured at 690 nm, indicating the photochemical turnover of chlorophyll-a_II (Chl-αn), and at 480 nm and 513 nm respectively, reflecting via electrochromic effect the formation of a transmembrane electric field. The data are compared with measurements of oxygen evolution. In chloroplasts with practically fully intact oxygen evolving capacity it was found: 1. The initial amplitude of the 690 nm absorption change induced by the second flash as a function of the time t_v between the first and second flash of a group increases with a half life of about 35 µs. On the other hand, the average oxygen yield due to the second flash as a function of the time t_v rises with a half life of about 600 µs (and a kinetics in the ms-range of minor extent), confirming the data of Vater et al. (1968). 2. Under far red background illumination, where contributions due to PS I in the µs-range can be excluded, the difference spectrum in the red of the absorption changes induced by the first flash corresponds with that of the absorption changes induced by the second flash fired 200 µ after the first flash. 3. The pattern of the absorption changes at 690 nm induced by repetitive double flash groups at t_v= 200 µs does not markedly change in normal chloroplasts by the presence of DBMIB?. Similar 690 nm absorption changes occur in trypsin treated chloroplasts, independent of the presence of DCMU. 4. The fast regeneration in the µs-range of Chl-a_n is also observed in the third flash of a triple flash group at a time t_v= 200 µs between the flashes of the group. 5. The initial amplitudes of the absorption changes with a decay kinetics slower than 100 µs induced by the second flash at 480 nm and 513 nm, respectively, as a function of the time t, between the first and second flash of a group, are characterized by a recovery half-time of about 600 µs, confirming earlier measurements at 520 nm (Witt and Zickler, 1974). On the basis of these results it is inferred that there does exist a photoreaction of Chl-α_n., with an electron acceptor, referred to as X_a, other than the ‘primary’ plastoquinone acceptor X320, if X320 persists in its reduced state. Under conditions of X320 being in the reduced state, this photochemical reaction was shown to be highly dissipative with respect to charging up the watersplitting enzyme system Y. Furthermore, this Chl-a_n-photoreaction with X_a does not lead to a vectorial transmembrane charge separation, which is stable for more than a few microseconds. Different models for the functional and structural organization of PS II are discussed.     

STEPS IN THE POPULATION OF THE EMITTER IN THE BACTERIAL LUCIFERASE REACTION

Abstract— The reaction of luciferase-bound flavin hydroperoxide with both I-¹H and 1–²H decanal has been examined at 2°C in both low (0.01 M ) and high (0.35 M ) phosphate buffer, pH 7, where the kinetics and deuterium isotope effects are quite different. Upon reaction in both buffers there are rapid (<2 ms) increases in absorption at both 380 and W nm, followed by decay over the subsequent seconds and minutes. The changes at 380 nm exhibit a primary isotope effect and are rapid compared to bioluminescence, indicating that the scission of the aldehyde C — H bond occurs prior to the step responsible for populating the electronically excited state. However, the final absorbance change at 600 nm decays in parallel to bioluminescence under the several different conditions studied, suggesting the involvement of a long-wavelength absorbing flavin species in the production of, the excited state. Evidence is also presented indicating that under certain conditions there may be two (sequential) steps, each of which exhibits a primary isotope effect involving the same H atom.     

ON THE NATURE OF THE INTERMEDIATE ELECTRON ACCEPTOR (A1) IN THE PHOTOSYSTEM I REACTION CENTER

Abstract— A sodium dodecyl sulfate-Photosystem I (PSI) complex has been prepared and characterized with respect to its electron acceptors. Component X and iron-sulfur centers A and B are absent from this preparation but the intermediate electron acceptor (A₁) is present. Flash-induced absorbance changes at 25°C show charge separation, followed by a back-reaction with a half-time of 5 µs. The spectrum of the flash-induced change from 350 to 550 nm indicates a contribution from the intermediate electron acceptor, A₁, as well as from P700⁺. EPR studies show that A₁ is associated with a free-radical signal having a g-value of 2.0025 and a linewidth of 12 gauss. A, would appear to be associated with a monomeric form of either Chi α or pheophytin a.     

A NEW SENSITIZATION EFFECT OF PHOTOCONDUCTIVITY IN THE SYSTEM POLY-N-VINYL-CARBAZOLE/ACCEPTOR/DYE*

Abstract— In three-component systems, consisting of poly-N-vinyl-carbazole/acceptor/dye, besides the polymer band and the dye band, an additional peak in the photoconduction spectrum is formed. By comparison with the absorption spectrum, it is shown that the additional peak in the photoconduction spectrum cannot be related to the charge-transfer band between the polymer and the acceptor nor to the charge-transfer band between dye and acceptor. As an explanation, a cooperation of acceptor radicals — which are formed in the dark between poly-N-vinyl-carbazole and acceptor molecules — and dye is discussed.     

ENERGY TRANSFER AND PHOTOOXIDATION KINETICS IN REACTION CENTERS ON THE PICOSECOND TIME SCALE

Abstract— Picosecond 530 nm actinic and 1242 nm probe light pulses have been used to measure the kinetics of energy transfer and photooxidation in Rhodopseudomonas sphaeroides R-26 reaction centers. The energy transfer rate between bacteriopheophytin and the bacteriochlorophyll dimer is 1.0 ± 0.3 ± 10¹¹s^-land photooxidation of the dimer occurs within 5 ps after the dimer reaches the first excited singlet state. Using these parameters in a simple model we are able to explain the odd result that the number of reaction centers oxidized by a saturating 530 nm actinic picopulse is only 60% of the number oxidized by a saturating CW light source.     

PHOTOINDUCED CHARGE TRANSFER POLYMERIZATION OF STYRENE INITIATED BY ELECTRON ACCEPTOR

Photoinduced charge transfer polymerization of styrene (St) with electron acceptor asinitiator was investigated. In case of fumaronitrile (FN) or maleic anhydride (MA) asinitiator the polymerization takes place regularly, whereas the tetrachloro-1, 4-benzenequinone (TCQ), 2, 3-dichloro-5, 6-dicyano-1, 4-benzenequinone (DDQ). ortetracyano ethylene (TCNE) as initiator the polymerization proceeds reluctantly only afterthe photoaddition reaction. A mechanism was proposed that free radicals would be formedfollowing the charge and proton transfer in the exciplex formed between St and electronacceptors.     

SENSITIZATION BY LUMAZINE PROTEINS OF THE BIOLUMINESCENCE EMISSION FROM THE REACTION OF BACTERIAL LUCIFERASES

Abstract— Lumazine protein from Photobacterium phosphoreurn blue shifts the in vitro bioluminescence spectra in the reactions using each of the 4 main types of bacterial luciferases: P. phosphoreum, P. leiognathi, Vibrio harveyi and V. fischeri . For the reaction initiated with FMNH₂ and tetradecanal at 2°C, this "sensitizing" property of lumazine protein differs quantitatively between the luciferases. An interaction constant characterizing each type of luciferase may be derived from a reciprocal plot of the spectral shift against the lurnazine protein concentration. The weakest interaction constant is in the V. fischeri reaction, 180 μM. For the V. harveyi reaction the interaction is in the range 6–9 μ M , and for both Photobacterium reactions it is 2–3 μM. A concentration of only 0.6 μ M of lumazine protein is sufficient to cause an observable change in the Photobacterium bioluminescence spectra. For the V. harveyi case the interaction constant is near to the equilibrium K _d for the luciferase-lumazine protein complex, observed directly by Visser and Lee. Both constants are decreased markedly by increase in phosphate concentration so that it is concluded that, with V. harveyi luciferase, sensitization occurs within this protein-protein complex. For P. phosphoreum luciferase, however, the equilibrium complex is too weak to correspond to the sensitizing interaction and it is concluded that the rate-limiting process is a protein-protein bimolecular collision. As judged from their molecular weight around 20000, spectral properties, and ability to blue shift the bioluminescence spectra, lumazine proteins are identified in a second strain of P. phosphoreum and in P. leiognathi .     

TRIPLET FORMATION AND TRIPLET DECAY IN REACTION CENTERS FROM THE PHOTOSYNTHETIC BACTERIUM Rhodopseudomonas sphaeroides

Abstract— In the reaction center of photosynthetic bacteria, with the primary ubiquinone reduced, the triplet state P^R of the primary electron donor (a pair of bacteriochlorophylls named P) is PO ulated with a takes place in a few ns. We measured by flash absorption spectroscopy the influence of temperature on formation and decay kinetics of P^R and ³Car in the reaction center of several strains of R. sphaeroides . The rate of triplet energy transfer, measured as the decay of P^R after a flash, decreases when the temperature is lowered. Between 60 and 30 K the half-time of energy transfer becomes longer than the ³Car half-time decay (about 6 μs) and below 20 K the transfer is slower than the internal decay of P^R (about 100 μs). In several cases it is clear that P^R and ³Car decay independently and are not in thermal equilibrium. The singlet energy transfer from carotenoid to P occurs with a high efficiency at all temperatures.
The data can be accounted for on the basis of estimated energy levels of P^R and ³Car, in the context of the equilibrium ³P ←³D where ³P is the localized triplet state of P-870 and ³D is another triplet state. A reasonable kinetic scheme leads us to estimate that ³D is 0.0025 ± 0.005 eV above ³P. ³D may thus be the state observed by Shuvalov and Parson (1981). We propose that both triplet and singlet energy transfer between P and the carotenoid occur via a bacteriochlorophyll, to which the carotenoid should be tightly coupled via exchange interaction.     

A PHOTOSYNTHETIC PHOTOELECTROCHEMICAL CELL USING FLAVIN MONONUCLEOTIDE AS THE ELECTRON ACCEPTOR

Abstract— We have been developing a solar cell using the photochemistry of photosystem 1 particles and flavin mononucleotide. We have studied the effect of changing various parameters including the nature of the electron donor, its redox poise, the pH of the medium and the salt concentration. When we put all of the improvements together, we obtain 1900 μW of power (2.4 W/m₂),15 mA short circuit current,and an open circuit potential of 640 mV. The maximum power conversion efficiency was 1%. Several light-dark cycles could be observed. One of the important properties of this cell is that it can store energy in the form of chemical potential.     

硫氰酸铵—结晶紫—H2O体系浮选分离铜

研究了硫氰酸铵－结晶紫－H2O体系浮选分离铜的行为及其与常见离子分离的条件。试验表明,在水溶液中Cu(Ⅱ）与硫氰酸铵、结晶紫形成不溶于水的三元缔合物,经过滤,此三元缔合物沉淀和水完全分离。在一定条件下,控制pH3．0－4．0,能使Cu( Ⅱ）与常见离子Zn(Ⅱ）、Fe( Ⅲ）、Al（Ⅲ）、Cd（Ⅱ）、 Ni（Ⅱ）、Mn（Ⅱ）分离。     

A METHOD FOR THE ELECTROCHEMICAL INITIATION OF THE in vitro BACTERIAL LUCIFERASE REACTION

Abstract— We have developed an electrochemical initiation method for the in vitro bacterial luciferase reaction using Vibrio harveyi luciferase. The cathodic conversion of oxidized riboflavin 5'-phosphate (FMN) to its reduced form (FMNH₂), on which the kinetics for the overall light-emitting reaction depend, is followed by the bacterial luciferase reaction. The electrochemical initiation method can be used for both turnover and nonturnover luciferase reactions, depending on the FMN electroreduction methods.     

电镜法对硫敏化中心的直接观察

敏化中心的形成是Ag₂S的聚集过程, 一般包括两步^[1]. 在第一步中, 增感剂中的硫离子与银离子在AgX晶体表面生成Ag₂S分子, 然后, 多个Ag₂S分子接合(coalescence)形成敏化中心.     

THE FUNCTION OF P700 AND CYTOCHROME f IN THE PHOTOSYNTHETIC REACTION CENTER OF SYSTEM 1 IN RED ALGAE

Abstract— Kinetics and quantum yields of light-induced oxidation of P700 and the f -type cytochrome were measured in marine red algae from absorbance changes in the region 420, 435 and 705 nm. The quantum yield for cytochrome oxidation in Iridaea splendens and Schizymenia pacifica was 0.5-0.65 in far-red light, at 20 and at 0C.
Oxidation rates of P700 measured when varying amounts of cytochrome were in the oxidized state indicated that a reaction center of system 1 in Iridaea contains 1 P700 and 4 cytochrome molecules. Oxidized P700 only accumulates when all 4 cytochromes are oxidized. The rate of photochemistry of system 1, measured as the sum of the rates of P700 and cytochrome oxidation, was independent of the oxidation level of cytochrome, but decreased with accumulation of oxidized P700. This decrease was less than proportional to the fraction of P700 that was in the oxidized state, which suggested transfer of excitation energy between reaction centers.
The quantum yield for cytochrome oxidation after dark periods of 1 min or more was only about 0.2. This effect was tentatively ascribed to a dark reduction of the cytochrome coupled to phosphorylation.