Picosecond dynamics of primary electron-transfer processes in bacterial photosynthesis.

The primary electron transfer processes in Rhodopseudomonas sphaeroides R-26 were studied as a function of temperature by means of picosecond spectroscopy. The first chemical step of the bacterial photosynthesis involves an electron transfer from the excited state of a bacteriochlorophyll a dimer, (BChl)2, to a bacteriopheophytin (BPh) to form the radical ion pair (BChl)2+. BPh-.. The upper limit for the formation time of this ion-pair was found to be 10 ps, at temperatures in the range 300-4.2 degree K. Similarly, the second chemical step, involving electron transfer from BPh-. to an ubiquinone-iron complex (QFe), was found to have a lifetime of approximately 150 ps, also independent of temperature in the same range. We interpret the absence of temperature dependence as indicating that process 2 proceeds via a tunneling mechanism. Utilizing our results in conjunction with electron tunneling theories, we calculate the distance between BPh-. and Q(Fe) to be 9--13 A. Our results also imply a closer proximity between (BChl)2 and BPh.     

Regulation of the primary photosynthesis processes

The mechanisms of primary processes of photosynthesis and macromolecular conformational changes that control the efficiency of primary energy transformation in photosynthesis are discussed. Special attention is focused on the analysis of chlorophyll fluorescence as an integrated parameter indicative of the efficiency and dynamics of primary steps of photosynthesis. Sharp changes in environmental conditions and other unfavorable factors may lead to the distortions of the coupling between consecutive electron transfer steps. As a result, an excess of electrons and/or electronic excitation energy may form at some sites of the electron transport chain. This may lead to the generation of reactive oxygen species responsible for the subsequent oxidative stress. The results of the application of these data in the areas of biotechnology and ecology are demonstrated.     

The orientation of the primary donor in bacterial photosynthesis.

The triplet state EPR spectra of magnetically aligned whole cells of Rhodopseudomonas viridis and Rhodopseudomonas palustris display a marked dependence on the orientation of the static EPR field with respect to the alignment field direction. This observation implies that the primary donor species on which the triplets are localized are ordered within the membranes. We have developed a theoretical model for the system to enable calculation of the orientation of the magnetic axes of the primary donor species with respect to the membranes in which they reside. The triplet state spectra are generated by an ensemble of partially ordered magnetic systems and a computer simulation of the experimental results. The triplet orientation is very similar for the two organisms studied, where one axis lies predominantly in the plane of the membrane and the other two axes have approximately equal projections onto the normal to the membrane.     

Energy upconversion theory of the primary photochemical reaction in plant photosynthesis

In this paper we suggest a basic mechanism for the utilization of light quanta in photosynthesis. Through interactions between the lowest lying triplet state of the reaction-center chlorophylls and the first excited singlet state of the antenna chlorophylls, absorbed light quanta are upconverted to a higher-lying charge transfer state of the reaction-center Chl molecules. It is shown that the efficiency of the upconversion process is maximized by the parallel configuration of the two Chl porphyrin rings in the reaction-center water adduct proposed by the writer. Steady-state solutions are obtained, and the theoretical results are shown to account for a variety of crucial experimental observations including (1) the doubling (in whole cells) of in vivo fluorescence quantum yield of system II in strong light, (2) the observation by Dutton et al. of the light-induced triplet-state reaction-center bacteriochlorophyll when the primary electron acceptor is reduced and (3) despite the apparent involvement of two excitations in the energy upconversion process, only one quantum is needed for the transfer of one electron in the primary photo-chemical reaction, satisfying the eight-quanta requirement for the evolution of one O₂ molecule in photosynthesis.     

The reaction between primary and secondary electron acceptors in bacterial photosynthesis

Following a 20-nsec actinic flash, which causes oxidation of P870 and cytochrome C422, Chromatium chromatophores enter a refractory state. While the chromatophores are in this state, a second flash does not cause further oxidation of P870 or cytochrome C422. The quanta of the second flash are wasted as fluorescence (and heat); apparently they do not energize an alternative photochemical reaction. The refractory state probably reflects the accumulation of the primary electron acceptor in a reduced form. By following the reappearance of the capacity for photochemistry, one can measure the kinetics of electron transfer between the primary electron acceptor and the secondary agent which reoxidizes it. In Chromatium chromatophores, this process requires about 60 μsec to proceed half-way to completion at pH 7, and 80 μsec at pH 8. The rate of the reaction increases with decreasing pH, but not in direct proportion to the proton concentration. It increases with temperature, with an E_a of about 8.3 kcal/mole. The kinetics are approximately second order in the concentration of the reduced acceptor.     

Theoretical study of the effect of several reverse reaction on the kinetics of the primary processes of photosynthesis

A theoretical model of energy migration and electron transport in photosynthesis of higher plants was considered. The set of different equations describing these processes takes into consideration the states of 4 components of electron transport chain and back reactions of electron transfer from the reduced acceptors to the oxidized reaction centres. The numerical integration of these equations was made for various kinetics parameters characterizing the electron transport chain.     

The effect of glutaraldehyde fixation on the elucidation of the morphogenesis of annulate lamellae in oocytes ofRana pipiens

Summary Primary fixation of the frog oocyte with glutaraldehyde, compared to osmium tetroxide, alters the appearance of components involved in the morphogenesis of annulate lamellae. With glutaraldehyde, the outer layer of the nuclear envelope is connected with membranous laminae of variable length. Rounded blebs of the outer layer of the nuclear envelope are infrequently observed. Further, rather than clusters or rows of cytoplasmic vesicles as are observed in osmium tetroxide-fixed cells; numerous and long, smooth-surfaced lamellae are present in the ooplasm after glutaraldehyde fixation. The long membranous laminae then become concentrated in several ooplasmic packets. This is followed by the progressive alignment or orientation of the laminae within the packet. Eventually, those aligned and formerly smooth-surfaced lamellae are converted into annulate lamellae.This study was supported by research grants (HD-00699, GM-09229) and a Career Development Award from the National Institutes of Health, U.S. Public Health Service.     

Immobilization of thylakoids in porous particles and stabilization of the photochemical processes by glutaraldehyde action at subzero temperature

Summary Lettuce thylakoids were immobilized by the action of glutaraldehyde at sub-zero temperature in presence of albumin. Particles with sponge structure and good mechanical properties were obtained. The activity yield after immobilization was found equal to 70% for the oxygen production. Photosystems I and II were both active after immobilization. The yield for the ATP production was 27%. An increase of stability of the immobilized thylakoids was observed when stored under illumination.     

The balance between primary forward and back reactions in bacterial photosynthesis.

The temperature dependence of the bacteriochlorophyll fluorescence and reaction center triplet yield in while cells of Rhodopseudomonas sphaeroides strain 2.4.1 and of the magnetic field-induced fluorescence increase are calculated, taking into account rate constants of losses in the antenna system and of charge separation and recombination in the reaction center. Triplet and singlet yield after recombination in the reaction center are described by the radical pair mechanism. Good fits of the theoretically calculated temperature dependence with published experimental results could be obtained, assuming that ks, the rate constant for recombination of the charges on the primary donor P+ and the reduced intermediate acceptor I- to the lowest excited singlet state P*I of the reaction center bacteriochlorophyll, is temperature-dependent via the Boltzmann factor Kso exp(-delta E/kT), where delta E is the energy difference between P*I and P+I- and kso is the frequency factor. kg and/or kt, the rate constants for recombination to the singlet ground and triplet states, respectively, were assumed to be temperature-independent, or temperature-dependent via their exothermicity factors ki = CiT-1/2 exp(-Ei/kT) with i = g, t. Depending on the particular choice for the temperature dependence of kg and kt, best fits were obtained for delta E = 45-75 meV and recombination rate constants at 300 K of ks = 0.4-0.8 ns-1, kg = 0.08-0.12 ns-1, and kt = 0.3-0.5 ns-1. The model predicts a lifetime of the radical pair P+I- that is somewhat larger than that of delayed fluorescence; a magnetic field increases both.     

The Feulgen reaction after glutaraldehyde fixation.

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH4 in 1% NaH2PO4 for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCl for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

Effect of methylmercury on primary photosynthesis processes in green microalgae Chlamydomonas reinhardtii

The sensitivity of green microalgae Chlamydomonas reinhardtii to methylmercury chloride (MeHg) and chloride mercury (HgCl2) was evaluated by measuring chlorophyll fluorescence parameters by the pulse-amplitude-modulation (PAM) fluorometry. It was shown that MeHg at concentrations above 1 microM decreased the Fv/Fm ratio, which characterizes the maximal efficiency of energy utilization in photosystem II. The degree of inhibition depended on the time of treatment and was always higher under illumination conditions (50 microE.m-2.s-1) than under dark conditions. A similar regularity was observed for the delta F/Fm' ratio, which characterizes the real efficiency of energy storage at the given intensity of the photosynthesis-exciting light. Incubation with 5 microM HgCl2 for 5 h did not affect both ratios. The decrease in Fm at constant F0 as well as changes in the fast fluorescence kinetics after MeHg treatment of algae cells indicated the damage on the donor side of photosystem II and the damage of the electron transfer from QA to QB. The reduction of photochemical fluorescence quenching (qN) under MeHg treatment is also evidence of the increase in the fraction of closed reaction centers (QA-). At the same time, increase in the steady-state level of P700 photooxidation indicated a disturbance of electron transfer between photosystems. The present study demonstrates that methylmercury treatment damaged the photosynthetic electron transfer chain at several sites. The inhibitory effect of methylmercury is much stronger than the effect of mercury chloride on photosynthetic processes.