Eugene L. Barsky

A study on the membrane potential and pH gradient in chromatophores and intact cells of photosynthetic bacteria.

Generation of membrane potential (delta psi) and transmembrane pH difference (delta pH) was studied in PPi-energized chromatophores of Rhodospirillum rubrum by means of measurements of carotenoid and bacteriochlorophyll absorption changes, atebrin and 8-anilinonaphthalene-1-sulphonate fluorescence responses, and phenyldicarbaundecaborane transport. The data obtained are consistent with the suggestion that carotenoid, bacteriochlorophyll and phenyldicarbaundecaborane responses are indicators of delta psi, while an atebrin response is an indicator of delta pH. The fluorescence of 8-anilinonaphthalene-1-sulphonate is affected both by delta psi and delta pH.

Determination of the quantum yields of the primary photosynthesis events and the photosynthetic unit types in purple bacteria

Summary1.Photoinduced changes in absorbancy and fluorescence yields were studied inChromatium minutissium andEctothiorhodospira Shaposhnikovii suspensions. These parameters were examined as functions of light intensity in the region of 750–950 nm under aerobic and anaerobic conditions.2.The fluorescence was shown to consist of two spectrally indistinguishable emissions: “photosynthetic” emission with a yield correlating with the state of reaction centres (P890) and “background” emission with constant yield.3.The “photosynthetic” fluorescence dependence on the portion of nonactive P890 was consistent with the multicentral (statistic) model of photosynthetic unit organization.4.In anaerobic conditions the amplitude of the photoinduced absorbancy increase around 910 nm was proportional to the portion of nonactive P890.5.A precise method for determinating the quantum yield of the primary electron donation was proposed. ForChr. minutissimum andE. shaposhnikovii under aerobic conditions these yields were found to be equal to 0·91±0·02 and 0·93±0·02 respectively.

Light-induced proton translocation through thylakoid and cytoplasmic membranes of Plectonema boryanum

Light-induced fluorescence changes of 9-aminoacridine, an indicator of proton gradient in energy-transducing membranes, were studied in Plectonema boryanum and other cyanobacteria. The fluorescence changes observed in cell suspensions resulted from a superposition of fluorescence quenching and enhancement as the analysis of the kinetic data shows. Both components of the fluorescence changes are abolished by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and m-chlorocarbonylcyanide phenylhydrazone. The inhibitory effect of DCMU is removed by 2,3,5,6- or N,N,N′,N′-tetramethyl-p-phenylenediamine. The fluorescence quenching sensitive to substrates and uncouplers of the photophosphorylation is only observed in membrane vesicles obtained by osmotic shock of P. boryanum spheroplasts. Presumably, light-induced quenching of the dye fluorescence in the cells of cyanobacteria is due to the proton transport from the cytoplasm in the inner space of thylakoids while fluorescence enhancement is due to the proton efflux from the cytoplasm into the incubation medium.

Absorption changes of carotenoids and bacteriochlorophyll in energized chromatophores of Rhodospirillum rubrum

Abstract The energization of Rhodospirillum rubrum chromatophores by the light, ATP, PPi, by dark electron transfer via energy-coupling sites of the redox chain, by the combination of KC1 and valinomycin causes absorption changes of carotenoids and bacteriochlorophyll. These changes due to the absorption-band shifts of the pigments are sensitive to the uncoupler p-trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP) but not to the combination of KC1 and nigericin, which abolishes fluorescence changes of atebrin. Dithionite and ferricyanide depress the light-induced absorption changes of bacteriochlorophyll but have no inhibitory effect on the PPi-induced changes. Analysis of bacteriochlorophyll absorption changes in the infra-red region shows that the photooxidation of bacteriochlorophyll reaction centers with the negative peak in the region of 890 nm is accompanied by red and blue shifts of bacteriochlorophyll absorption bands. These shifts are due to a transmembrane electrochemical gradient of H+ and a local electric field arising as a result of oxidation of the reaction centers. It appears that the superposition of the (1) red shift which is characterized by negative and positive peaks at 865 and 895 nm, respectively, and (2) photobleaching of bacteriochlorophyll reaction centers in the region of 890 nm cause overall absorption changes with the negative peak at 865 nm.

Inhibition of photosynthetic oxygen evolution by protonophoric uncouplers

The protonophoric uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,3,4,5,6-pentachlorophenol (PCP) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) inhibited the Hill reaction with K3[Fe(CN)6] (but not with SiMo) in chloroplast and cyanobacterial membranes (the I50 values were approx. 1–2, 4–6 and 0.04–0.10 μM, respectively). The inhibition is due to oxidation of the uncouplers on the Photosystem II donor side (ADRY effect) and their subsequent reduction on the acceptor side, ie. to the formation of a cyclic electron transfer chain around Photosystem II involving the uncouplers as redox carriers. The relative amplitude of nanosecond chlorophyll fluorescence in chloroplasts was increased by DCMU or HQNO and did not change upon addition of uncouplers, DBMIB or DNP-INT; the HQNO effect was not removed by the uncouplers. The uncouplers did not inhibit the electron transfer from reduced TMPD or duroquinol to methylviologen which is driven by Photosystem I. These data show that CCCP, PCP and TTFB oxidized on the Photosystem II donor side are reduced by the membrane pool of plastoquinone (Qp) which is also the electron donor for K3 [Fe(CN)6] in the Hill reaction as deduced from the data obtained in the presence of inhibitors. Inhibition of the Hill reaction by the uncouplers was maximum at the pH values corresponding to the pK of these compounds. It is suggested that the tested uncouplers serve as proton donors, and not merely as electron donors on the oxidizing side of Photosystem II.

Interaction of carbonyl cyanide m-chlorophenylhydrazone with the photosystem II acceptor side

We show that CCCP, known as an uncoupler of photophosphorylation and an ADRY agent, inhibits FeCy photoreduction and coupled O2 evolution by isolated chloroplasts equally (150 ∼ 2μM), but is practically without effect on the O2 evolution coupled with SiMo reduction within the 0.2–10μM concentration range. CCCP has no effect on the nanosecond chlorophyll fluorescence in chloroplasts incubated at low light intensity, but decreases it at high light intensity. The electron transfer from reduced TMPD or duroquinol to methylviologen is resistant to CCCP. The efficiency of the CCCP inhibitory action on the FeCy photoreduction depends on the rate of electron flow, which is controlled by the light intensity. The data obtained show that CCCP is oxidized by the photosystem II donor side and is reduced by Qp, competing for electrons with FeCy and the cytochrome blf complex.

Surface charge on thylakoid membranes: regulation of photosynthetic electron transfer in cyanobacteria

The establishment of the steady-state rate of photosynthetic O2 evolution by cells of Anabaena variabilis and other cyanobacteria was found to be preceded by a lag-phase the duration of which depended on the time of cell preincubation in the dark. Electron acceptors (benzoquinone, N,N,N′,N′-tetramethyl-p-phenylenediamine, 2,3,5,6-tetramethyl-p-phenylenediamine or 2,6-dichlorophenolindophenol) abolished the lag-phase as well as the inhibitory effect of cyanide on electron transfer. Mono-, di-and trivalent cations added to the cell suspension markedly reduced the lag-phase. As cation concentrations were increased, acceleration and subsequent deceleration of the O2 evolution rate were observed. The efficient concentrations of cations decreased as their valency increased. The lag-phase and the rate of photosynthetic O2 evolution by the blue-green algae are suggested to depend on the value of the membrane surface charge governing the electrostatic interaction between unidentified membrane-bound redox components. The combination of valinomycin and nigericin as well as gramicidin D enhanced the duration of the lagphase by deenergization of thylakoid membrane.

Photoreduction of silicomolybdate in chloroplasts by agents accelerating the deactivation reactions of the water‐oxidizing system

Uncouplers of photosynthetic phosphorylation, CCCP, TTFB and PCP, inhibited light‐induced O2 evolution in the Hill reaction with SiMo (I 50 ∼20, 3 and 45 μM, respectively), but only insignificantly diminished SiMo photoreduction by pea chloroplasts. The same properties were exhibited by the ADRY agent ANT2p. CCCP, TTFB and PCP are oxidizable compounds with redox potentials of +1.17, +1.18 and +1.09 V (pH 6.0), as determined by cyclic voltammetry. Similarly to NH2OH, the tested uncouplers can apparently serve as electron donors for photosystem II.

Quenching of chlorophyll fluorescence by quinones.

Quinones caused quenching of Chl a fluorescence in native and model systems. Menadione quenched twofold the fluorescence of Chl a and BChl a in pea chloroplasts, chromatophores of purple bacteria, and liposomes at concentrations of 50‐80 μM. To obtain twofold quenching in Triton X‐100 micelles and in ethanol, the addition of 1.3 mM and 11 mM menadione was required, respectively. A proportional decrease in the lifetime and yield of Chl a fluorescence in chloroplasts, observed as the menadione concentration increased, is indicative of the efficient excitation energy transfer from bulk Chl to menadione. The decrease in the lifetime and yield of fluorescence was close to proportional in liposomes, but not in detergent micelles. The insensitivity of the menadione quenching effect to DCMU in chloroplasts, and similarity of its action in chloroplasts and liposomes indicate that menadione in chloroplasts interacts with antenna Chl, i. e., nonphotochemical quenching of fluorescence occurs.

Nanosecond fluorescence of chloroplasts as a probe for electron transfer disruption in photosystem II

Abstract The relative amplitude of fluorescence (lifetime, 1.2 ns) of wheat chloroplasts illuminated at low light intensity is increased 35–50 times on addition of inhibitors of electron transfer on the acceptor side of photosystem II (PS II) (diuron, dinoseb and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO)), but is insensitive to cytochrome b6f complex inhibitors (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and 2-iodo-6-isopropyl-3-methyl-2,4,4′-trinitrodiphenyl ether (DNP-INT)) and to photophosphorylation uncouplers. At high light intensities, the relative amplitude of the 1.2 ns fluorescence component increases to the same extent as observed at low intensities in the presence of diuron. The nanosecond fluorescence of chloroplasts exposed to high intensity light is decreased 3–4-fold by carbonylcyanide m-chlorophenylhydrazone (CCCP). The data obtained show that the PS II fluorescence of chloroplasts in the nanosecond region is sensitive to a change in the electron transport rate at the level of the quinone acceptors of PS II. The nanosecond fluorescence of chloroplasts illuminated at high light intensity reflects the state of the electron transport system on the donor side of PS II.