Reverse electron flow in chloroplasts

Energy dependent reverse electron flow reactions in isolated thylakoids provide a unique tool to study, in the dark, the coupling between the ATP synthase, proton transport and the electron transfer system. Appropriate experimental conditions have been established to follow experimentally the following reactions:

1. ATP driven proton uptake into the inner-thylakoid space, which requires preactivation of the ATP synthase.
2. ATP driven reverse electron transport, which involves proton transport as an intermediate, and results in the reduction of Q_A by an externally added electron donor.
3. ATP driven luminescence, which requires the presence of an oxidized partner on the water side of photosystem II, and involves electron transport from Q_B to Q_A.
4. ΔpH driven reverse electron flow, which does not require the participation of the ATP synthase, and uses reduced intermediates between the two photosystems as electron donors for the reduction of Q_A.
5. ΔpH driven luminescence which again uses reduced intermdiates between the two photosystems as electron donors for Q_A reduction, and requires the presence of an oxidized partner on the water side of photosystem II.

Several of these reactions have been shown to occur in intact chloroplasts and may provide an important regulatory mechanism in vivo.     

Properties of ATP-driven reverse electron flow in chloroplasts

1. The reverse reactions induced by coupled ATP hydrolysis were studied in spinach chloroplasts by measurements of the ATP-induced increase in chlorophyll fluorescence reflecting reverse electron flow, and of the ATP-induced decrease in 9-aminoacridine fluorescence, representing formation of the transthylakoidal proton gradient (ΔpH). ATP-driven reverse electron flow was kinetically analysed into three phases, of which only the second and third one were paralleled by corresponding phases in ΔpH formation. The rapid first phase and formation of a ΔpH occur also in the absence of the electron transfer mediator phenazine methosulfate.2. The rate and extent of the reverse reactions were measured at temperatures in the range from 0 to 30°C. The rate of formation of ΔpH and of reverse electron flow were faster at high temperatures, but the maximal extent of ΔpH and chlorophyll fluorescence increase were observed at the lowest temperature. Considering rate and extent of the ATP-stimulated reactions, a temperature optimum around 15°C was found. Light activation of the ATPase occurred throughout the range studied. At 0°C and in the presence of inorganic phosphate the activated state for ATPase was maintained for more then 10 min.3. The ATP-induced rise in chlorophyll fluorescence yield was found to be of similar magnitude as the rise induced by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), when both were measured with an extremely weak measuring beam. It is concluded, that both effects, although derived via distinctly different pathways, are limited by the same electron donating or electron accepting pool.     

Free flow electrophoresis as a tool for enrichment of mutants with temperature-dependent lethal mutations in lipid A synthesis

Free flow electrophoresis was shown to be a useful tool to enrich for mutants conditionally defective in lipid A synthesis. The method was based on the observation that electrophoretic mobility of bacterial cells is dependent on the structure of lipopolysaccharides and is influenced by lesions in the synthesis of the O-specific chains as well as by lesion in the synthesis of the complete 3-deoxy-D-manno-octulosonic acid (dOclA) lipid A region. Using this procedure a new mutant conditionally defective in dOclA-8-P synthesis was isolated (mutant Ts5). Following a shift to nonpermissive conditions it accumulates a mixture of at least two equally represented lipid A precursor structures. One is made up of glucosamine, phosphate and 3-hydroxymyristic acid in a molar ratio 1.0:1.0:2.0 and lacks dOclA and the nonhydroxylated fatty acids lauric, myristic and palmitic acid. The precursor preparation derived from mutant Ts5 thus differs from previously described lipid A intermediates by the relatively high substitution by palmitic acid. The implications of the above findings to the biosynthesis of lipid A are discussed.     

Properties of ATP-driven reverse electron flow in chloroplasts.

1. The reverse reactions induced by coupled ATP hydrolysis were studied in spinach chloroplasts by measurements of the ATP-induced increase in chlorophyll fluorescence reflecting reverse electron flow, and of the ATP-induced decrease in 9-aminoacridine fluorescence, representing formation of the transthylakoidal proton gradient (deltapH). ATP-induced reverse electron flow was kinetically analysed into three phases, of which only the second and third one were paralleled by corresponding phases in deltapH formation. The rapid first phase and formation of a deltapH occur also in the absence of the electron transfer mediator phenazine methosulfate. 2. The rate and extent of the reverse reactions were measured at temperatures in the range from 0 to 30 degrees C. The rate of formation of delta pH and of reverse electron flow were faster at high temperatures, but the maximal extent of delta pH and chlorophyll fluorescence increase were observed at the lowest temperature. Considering rate and extent of the ATP-stimulated reactions, a temperature optimum around 15 degrees C was found. Light activation of the ATPase occurred throughout the range studied. At 0 degrees C and in the presence of inorganic phosphate the activated state for ATPase was maintained for more than 10 min. 3. The ATP-induced rise in chlorophyll fluorescence yield was found to be of similar magnitude as the rise induced by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU), when both were measured with an extremely weak measuring beam. It is concluded, that both effects, although derived via distinctly different pathways, are limited by the same electron donating or electron accepting pool.     

Purification of intact chloroplasts from spruce (Picea abies) by free-flow electrophoresis

Highly purified chloroplasts were isolated from spruce ( Picea abies [L.] Karst.) needles by free-flow electrophoresis. The chloroplast crude suspension was separated into two structurally different populations. The two populations could not be distinguished according to their protein/chlorophyll ratios and protein patterns from two-dimensional gel electrophoresis. The O² evolution, however, showed differences: the chloroplast population deflected towards the cathode contained a major part of structurally intact chloroplasts in contrast to the population deflected towards the anode. The two populations were not contaminated by endoplasmatic membranes or mitochondria.     

The NADPH-dependent electron flow in chloroplasts of the higher plants

The NADPH-dependent reduction of some photosynthetic electron carriers in the dark, and the reduction of NADP+ associated with the glycolytic sequence and the oxidative pentose phosphate pathway in chloroplasts are reviewed. The postulated pathways of electron transports sensitive and insensitive to antimycin A are also evaluated. It is proposed that the electron flow, predominantly through cytochrome bf complex, may be also involved in the pathway of NADPH-dependent and antimycin A-insensitive back electron transport. An information on the chlororespiration in higher plants is also included.     

Flash spectroscopic studies of cyclic electron flow in intact chloroplasts

The kinetic behaviours of cytochrome $\text{b}$-563 and cytochrome $\text{f}$ are shown to be consistent with their participation in coupled cyclic electron flow in intact chloroplasts. Electron transfer between cytochromes $\text{b}$-563 and cytochrome $\text{f}$ is antimycin sensitive. Fluorescence induction studies indicate that plastoquinone may function in a coupled step between the cytochromes.     

Hyphenated thermal field flow fractionation--capillary electrophoresis

The possibility was considered to use the transverse thermophoresis of analytes in the capillary for capillary electrophoresis (CE) to control the separation process, decrease the peak width due to thermal effects and provide new separation parameters in CE. As the examination has shown, in non-aqueous buffers the Joule heating in the capillary for CE can provide transverse temperature gradients comparable with the temperature gradients in conventional devices for thermal field flow Fractionation (ThFFF). It was proposed to use the non-uniform velocity profile of analytes caused by the transverse temperature gradient and the temperature dependence of the buffer viscosity for the FFF-like separation of analytes besides CE separation. The expressions for the peak parameters have been derived, where the non-uniform transverse analyte concentration distribution due to the thermophoresis is taken into account, and the possibilities based on FFF-CE principles are discussed. As possible objects of this hyphenated technique, macromolecules and particles are considered.     

The NADPH-dependent electron flow in chloroplasts of the higher plants

The NADPH-dependent reduction of some photosynthetic electron carriers in the dark, and the reduction of NADP+ associated with the glycolytic sequence and the oxidative pentose phosphate pathway in chloroplasts are reviewed. The postulated pathways of electron transports sensitive and insensitive to antimycin A are also evaluated. It is proposed that the electron flow, predominantly through cytochrome bf complex, may be also involved in the pathway of NADPH-dependent and antimycin A-insensitive back electron transport. An information on the chlororespiration in higher plants is also included. This revised version was published online in June 2006 with corrections to the Cover Date.     

The purification of beta-galactosidase by flow electrophoresis

A plant has been constructed for the free-flow electrophoresis. It permits carrying out electrophoretic separation both in a free flow and with the use of neutral grained fillers. Beta-galactosidase has been purified from the culture liquid of the Escherichia coli cell phage lysate. Its content in the purified material amounted to 81.2     

Resolution of microbial mixtures by free flow electrophoresis

Summary The resolution of bacterial mixtures by free flow electrophoresis (FFE) was not affected by the position of the microbes on the growth curve and approximately 70% of the individual cells applied were recovered as viable cells. The dependence of bacterial electrophoretic mobility on the pH, salt concentration, and viscosity of the electrolyte was determined. Suspending media and running electrolyte were developed which allowed collection of samples of>99% purity within two minutes of introduction of a mixture of Escherichia coli and Staphylococcus aureus. Most bacterial strains migrated in a single band, although some migrated in more than one band. Escherichia coli was resolved from each of 10 different species. The considerable variation in mobility found in 21 different E. coli strains, however, appears to preclude use of FFE as a method of species identification.