The reduction of artificial electron acceptors at subzero temperatures by chloroplasts suspended in fluid media

1. 1. Chloroplasts can be suspended in aqueous/organic mixtures which are liquid at sub-zero temperatures with a good retention of the ability to reduce artificial electron acceptors. The reduction of ferricyanide and 2,6-dichlorophenolindophenol at temperatures above 0δC is about 50% inhibited by 50% (v/v) ethylene glycol. Higher concentrations cause more extensive inhibition.

2. 2. Different solvents were compared on the basis of their ability to cause a given depression of the freezing point of an aqueous solution. Ethylene glycol caused less inhibition of electron transport than glycerol, which in its turn was found to be superior to methanol.

3. 3. The reduction of oxidised 2,3,5,6-tetramethyl-p-phenylenediamine could be measured at −25δC in 40% (v/v) ethylene glycol. Using an acceptor with a high extinction coefficient, methyl purple (a derivative of 2,6-dichlorophenolindophenol) it was possible to observe electron flow at temperatures as low as −40δC in 50% (v/v) ethylene glycol.

4. 4. From studies of the effects of the inhibitors 3(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone it is suggested that electron flow from the donor side of Photosystem II to the acceptor side of Photosystem I can occur at temperatures at least as low as −25δC. The ultimate electron donor is presumably water but it was not possible to demonstrate this directly.

Light-induced proton transport by chloroplasts suspended in fluid media at sub-zero temperatures: kinetics and stoichiometry.

1. Chloroplasts suspended in a medium containing ethanediol and water (1 : 1, v/v) at -16 degrees C show light-induced proton uptake and subsequent dark efflux. Proton uptake in continuous light showed biphasic kinetics. 2. A 1 ms flash caused a single turnover of the photochemical centres at -16 degrees C. Under the same conditions 3H+ were taken up from the external medium in the presence of methyl viologen as electron acceptor. 3. The flash-induced proton uptake was exponential and monophasic with t1/2 = 3 s. The flash-induced proton release into the thylakoid interior was biphasic, with half-times of less than 0.1 s and 3 s. The fast phase represented approximately 30% of the total release and may be correlated with the oxidation of water. 4. The half-time of reduction of cytochrome f in the dark following illumination in the presence of 2 mM NH4Cl (2.5 s) is similar to the half-time of the slow phase of proton release, suggesting a correlation between the kinetics of cytochrome f reduction and plastoquinol oxidation.     

The properties of cytochrome f and P700 in chloroplasts suspended in fluid media at sub-zero temperatures.

1. The properties of P700 and cytochrome f have been studied at sub-zero temperatures in chloroplasts suspended in a medium containing 50% (v/v) ethylene glycol. The dark reduction of these components after a period of illumination provided information about the rate-limiting step of photosynthetic electron transport under these conditions. 2. The oxidation of P700 on illumination in the presence of methyl viologen and its subsequent dark reduction can be observed at -35 degrees C. This cycle of reactions could be repeated many times. The rate of reduction was increased by NH4Cl and reduction was inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea. 3. The oxidation and reduction of cytochrome f could also be observed under similar conditions. The activation energies for the reduction of cytochrome f and P700 are similar (about 75 kJ mol-1) and the reduction of cytochrome f is also inhibited by dichlorophenyldimethylurea and stimulated by NH4Cl. 4. The reduction of both cytochrome f and P700 seemed to follow first-order kinetics, but the t1/2 for the redcution of the cytochrome was at least three times that for the reduction of P700 at the same temperature. It was concluded that the results were only compatible with a model in which the main pathway of electrons from plastoquinone to P700 involved cytochrome f if the equilibrium constant between the cytochrome and P700 was very much less than that expected from their redox potentials.     

Transient kinetics of the electron transfer between P-700, plastocyanin and cytochrome f in chloroplasts suspended in fluid media at sub-zero temperatures

The kinetics of redox changes of P-700, plastocyanin and cytochrome f in chloroplasts suspended in a fluid medium at sub-zero temperatures have been studied following excitation of the chloroplasts with either a single-turnover flash, a series of flashes or continuous light. The results show that: (1) The kinetics of reduction of P-700⁺ and those of oxidation of plastocyanin are consistent with a bimolecular reaction between these two components as previously suggested (Olsen, L.F., Cox, R.P. and Barber, J. (1980) FEBS Lett. 122, 13–16). (2) Cytochrome f shows heterogeneity with respect to its kinetics of oxidation by Photosystem I. (3) In contrast to the situation when plastoquinol is the electron donor, reduction of cytochrome f by electrons derived from diaminodurene occurs with sigmoidal kinetics that shows a good fit to an apparent equilibrium constant of 12 between the cytochrome and P-700. (4) The rate of electron transfer from plastoquinol to Photosystem I depends on the redox state of the plastoquinone pool. (5) In relation to current ideas about the lateral heterogeneity of Photosystem I and Photosystem II in the thylakoid membrane, the results are consistent with the function of plastocyanin as a mobile carrier of electrons in the intrathylakoid space.     

Photosynthetic electron transport and electrochromic effects at sub-zero temperatures.

Spinach chloroplasts, suspended in a liquid medium containing ethyleneglycol, showed reversible absorbance changes near 700 and 518 nm due to P-700 and "P-518" in the region from -35 to -50 degrees C upon illumination. The kinetics were the same at both wavelengths, provided absorbance changes due to Photosystem II were suppressed. At both wavelengths, the decay was slowed down considerably, not only by the System I electron acceptor methyl viologen, but also by silicomolybdate. The effect of the latter compound is probably not due to the oxidation of the reduced acceptor of Photosystem I by silicomolybdate, but to the enhanced accessibility of the acceptor to some other oxidant. In the presence of both an electron donor and acceptor for System I, a strong stimulation of the extent of the light-induced absorbance increase at 518 nm was observed. The most effective donor tested was reduced N-methylphenazonium methosulphate (PMS). The light-induced difference spectrum was similar to spectra obtained earlier at room temperature, and indicated electrochromic band shifts of chlorophylls a and b and carotenoid, due to a large potential over the thylakoid membrane, caused by sustained electron transport. It was estimated that steady-state potentials of up to nearly 500 mV were obtained in this way; the potentials reversed only slowly in the dark, indicating a low conductance of the membrane. This decay was accelerated by gramicidin D. The absorbance changes were linearly proportional to the membrane potential.     

Photosynthetic electron transport and electrochromic effects at sub-zero temperatures

Spinach chloroplasts, suspended in a liquid medium containing ethyleneglycol, showed reversible absorbance changes near 700 and 518 nm due to P-700 and “P-518” in the region from ?35 to ?50 °C upon illumination. The kinetics were the same at both wavelengths, provided absorbance changes due to Photosystem II were suppressed. At both wavelengths, the decay was slowed down considerably, not only by the System I electron acceptor methyl viologen, but also by silicomolybdate. The effect of the latter compound is probably not due to the oxidation of the reduced acceptor of Photosystem I by silicomolybdate, but to the enhanced accessibility of the acceptor to some other oxidant.In the presence of both an electron donor and acceptor for System I, a strong stimulation of the extent of the light-induced absorbance increase at 518 nm was observed. The most effective donor tested was reduced N-methylphenazonium methosulphate (PMS). The light-induced difference spectrum was similar to spectra obtained earlier at room temperature, and indicated electrochromic band shifts of chlorophylls a and b and carotenoid, due to a large potential over the thylakoid membrane, caused by sustained electron transport. It was estimated that steady-state potentials of up to nearly 500 mV were obtained in this way; the potentials reversed only slowly in the dark, indicating a low conductance of the membrane. This decay was accelerated by gramicidin D. The absorbance changes were linearly proportional to the membrane potential.     

Photosynthetic control in isolated spinach chloroplasts with endogenous and artificial electron acceptors

Spinach chloroplasts, isolated rapidly in isotonic media will reproducibly give photosynthetic control rates (State 3/State 4) of 4–6, and ADP/O ratios (equivalent to ATP/2e⁻) of 1.4 – 2.1 when assayed in slightly hypotonic media. Photosynthetic control can be followed as oxygen evolution with either ferricyanide or NADP as electron acceptors, or as oxygen uptake in the presence of azide, which blocks chloroplast catalase, either alone (endogenous catalyst) or with added methyl viologen. This control can be triggered either by added ADP or by added Pi in all cases. Optimum concentrations of Mg, Pi and EDTA are required; the pH is also critical. Excess EDTA results in an inhibition of electron transport on addition of ADP.     

Temperature dependence of chlorophyll {alpha} fluorescence in lettuce and spinach chloroplasts at sub-zero temperatures

The temperature dependence of chlorophyll fluorescence wasmeasured in spinach and lettuce chloroplasts at sub-zero temperaturesin the presence of 50% ethylene glycol. In the presence of 5mM Mg²⁺, a fluorescence maximum appeared at –31?C in boththe spinach and lettuce chloroplasts, while in the presenceof only 5 mM Na⁺ as cations the maximum shifted to –20?Cin the spinach chloroplasts and to –11?C in the lettucechloroplasts. Since the occurrence of a maximum in the temperatureversus fluorescence curve is an indication for the transitionof the physical phase of thylakoid membrane lipids between theliquid crystalline and the phase-separation state (16, 18),these findings suggest that the (major) phase transition ofmembrane lipids occurs at these low temperatures in chloroplastsof higher plants and also that the phase transition temperatureis markedly lowered by the presence of divalent cations. Ethylene glycol at a concentration of 50% had almost no effecton the temperature dependence of chlorophyll fluorescence ina lamellar membrane preparation of Anabaena variabilis. In awater suspension of dimyristoylphosphatidylcholine, the additionof ethylene glycol to 50% did not alter the characteristic featureof the temperature dependence of fluorescence of 1-anilinonaphthalene-8-sulfonate.These findings suggest that 50% ethylene glycol does not affectthe temperature of the transition of the physical phase of membranelipids. ¹ C.I.W.-D.P.B. Publication No. 592. ² Present Address: Department of Biophysics and Biochemistry,Faculty of Science, University of Tokyo, Hongo 113, Tokyo, Japan. (Received June 22, 1977; )     

Heterogeneity of system II secondary electron acceptors in Tris-washed chloroplasts

Tris-washed chloroplasts were submitted to saturating short flashes, and then rapidly mixed with dichlorophenyldimethylurea (DCMU). The amount of singly reduced secondary acceptor was estimated from the DCMU-induced increase in fluorescence, caused by the reverse electron flow from secondary to primary acceptor. The back-transfer from the singly reduced secondary acceptor to the primary acceptor Q induced by DCMU addition affects only a part (60%) of the variable fluorescence (ΔF_max). As previously shown, the quenchers involved in this phenomenon, ‘B-type’ quenchers, are different from those controlling the complementary part of the fluorescence, the non-B-type. In this report, we show that at pH 8.5 in the B-type systems, there exist two kinds of secondary electron acceptors: B, a two-electron acceptor, the corresponding Q accounting for 40% of the variable fluorescence; B′, a one-electron acceptor, the corresponding Q accounting for 20% of the variable fluorescence. The lifetimes of B^? and B′^? in the absence of DCMU are 40 and 1 s, respectively. The primary acceptors of the B and B′ systems can be considered as corresponding to the Q₁s defined previously (Joliot, P. and Joliot, A. (1981) in Proceedings of the 5th International Congress on Photosynthesis (Akoynoglou, G., ed.), pp. 885–899, Balaban International Science Services, Philadelphia). The B′ centers seems to be equivalent to the Q_β centers as defined by other workers (Van Gorkom, H.J., Thielen, A.P.G.M. and Gorren, A.C.F. (1982) in The Function of Quinones in Energy Conserving Systems (Trumpower, B.L., ed.), Academic Press, New York, in the press).     

Water permeability of yeast cells at sub-zero temperatures

Summary A combined cryomicroscopic-multiple nonlinear regression analysis technique has been used to determine the water permeability of the yeast cellSaccharomyces cerevisiae during freezing. The time rate of change in volume of supercooled yeast cells was photographically monitored using a cryomicroscope which is capable of controlling in a programmable manner both the temperature and the time rate of change in temperature of the cell suspension being studied. Multiple nonlinear regression analysis together with a thermodynamic model of cell water transport during freezing was then used to statistically deduce the subzero temperature dependence of the cell water permeability. The water permeability process forS. cerevisiae being cooled at subzero temperatures was found to be rate-limited by the passage of water through either the plasmalemma, the cell wall, or a combination of these two permeability barriers. The hydraulic water permeability coefficient for yeast at 20°C is approximately 1–2×10^–13 cm³/dyne sec, if extrapolation from subzero temperatures to room temperature is permissible, while the apparent activation energy governing the permeability process at subzero temperatures is approximately 45–68 kJ/mol (11–16 kcal/mol). Appendix I: Volumetric Changes in Yeast Cells during Freezing at Constant Cooling Rates     

Desiccation stress at sub-zero temperatures in polar terrestrial arthropods

Cold tolerant polar terrestrial arthropods have evolved a range of survival strategies which enable them to survive the most extreme environmental conditions (cold and drought) they are likely to encounter. Some species are classified as being freeze tolerant but the majority of those found in the Antarctic survive sub-zero temperatures by avoiding freezing by supercooling. For many arthropods, not just polar species, survival of desiccating conditions is equally important to survival of low temperatures. At sub-zero temperatures freeze avoiding arthropods are susceptible to desiccation and may lose water due to a vapour diffusion gradient between their supercooled body fluids and ice in their surroundings. This process ceases once the body fluids are frozen and so is not a problem for freeze tolerant species. This paper compares five polar arthropods, which have evolved different low temperature survival strategies, and the effects of exposure to sub-zero temperatures on their supercooling points (SCP) and water contents. The Antarctic oribatid mite (Alaskozetes antarcticus) reduced its supercooling point temperature from -6 to -30 degrees C, when exposed to decreasing sub-zero temperatures (cooled from 5 to -10 degrees C over 42 days) with little loss of body water during that period. However, Cryptopygus antarcticus, a springtail which occupies similar habitats in the Antarctic, showed a decrease in both water content and supercooling ability when exposed to the same experimental protocol. Both these Antarctic arthropods have evolved a freeze avoiding survival strategy. The Arctic springtail (Onychiurus arcticus), which is also freeze avoiding, dehydrated (from 2.4 to 0.7 g water g(-1) dry weight) at sub-zero temperatures and its SCP was lowered from c. -3 to below -15 degrees C in direct response to temperature (5 to -5.5 degrees C). In contrast, the freeze tolerant larvae of an Arctic fly (Heleomyza borealis) froze at c. -7 degrees C with little change in water content or SCP during further cold exposure and survived frozen to -60 degrees C. The partially freeze tolerant sub-Antarctic beetle Hydromedion sparsutum froze at c. -2 degrees C and is known to survive frozen to -8 degrees C. During the sub-zero temperature treatment, its water content reduced until it froze and then remained constant. The survival strategies of such freeze tolerant and freeze avoiding arthropods are discussed in relation to desiccation at sub-zero temperatures and the evolution of strategies of cold tolerance.     

Inactivation of unbound rat liver glucocorticoid receptor by N-alkylmaleimides at sub-zero temperatures

Biosynthetically radiolabelled heparan sulphate proteoglycans have been isolated from the growth medium and the cell lysate of a human neuroblastoma cell line (CHP100). Chromatography on Sepharose CL-4B identified two heparan sulphate proteoglycans in the medium (Kav 0.220 and 0.389), whereas in the cell lysate the major proteoglycan species were more heterogenous and of a smaller overall molecular size (Kav 0.407) than the medium-derived counterparts. Chromatography on Sepharose CL-6B of free heparan sulphate glycosaminoglycan chains showed that the majority of cell-layer-derived material heparan sulphate 2, Kav = 0.509) was smaller than medium heparan sulphates (heparan sulphate 1 and heparan sulphate 2, Kav 0.230 and 0.317). Analysis of the patterns of polymer sulphation by nitrous acid treatment, gel chromatography and high-voltage electrophoresis established that in each heparan sulphate fraction there was on average 1.1 sulphate residues per disaccharide with an N:O sulphate ratio of 1.1. Heparan sulphate in the medium had a high proportion of di-O-sulphated disaccharides in regions of the chain with repeat disaccharide sequences of structure GlcA-GlcNSO3, whereas cell-associated material was enriched in di-O-sulphated tetrasaccharides of alternating sequences GlcA-GlcNAc-GlcA-GlcNSO3. The identification of several populations of heparan sulphate proteoglycans differing in molecular size and glycosaminoglycan fine structure may reflect the functional diversity of this family of macromolecules in the nervous system.     

Protein crystallography at sub-zero temperatures: lysozyme-substrate complexes in cooled mixed solvents.

To determine the feasibility of direct X-ray crystallographic structure determination of productive enzyme-substrate complexes and to ascertain the best conditions for such studies, the hydrolysis of bacterial cell walls and oligosaccharides by human leukaemic lysozyme was investigated in mixed aqueous/organic solvents and high salt solutions. Although high salt solutions modify the enzymic reaction, hydrolysis in mixed solvents appears to proceed by the same mechanism as in aqueous solution. At low temperatures the reaction is slowed progressively, and at −25 °C the enzyme-substrate complex in mixed solvents is stable indefinitely. The conformation of the enzyme is not significantly altered in these solvents, and the enzyme-substrate complex can be formed by direct addition of substrate to the enzyme at sub-zero temperatures, as required for crystallographic studies. The pH profile of the reaction in mixed solvents allows conditions of optimal binding to be selected. These studies in solution demonstrate that low-temperature protein crystallography may indeed permit the direct determination of the three-dimensional structure of enzyme-substrate complexes. They also delineate the precise conditions of pH, temperature and solvent to use in the crystallographic experiments.     

Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration

An extension of the respiratory chain to the cell surface is necessary to reduce extracellular electron acceptors like ferric iron or manganese oxides. In the past few years, more and more compounds were revealed to be reduced at the surface of the outer membrane of Gram-negative bacteria, and the list does not seem to have an end so far. Shewanella as well as Geobacter strains are model organisms to discover the biochemistry that enables the dissimilatory reduction of extracellular electron acceptors. In both cases, c-type cytochromes are essential electron-transferring proteins. They make the journey of respiratory electrons from the cytoplasmic membrane through periplasm and over the outer membrane possible. Outer membrane cytochromes have the ability to catalyze the last step of the respiratory chains. Still, recent discoveries provided evidence that they are accompanied by further factors that allow or at least facilitate extracellular reduction. This review gives a condensed overview of our current knowledge of extracellular respiration, highlights recent discoveries, and discusses critically the influence of different strategies for terminal electron transfer reactions.     

The cold-induced denaturation of lactate dehydrogenase at sub-zero temperatures in the absence of perturbants

The cold-induced denaturation of lactate dehydrogenase has been determined in an unfrozen, cryoprotectant free solution at sub-zero temperatures. The cold-induced denaturation temperature (TL) has been found to be -28 degrees C. The results for the first time clearly establish that temperature alone can induce denaturation in a cooled protein solution. The validity of earlier data, obtained in the presence of perturbants (particularly pH or guanidinium chloride), is discussed.     

Growth at sub-zero temperatures of black spot fungi from meat

Glycerol can prevent both freezing and desiccation of micro-organisms growing at sub-zero temperatures. On media containing glycerol, at concentrations readily tolerated by the organisms at ambient temperatures, three species of fungi isolated from black spot spoilt meat failed to grow at temperatures much below -5°C. This would, therefore, seem to be the minimum possible growth temperature of these organisms. Although the fungi could grow on frozen media, their rates of growth were such that, on frozen meat, several months would be required for colonies to become barely visible. It therefore seems that significant black spot spoilage will only develop on frozen meat if it is held at temperatures within 2–3° below the freezing point for prolonged periods, or if the meat surface reaches higher temperatures with surface drying inhibiting bacterial growth. There has been little study during the last 50 years of mould spoilage of meat, although it is still of importance in the international trade in frozen meats. Because moulds grow relatively slowly, they only spoil meat if the storage conditions prevent bacterial growth, but there are few firm data on the time and temperature requirements for visible mould growth to develop in the absence of bacterial spoilage. Such data are necessary if the causes of particular outbreaks of fungal spoilage are to be assessed correctly.