Membrane lipid composition, fluidity, and surface charge changes in response to growth of the fresh water cyanobacterium Synechococcus 6311 under high salinity.

The effect of adaptation to saline growth of a fresh water cyanobacterium Synechococcus 6311 on components of the cytoplasmic membranes and thylakoids was investigated. Significant changes in membrane surface charge, lipid, fatty acid, and carotenoid composition were observed upon transfer of the cells from a low salt (0.015 M NaCl) to a high salt (0.50 M NaCl) growth medium. Very similar changes in the polar lipid classes and fatty acid composition were observed in both membranes, but changes in fluidity and surface charge and a significant shift in the protein to lipid ratio were only apparent in the cytoplasmic membranes. The fluidity and surface charge data correlate well with functional studies and we can attribute the cytoplasmic membrane as the major site of interaction and adaptation to the saline environment.

Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans.

Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [35S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min-1 (mg of protein)-1, respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa3-type enzyme from the properties of its redox-active and EDTA-resistant Cu2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa3-type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa3-type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme (EC 1.9.3.1).

Application of photosynthetic N2-fixing cyanobacteria to the CELSS program.

The feasibility of using photosynthetic microalgae (cyanobacteria) as a subsystem component for the CELSS program, with particular emphasis on the manipulation of the biomass (protein/carbohydrate) has been addressed. Using factors which retard growth rates, but not photosynthetic electron flux, the partitioning of photosynthetically derived reductant may be dictated towards CO2 fixation (carbohydrate formation) and away from N2 fixation (protein formation). Cold shock treatment of fairly dense cultures markedly increases the glycogen content from 1% to 35% (dry weight), and presents a useful technique to change the protein/carbohydrate ratio of these organisms to a more nutritionally acceptable form.

Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase.

Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate.

Sulfate-dependent iron oxidation by Thiobacillus ferrooxidans: characterization of a new EPR detectable electron transport component on the reducing side of rusticyanin.

Iron(II) oxidation by pH 2.5 HCl-washed cells of Thiobacillus ferrooxidans is known to be sulfate dependent. Sulfate dependence of the autooxidation of a novel component in the electron transport pathway is demonstrated. This component exhibits an electron paramagnetic resonance (EPR) signal in the oxidized state at g = 2.005 distinguishable from the g = 2.08 signal attributed to rusticyanin. The novel component is proposed to be a three-iron-sulfur cluster based upon the g value, lineshape, and temperature dependence. Oxyanion specificity for the EPR signal has the same dependence on sulfate as does iron(II) oxidation. By using azide to inhibit electron transfer to oxygen, sulfate was shown to be involved in electron transfer from the g = 2.005 component to the copper of rusticyanin.

The role of respiration during adaptation of the freshwater cyanobacterium Synechococcus 6311 to salinity.

Growth of the freshwater cyanobacterium Synechococcus 6311 under saline conditions stimulated respiration tenfold during the first 24 h, while growth and photosynthesis were inhibited. The elevated respiration rate was seen under both light and dark conditions, was uncoupler and cyanide sensitive, and did not decrease upon salt removal. Membrane preparations from salt-grown cells exhibited a tenfold increase in cytochrome oxidase activity, while electron transfer rates from NADPH to cytochrome c only increased threefold. Cytochrome oxidase activities were correlated with levels of EPR detectable Cu2+ in the salt and control membranes. Sodium-driven proton (antiproter) gradients in salt-grown cells were sensitive to cyanide but not dicyclohexylcarbodiimide, indicating the direct role of respiratory electron transport in maintaining low intracellular sodium levels.

EPR signals of redox active copper in EDTA washed membranes of the cyanobacterium Synechococcus 6311.

A signal of Cu2+ (g = 2.03) was detected by electron paramagmetic resonance spectroscopy in oxidized membrane preparations of Synechococcus 6311. The membranes were prepared and washed in the presence of EDTA (10mM, pH 8.0) and, hence, were depleted of adventitious copper; the treatment also would remove any membrane-associated soluble redox proteins and other paramagnetic metal ions. 0.1% Triton X-100 facilitated detection of the Cu2+ signal which was fully reduced by dithionite or ascorbate plus N,N,N',N',-tetramethyl-p-phenylenediamine, and partially reduced NADPH and NADH, which are known to donate electrons to the terminal oxidase of cyanobacteria via the respiratory chain. Using temperature dependence and power saturation of the EPR copper signal, we conclude that copper is a firmly bound constituent of the terminal oxidase in an environment which is very similar if not identical to other cytochrome c oxidase preparations.

Kinetics of leaf nitrite reductase with Methyl Viologen and ferredoxin under controlled redox conditions.

Some factors that influence the activity of nitrite reductase (EC 1.7.7.1) were investigated, the enzyme from Curcurbita pepo (vegetable marrow) being used. The activity with ferredoxin or Methyl Viologen as electron donor was inhibited by certain salts, including NaCl. The steady-state kinetic parameters measured in a commonly used open-tube (aerobic) system were compared with a closed-cell (anaerobic) system in which the redox potential, and thus the concentrations of oxidized and reduced donor, could be controlled. This showed that in the open-tube system the apparent Km values determined were overestimated (by a factor of 10 for reduced Methyl Viologen), owing to incomplete mediator reduction and competitive inhibition by the oxidized form of the mediator.

EPR spectra of photosystem I and other iron protein components in intact cells of cyanobacteria.

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe3+ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor 'X' of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 +/- 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe3+, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.