Herbert Böhme

Regulation of nitrogen fixation in heterocyst-forming cyanobacteria

Abstract Some cyanobacteria are able to reduce atmospheric dinitrogen to ammonia—a process where oxygen evolved by photosynthetic activity in the same cell is detrimental to nitrogen fixation. Strategies to avoid oxygen range from temporal separation of nitrogen fixation and oxygen evolution (in unicellular and filamentous, non-heterocystous strains) to spatial separation and cellular differentiation into nitrogen fixing heterocysts (in filamentous cyanobacteria). Recent research has begun to clarify the genes involved in nitrogen fixation, the mechanisms that regulate the expression of proteins involved in the assimilation of nitrogen from different sources and the way in which the cell is able to sense its nitrogen status.

A distinct ferredoxin for nitrogen fixation isolated from heterocysts of the cyanobacterium Anabaena variabilis

Ferredoxin from heterocysts of Anabaena variabilis has been isolated and its biological activity compared to ferredoxin obtained from vegetative cells of the same organism. Both ferredoxins catalyze equally effective NADP+ photoreduction by heterocyst thylakoids. When photoreduced, both ferredoxins transfer electrons to nitrogenase from A. variabilis, with heterocyst ferredoxin being twice as active as vegetative cell ferredoxin. In the dark, however, only heterocyst ferredoxin is able to link reducing power, generated by soluble systems, such as H2/hydrogenase (from Clostridium pasteurianum) and NADPH/ferredoxin:NADP+ oxidoreductase (from A. variabilis), to the cyanobacterial nitrogenase. Using heterocyst homogenates with glucose 6‐phosphate as electron donor, only ferredoxin from heterocysts is able to stimulate nitrogenase activity further.

Components and activity of the photosynthetic electron transport system of intact heterocysts isolated from the blue-green alga Nostoc muscorum

Heterocysts of the blue-green alga Nostoc muscorum have been isolated by prolonged treatment with lysozyme. Quantitative data are presented which show the occurrence of cytochromes c-553, f-557 and b-563 in heterocysts in amounts comparable to vegetative cells. Particularly the content of the water-soluble cytochrome c-553 can be used to evaluate the intactness of a heterocyst preparation. Cytochrome f-557 has been partially purified and found to be a c-type cytochrome corresponding to cytochrome f of higher plants and other algae. Cytochrome b-559 is present in vegetative cells but not in heterocysts. The content of plastoquinone in heterocysts is reduced to 42% of the amount present in vegetative cells. These data suggest a degradation of Photosystem II during heterocyst differentiation. Measurements of photosynthetic electron transport in heterocysts proved the inability of the photosynthetic apparatus to carry out electron transport with electrons donated by water or diphenylcarbazide. In Tris-washed thylakoids from vegetative cells, however, diphenylcarbazide can act as an electron donor to Photosystem II.

In vitro studies on pathways and regulation of electron transport to nitrogenase with a cell-free extract from heterocysts of Anabaena variabilis

A cell-free preparation of heterocysts from Anabaena variabilis showed high nitrogenase activities with several physiological electron donors, dependent on addition of an ATP-generating system. Light-induced acetylene reduction with the artificial electron donor to photosystem I, diaminodurol, exhibited the same light saturation as with hydrogen as donor. Inhibitors of electron flow through plastoquinone affected light-induced, hydrogen- or NADH-dependent nitrogenase activity in a similar way. Several uncoupling agents were without effect, indicating that energized membranes are not a prerequisite for nitrogen fixation. We conclude that NADH or hydrogen deliver electrons to nitrogenase via photosystem I and ferredoxin, feeding in at the plastoquinone site.In the light, addition of NADP induced a lag in H2- or NADH-supported acetylene reduction apparently by competing with nitrogenase for electrons at the reducing side of photosystem I. Time reversal of this inibition reflects a regulation of photosystem I-dependent nitrogenase activity by the NADPH/NADP ratio in the cell. This was directly demonstrated by differently adjusted NADPH/NADP ratios.NADPH donates electrons to nitrogenase in the dark and in the light, the light reaction being DBMIB-sensitive. NADPH-supported acetylene reduction was inhibited by NADP. This inhibition was not reversed with time, pointing to an involvement of ferredoxin: NADP oxidoreductase (EC 1.18.1.2) in this pathway. Apparently, in the dark, this enzyme is able to directly reduce ferredoxin, whereas in the light electrons from NADPH first have to pass through photosystem I before reducing ferredoxin, hence nitrogenase.Intermediates of glycolysis, like glucose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate supported nitrogenase activity in the dark, each with catalytic amounts of both NAD and NADP as equally effective cofactors.We conclude that in heterocysts electrons for nitrogen fixation are essentially supplied by dark reactions, mainly by glycolysis. NADH (and hydrogen) contribute electrons via photosystem I in the light, whereas the NADPH/NADP ratio regulates linear and cyclic electron flow at the reducing side of photosystem I to provide a ratio of ATP/electrons most effective for nitrogenase.

Reciprocal formation of plastocyanin and cytochrome c-553 and the influence of cupric ions on photosynthetic electron transport.

The green alga Scenedesmus acutus is able to synthesize plastocyanin and cytochrome c-553. The concentrations of plastocyanin and cytochrome c-553 vary inversely in response to the cupric-ion concentrations of the growth medium (Bohner, H. and Böger, P. (1978) FEBS Lett. 85, 337-339). Both proteins form a homogeneous donor pool to the reaction center of Photosystem I. This donor pool can be varied quantitatively and qualitatively by different growth conditions. These variations have no influence on algal growth or photosynthetic electron transport as measured in vivo by oxygen evolution, fluorescence induction and cytochrome f-553 and c-553 redox reactions using Cu2+ concentrations of less than 10 microM in the culture medium. At higher cupric-ion concentrations, which already retard algal growth, specific sites of the photosynthetic electron-transport chain are affected: the oxidizing side of Photosystem II and the reducing side of Photosystem I.

The heterocyst-specific fdxH gene product of the cyanobacterium Anabaena sp. PCC 7120 is important but not essential for nitrogen fixation

Abstract To clarify the role of the heterocyst-specific [2Fe-2S] ferredoxin in cyanobacterial nitrogen fixation, mutational analysis of the Anabaena 7120 fdxH gene region was carried out. First, the DNA sequence of the wild-type 3509-bp EcoRI fragment downstream of the fdxH gene was determined. Genes homologous to ORF3 from the fdxH gene regions of A. variabilis and Plectonemaboryanum, the mop genes of Clostridiumpasteurianum encoding molybdo-pterin binding proteins, and ORF3 from the A. variabilis hydrogenase gene cluster were identified within the sequenced region. For mutational analysis the Anabaena 7120 mutant strains LAK4, BMB92, and KSH10 were constructed. In LAK4 the fdxH coding region is disrupted by an interposon, whereas BMB92 is deleted for a 2799-bp NheI fragment encompassing fdxH, ORF3, mop, ORF4, and ORF5. Mutant strain KSH10 is a derivative of BMB92, complemented for fdxH but not for the other genes located further downstream. Analysis of the Nif phenotype of these mutant strains showed that FdxH is necessary for maximum nitrogenase activity and optimal growth under nitrogen-fixing conditions, but not absolutely essential for diazotrophic growth. The role of alternative electron donors for nitrogenase, which might substitute for FdxH, is discussed. Iron concentrations (1μM Fe) sufficient to induce synthesis of the vegetative cell flavodoxin did not stimulate diazotrophic growth of the fdxH mutant strains, suggesting that FdxH was not replaced by a NifJ-flavodoxin system. Comparison of LAK4 and BMB92 indicated that one of the genes located downstream of fdxH might also play a (minor) role in nitrogen fixation.

Isolation and characterization of soluble cytochrome c-553 and membrane-bound cytochrome f-553 from thylakoids of the green alga Scenedesmus acutus☆

Soluble cytochrome c-553 and membrane-bound cytochrome f-553 from the alga Scenedesmus acutus were purified to apparent homogeneity. The properties of cytochrome c-553 are comparable to preparations obtained from other eukaryotic algae, whereas the thylakoid-bound species resembles higher plant cytochrome f. Common characteristics are: 1. An asymmetrical alpha-band at 553 nm. 2. A midpoint redox potential of +38 MV (pH 7.0), with a pH dependency above pH 8.0 of -60mV/pH unit. 3. Formation of a pyridine hemochromogen with a maximum at 550 nm; no adducts with CN- or CO are observed. Distinguishing features are: 1. Cytochrome f-553 has a more complicated beta-band, with maxima at 531.5 and 524 nm, and hence a more complex low-temperature spectrum. Also the positions of the gamma- and delta-bank at 421.5 and 331 nm, respectively, distinguish cytochrome f-553 from cytochrome c-553, with gamma- and delta-bands at 416 and 318 nm. 2. The ferricytochrome c-553 spectrum exhibits a weak band at 692 nm, which is not observed with cytochrome f.

Phosphorylation and nitrogenase activity in isolated heterocysts from Anabaena variabilis (ATCC 29413)

Adenylate-pool composition, energy charge, and nitrogenase activity were examined in isolated heterocysts from Anabaena variabilis (ATCC 29413). ATP formation was detected as a light- or oxygen-induced increase in ATP concentration. No cofactors or substrates had to be added for photophosphorylation to occur, whereas oxidative phosphorylation was dependent on hydrogen and oxygen (Knallgas reaction). The increase in ATP concentration was reflected by a decrease in AMP concentration, accompanied by small changes in ADP levels. Thus, a regulation of the adenylate pool by a myokinase (adenylate kinase) has to be assumed. Upon dark-light transitions, the energy charge in heterocysts increased from values below 0.4 to values approaching 0.8. High energy-charge values, reached in the light only, allowed for high rates of acetylene reduction in the presence of hydrogen. The increase in the energy charge in the dark to approx. 0.64 by addition of oxygen (5% (vv) in the presence of hydrogen) resulted in low nitrogenase activities, generally not exceeding 1–3% of the light-induced rates. In the dark, oxygen concentrations above 10% were inhibitory to both ATP formation and acetylene reduction. Increasing light intensities led to a steep increase in energy charge followed by an increase in nitrogenase activity. Plotting enzyme activity versus energy charge, a nonlinear, asymptotic relationship was observed.

Electron donation to nitrogenase in a cell-free system from heterocysts of Anabaena variabilis

Abstract Glycogen degradation and intermediates of the oxidative pentose-phosphate cycle supported high rates of acetylene reduction in a cell-free heterocyst preparation. This activity could be further increased by addition of ferredoxin and nitrogenase, isolated from heterocysts; rates well over 400 μmol C2H4/mg Chl per h were observed. Under these conditions ferredoxin from vegetative cells was inactive. Light and especially dithiothreitol inhibited substrate-dependent nitrogenase activity, pointing to an obligate involvement of glucose-6-phosphate dehydrogenase in carbohydrate degradation as the main electron source for nitrogenase.

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates. It is suggested that besides the reaction sequence cytochrome 553 leads to plastocyanin leads to Photosystem I reaction center, a second pathway cytochrome 553 leads to Photosystem I reaction center may operate additionally.