Günter A. Peschek

The phototrophic prokaryotes

Historical Perspectives: Research on Purple Bacteria During Twenty-Four Years of International Symposia on Photosynthetic Prokaryotes G. Drews. Freiburg to Vienna-Looking at Cyanobacteria over the Twenty-Five Years N.G. Carr. Photosynthesis: Structural and Functional Analyses of Cyanobacterial Photosystem I: The Directionality of Electron Transfer F. Yang, et al. Electron Transport and Bioenergetics: Photosynthesis and Respiration of Cyanobacteria: Bioenergetic Significance and Molecular Interactions G.A. Peschek. Genome Analysis and Molecular Biology: From Plastid to Cyanobacterial Genomes M. Sugiura. Metabolism of N, C, H: Cyanobacterial Nitrogen Assimilation Genes and NtcA-Dependent Control of Gene Expression E. Flores, et al. Ecology and Symbiosis. Widening Perceptions of the Occurrence and Significance of Cyanobacterial Toxins G.A. Codd, et al. Phylogeny, Taxonomy, and Evolution: Identification of a Putative Gamma Linker Polypeptide Gene in the Marine Oxyphotobacterium Prochlorococcus Marinus: Implications for the Phylogeny of Prochlorococcus Phycoerythrins W.R. Hess, F. Partensky. 86 Additional Articles. Index.

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.

Increased levels of cytochrome oxidase and sodium-proton antiporter in the plasma membrane of Anacystis nidulans after growth in sodium-enriched media.

Typical rates of horse heart ferrocytochrome c oxidation by purified plasma membrane preparations of the cyanobacterium Anacystis nidulans after one week of growth at low (0.03 M) and high (0.4–0.5 M) Na+ concentrations were 15 and 170 per mg protein, respectively. Virtually no change was observed in the cytochrome oxidase activity of isolated and purified thylakoid membranes (approx. 3–5 nmol cyt per mg protein irrespective of Na+ concentration during growth). Concomitantly, the rate of whole cell respiration (oxygen uptake) and the capacity for Na+/H+ exchange (per mg dry wt cells) by 0.4 M NaCl‐grown cells were 7‐ and 5‐fold enhanced, respectively, compared with control cells. The data are in accordance with electron transport‐driven Na+ extrusion from respiring A. nidulans, primarily mediated by the plasma membrane‐bound cytochrome oxidase.

Occurrence, phylogeny, structure, and function of catalases and peroxidases in cyanobacteria

Cyanobacteria have evolved approximately 3x10(9) years ago from ancient phototrophic microorganisms that already lived on our planet Earth. By opening the era of an aerobic, oxygen-containing biosphere, they are the true pacemakers of geological and biological evolution. Cyanobacteria must have been among the first organisms to elaborate mechanisms for the detoxification of partially reduced oxygen species including (hydrogen) peroxide. Since there is still an suprising lack of knowledge on the type, role, and mechanism(s) of peroxide-degrading enzymes in these bacteria, all 44 fully or partially sequenced genomes for haem and non-haem catalases and peroxidases have been critically analysed based on well known structure-function relationships of the corresponding oxidoreductases. It is demonstrated that H(2)O(2)-dismutating enzymes are mainly represented by bifunctional (haem) catalase-peroxidases and (binuclear) manganese catalases, with the latter being almost exclusively found in diazotrophic species. Several strains even lack a gene that encodes an enzyme with catalase activity. Two groups of peroxidases are found. Genes encoding putative (primordial) haem peroxidases (with homology to corresponding mammalian enzymes) and vanadium-containing iodoperoxidases are found only in a few species, whereas genes encoding peroxiredoxins (1-Cys, 2-Cys, type II, and Q-type) are ubiquitous in cyanobacteria. In addition, approximately 70% contain NADPH-dependent glutathione peroxidase-like proteins. The occurrence and phylogeny of these enzymes is discussed, as well as the present knowledge of their physiological role(s).

Effect of distal cavity mutations on the formation of compound I in catalase-peroxidases.

Catalase-peroxidases have a predominant catalase activity but differ from monofunctional catalases in exhibiting a substantial peroxidase activity and in having different residues in the heme cavity. We present a kinetic study of the formation of the key intermediate compound I by probing the role of the conserved distal amino acid triad Arg-Trp-His of a recombinant catalase-peroxidase in its reaction with hydrogen peroxide, peroxoacetic acid, andm-chloroperbenzoic acid. Both the wild-type enzyme and six mutants (R119A, R119N, W122F, W122A, H123Q, H123E) have been investigated by steady-state and stopped-flow spectroscopy. The turnover number of catalase activity of R119A is 14.6%, R119N 0.5%, H123E 0.03%, and H123Q 0.02% of wild-type activity. Interestingly, W122F and W122A completely lost their catalase activity but retained their peroxidase activity. Bimolecular rate constants of compound I formation of the wild-type enzyme and the mutants have been determined. The Trp-122 mutants for the first time made it possible to follow the transition of the ferric enzyme to compound I by hydrogen peroxide spectroscopically underlining the important role of Trp-122 in catalase activity. The results demonstrate that the role of the distal His-Arg pair in catalase-peroxidases is important in the heterolytic cleavage of hydrogen peroxide (i.e. compound I formation), whereas the distal tryptophan is essential for compound I reduction by hydrogen peroxide.

Evidence for plastoquinol-cytochrome fb-563 reductase as a common electron donor to P700 and cytochrome oxidase in cyanobacteria

Abstract Membranes isolated from Nostoc sp. strain MAC and Anacystis nidulans displayed spectral changes in the cytochrome f b region when examined by reduced minus oxidized or dual wavelength spectrophotometry under physiological conditions. The same changes accompanied both light-induced (photosynthetic) and oxygen-induced (respiratory) electron transport. Physiological reduction of the cytochrome f b moiety was abolished after extraction of plastoquinone but reappeared on reconstitution of the depleted membranes with authentic plastoquinone. Moreover, a mutual inhibition of photosynthetic and respiratory activities could be directly demonstrated with the isolated membranes. From the results it is concluded that the membrane-bound plastoquinol-cytochrome f b reductase functions as a common electron donor to both P700 and the cytochrome oxidase in cyanobacteria.

Respiratory electron transport in plasma and thylakoid membrane preparations from the cyanobacterium Anacystis nidulans

Plasma and thylakoid membranes were separated and purified from cell‐free extracts of Anacystis nidulans by discontinuous sucrose density gradient centrifugation. Plasma membranes contained less than 0.05% (w/w) chlorophyll per protein. Both plasma and thylakoid membranes oxidized horse heart ferrocytochrome c (10 ± 4 and 3 ± 1 per mg protein, respectively), with strong inhibition by low concentrations of cyanide, sulfide, azide, carbon monoxide, and salicyl aldoxime. The activity was stimulated 125% by 3.5% Tween 80 and totally suppressed by 100 mM NaCl. Oxidized cytochrome c was reduced by both types of membranes in the presence of NAD(P)H. The results indicate the occurrence of a respiratory chain including cytochrome‐c oxidase in both plasma and thylakoid membranes of A. nidulans.

Catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803: cloning, overexpression in Escherichia coli, and kinetic characterization.

Abstract The Synechocystis PCC 6803 katG gene encodes a dual-functional catalase-peroxidase (EC 1.11.1.7). We have established a system for the high level expression of a fully active recombinant form of this enzyme. Its entire coding DNA was extended using a synthetic oligonucleotide encoding a hexa-histidine tag at the C-terminus and expressed in Escherichia coli [BL21-(DE3)pLysS] using the pET-3a vector. Hemin was added to the culture medium to ensure its proper association with KatG upon induction. The expressed protein was purified to homogeneity by two chromatography steps including a metal chelate affinity and hydrophobic interaction chromatography. The homodimeric acidic protein (pI = 5.4) had a molecular mass of 170 kDa and a Reinheitszahl (A406/A280) of 0.64. The recombinant protein contained high catalase activity (apparent K m = 4.9 ± 0.25 mM and apparent k cat = 3 500 s−1) and an appreciable peroxidase activity with o-dianisidine, guaiacol and pyrogallol, but not with NAD(P)H, ferrocytochrome c, ascorbate or glutathione as electron donors. By using both conventional and sequential stopped-flow spectroscopy, formation of compound I with peroxoacetic acid was calculated to be (8.74 ± 0.26) × 103 M−1 s−1, whereas compound I reduction by o-dianisidine, pyrogallol and ascorbate was determined to be (2.71 ± 0.03) × 106 M−1 s−1, (8.62 ± 0.21) × 104 M−1 s−1, and (5.43 ± 0.19) × 103 M−1 s−1, respectively. Cyanide binding studies on native and recombinant enzyme indicated that both have the same heme environment. An apparent second-order rate constant for cyanide binding of (4.8 ± 0.1) × 105 M−1 s−1 was obtained.

Isolated and purified plasma and thylakoid membranes of the cyanobacteriumAnacystis nidulans contain immunologically cross-reactive aa3-type cytochrome oxidase

Cell-free extracts ofAnacystis nidulans were fractionated by discontinuous sucrose density gradient centrifugation resulting in the separation of two distinct types of membranes, the heavier one containing the chlorophyll and the lighter one devoid of chlorophyll. Identity of the latter with plasma membrane was confirmed by labeling of intact cells with impermeant marker,35S-diazobenzenesulfonate, prior to cell disruption. Both membrane fractions were purified individually by repeated recentrifugation on identical gradients. Purified membranes were subjected to dissociating polyacrylamide gel electrophoresis, either type of membranes yielding a distinct polypeptide pattern. After transfer of the polypeptides to nitrocellulose by Western blotting, two of the proteins, with molecular weights of approximately 55,000 and 32,000, respectively, gave strong and specifically complementary cross-reactions with antibodies raised against subunits I and II of the aa3-type cytochrome oxidase fromParacoccus denitrificans. The findings will be discussed in terms of the presence of aa3-type cytochrome oxidase in both plasma and thylakoid membranes ofAnacystis nidulans.

Electron transport reactions in respiratory particles of hydrogenase-induced Anacystis nidulans

Respiratory particles from hydrogen-grown Anacystis nidulans were found to oxidize H2, NADPH, NADH, succinate and ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine at rates corresponding to 28, 15, 6, 2.5, and 70 nmol O2 taken up x mg protein−1xmin−1, respectively. The particles were isolated by brief sonication of lysozyme-pretreated cells. Respiratory activities were studied in terms of both substrate oxidation and O2 uptake. The stoichiometry between oxidation of H2, NADPH, NADH or succinate, and consumption of O2 was calculated to be 1.95+-0.1 with each substrate.Inhibitors of flavoproteins did not affect the oxyhydrogen reaction while 2-n-heptyl-8-hydroxyquinoline-N-oxide as well as compounds known to block the terminal oxidase impaired the oxidation of both H2 and of NAD(P)H or succinate in a parallel fashion. No additivity of O2 uptake was observed when NADPH, NADH or succinate was present in addition to H2. Instead, H2 uptake was depressed under such conditions, and also the oxidation of NAD(P)H or succinate was increasingly lowered by increasing H2 tensions.The results suggest that in Anacystis molecular hydrogen is oxidized through the same type of respiratory chain as are NAD(P)H and succinate. Moreover, the cyanide-resistant branch of respiratory O2 uptake will be discussed, and a few results obtained with particles prepared from thylakoid-free Anacystis will also be presented.