Impact of salt adaptation on esterified fatty acids and cytochrome oxidase in plasma and thylakoid membranes from the cyanobacterium Anacystis nidulans

During adaptation of photoautotrophically growing fresh water cyanobacterium Anacystis nidulans to high salinity the cells showed a pronounced increase of proton-sodium antiporter activity, and of cytochrome c oxidase in isolated and purified plasma membrane. At the same time the concentrations of plasma membrane-bound EDTA-resistant copper and iron (determined by inductively coupled plasma atomic emission spectrometry) rose proportionately, accompanied by an increase in whole cell respiration. In plasma membranes from salt adapted cells lipid/protein ratios were markedly higher than in control cells, levels of esterified saturated and long-chain fatty acids being significantly higher than the respective levels of unsaturated and short-chain fatty acids which explains the higher lipid-phase transition temperatures derived from Arrhenius plots. Immunoblotting of the membrane proteins with antisera raised against the cytochrome c oxidases from Paracoccus denitrificans and A. nidulans gave two cross-reacting bands with apparent molecular weights around 50000 and 30000 (subunits I and II, respectively) which were more pronounced in plasma membranes from salt adapted cells when compared to control cells. The protein pattern of plasma membranes from salt adapted cells also showed the appearance of bands at apparent molecular weights of 44000–48000 and 54000–56000 which might stem from the proton/sodium-antiporter in this membrane.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - cyt cytochrome - DCCD N,N-dicyclohexylcarbodiimide - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - ICP-AES inductively coupled plasma atomic emission spectrometry - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - EPR electron paramagnetic resonance spectrometry     

Did respiration or photosynthesis come first?

The similarity of the mechanisms in photosynthetic and in oxidative phosphorylation suggests a common origin (conversion hypothesis). It is proposed that an early form of electron flow with oxidative phosphorylation (“prerespiration”), to therminal electron acceptors available in a reducing biosphere, was supplemented by a photocatalyst capable of a redox reaction. In this way, cyclic photophosphorylation arose. Further stages in evolution were reverse electron flow, powered by ATP, to make NADH as a reductant for CO₂, and subsequently noncyclic electron flow. These processes concomitantly provided the oxidants indispensable for full development of oxidative phosphorylation, i.e. for normal respiration: sulphate, O₂, and, with participation of the nitrificants, nitrite and nitrate. Thus prerespiration preceded photosynthesis, and this preceded respiration. It is also suggested that nonredox photoprocesses of the Halobacterium type are not part of the mainstream of bioenergetic evolution. They do not lead to photoprocesses with electron flow.     

Nitrogen fixation as evidence for the reducing nature of the early biosphere

Probably the first nitrogen fixers were anaerobic, non-photosynthetic, bacteria, i.e. fermenters. During the evolution of N₂ fixation they still needed nitrogen on the oxidation level of ammonia. Because of the complexities in structure and function of nitrogenase this evolution must have required a long time. The photosynthetic and later the respiring bacteria inherited the capacity for N₂ fixation from the fermenters, but the process did not change a great deal when it was taken over.Because of the long need for NH₃, which is unstable in a redoxneutral atmosphere, a long-persisting reducing atmosphere was needed. The transition to a redoxneutral atmosphere, dominated by CO₂, H₂O and N₂, cannot have been rapid, and the NH₃ in it was recycled. Probably the atmosphere contained for a long time, as was suggested by Urey but is often denied now, a great deal of methane as a reductant. The recycling occurred with participation of intermediates like cyanide, through energy input as UV radiation or as electric discharges. A stationary state was set up.The hypothesis is recalled that coloured, photosynthetic, NH₃ bacteria, analogous to coloured sulphur bacteria, may have existed, or may still exist, in reducing conditions. A few remarks are made about the origin of nitrification in the later, oxidizing atmosphere.     

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.     

Catalase-peroxidase from synechocystis is capable of chlorination and bromination reactions

Catalase-peroxidases (KatGs) are multifunctional heme peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity of broad specificity. Here, we show that catalase-peroxidases are also haloperoxidases capable of oxidizing chloride, bromide, and iodide in a peroxide- and enzyme-dependent manner. Recombinant KatG and the variants R119A, W122F, and W122A from the cyanobacterium Synechocystis PCC 6803 have been tested for their halogenation activity. Halogenation of monochlorodimedon (MCD), formation of triiodide and tribromide, and bromide- and chloride-mediated oxidation of glutathione have been tested. Halogenation of MCD by chloride, bromide, and iodide was shown to be catalyzed by wild-type KatG and the variant R119A. Generally, rates of halogenation increased in the order Cl(-) < Br(-) < I(-) and/or by decreasing pH. The halogenation activity of R119A was about 7-9% that of the wild-type enzyme. Upon exchange of the distal Trp122 by Phe and Ala, both the catalase and halogenation activities were lost but the overall peroxidase activity was increased. The findings suggest that the same redox intermediate is involved in H(2)O(2) and halide oxidation and that distal Trp122 is involved in both two-electron reactions. That halides compete with H(2)O(2) for the same redox intermediate is also emphasized by the fact that the polarographically measured catalase activity is influenced by halides, with bromide being more effective than chloride.