Comment on "A bacterium that can grow by using arsenic instead of phosphorus".

Wolfe-Simon et al. (Research Articles, 3 June 2011, p. 1163; published online 2 December 2010) argued that the bacterial strain GFAJ-1 can vary the elemental composition of its biomolecules by substituting arsenic for phosphorus. Although their data show that GFAJ-1 is an extraordinary extremophile, consideration of arsenate redox chemistry undermines the suggestion that arsenate can replace the physiologic functions of phosphate.

XAS investigations of Fe(VI).

Recent attention has been given to a reexamination of results from the early Viking missions to Mars that suggested the presence of one or more strong oxidants in Martian soil. Since Fe is one of the main constituents of the Martian surface and Fe(VI) is known to be a highly reactive, strong oxidant, we have made XANES and EXAFS measurements of Fe(II), Fe(III), Fe(IV), and Fe(VI) in solid and solution forms. Results from these studies indicate a preedge XANES feature from Fe(VI) samples similar to that commonly seen from Cr(VI) samples. Results of first shell analysis indicate a linear relationship between the Fe-O bondlength and Fe valence state.

Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1.

Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.

Expression of a tetraheme protein, Desulfovibrio vulgaris Miyazaki F cytochrome c(3), in Shewanella oneidensis MR-1.

Cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F was successfully expressed in the facultative aerobe Shewanella oneidensis MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 0.5 mg of cytochrome/g of cells. A derivative of the broad-host-range plasmid pRK415 containing the cytochrome c(3) gene from D. vulgaris Miyazaki F was used for transformation of S. oneidensis MR-1, resulting in the production of protein product that was indistinguishable from that produced by D. vulgaris Miyazaki F, except for the presence of one extra alanine residue at the N terminus.

Reconstitution of the 2Fe-2S center and g = 1.89 electron paramagnetic resonance signal into overproduced Nostoc sp. PCC 7906 Rieske protein.

The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp. PCC 7906. A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske protein. Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies. The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonance signals at 15 K. Two methods of reconstitution yielded Rieske preparations in which 20-60% of the protein contained 2Fe-2S clusters as determined by EPR spin quantitation. The reconstituted Rieske protein was soluble and stable at 4 degrees C in buffers containing nonionic detergents and showed a redox midpoint potential of +321 mV at pH 7.0 as determined by optical circular dichroism (CD) spectroscopy. These data demonstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cluster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis.

Purification and properties of a low-redox-potential tetraheme cytochrome c3 from Shewanella putrefaciens.

Shewanella putrefaciens is a facultatively anaerobic bacterium in the gamma group of the proteobacteria, capable of utilizing a wide variety of anaerobic electron acceptors. An examination of its cytochrome content revealed the presence of a tetraheme, low-redox-potential (E'o = -233 mV), cytochrome c-type cytochrome with a molecular mass of 12,120 Da and a pI of 5.8. The electron spin resonance data indicate a bis-histidine coordination of heme groups. Reduction of ferric citrate was accompanied by oxidation of the cytochrome. The biochemical properties suggested that this protein was in the cytochrome c3 group, which is supported by N-terminal sequence data up to the first heme binding site.

[Change in certain physical characteristics of cells at various stages of the cell cycle]. / Izmenenie nekorotykh fizicheskikh kharakteristik kletok na raznykh stadiiakh kletochnogo tsikla.

Experiments on semisynchronized cultures of animal and plant cells, performed in our laboratory approximately 30 years ago, have demonstrated that there exist regular changes of certain physical parameters of the cells during the cell cycle. In particular on yeast cultures there has been observed the appearance of specific electron paramagnetic resonance (EPR) signals with simultaneous change of the static magnetic susceptibility of the cultures in the period immediately preceding the beginning of the intensive budding. On chlorella cultures grown at certain regimes of light and darkness it is possible to observe characteristic changes of the kinetics of the photoconduction signal at the frequency of 10 HHz (SHR-photoconduction). It can be conjectured that these effects are of a similar nature linked to some changes of the intracellular structures at certain stages of the cell cycle. The work performed much later makes it possible to link these changes to the appearance of spin, glass-like structures in macromolecular intracellular bodies.

[Transferrin iron-binding capacity in hypersideremia]. / K voprosu ob obshchei zhelezosviazyvaiushchei sposobnosti transferrina pri gipersideremiiakh.

Three methods for evaluation of serum iron-binding capacity have been described: biochemical, immunological based on transferrin assay, and biophysical based on electron-paramagnetic resonance (EPR) spectroscopy of transferrin. Interrelation has been shown between transferrin and general serum iron-binding capacity. Basing on the data presented it is suggested that in secondary hemochromatosis plasma contains an iron pool that is not specifically bound with transferrin, while in primary hemochromatosis such pool was not detected.

[Behavior of magnetic particles of metallic iron in animals]. / Povedenie magnitnykh chastits metallicheskogo zheleza v organizme zhivotnykh.

Intraperitoneal introduction of 5-8 mu ferromagnetic iron particles into mouse leads to their capsulation in the liver and preservation in the organism during no less than 1 month. We have observed activation of peroxide lipid oxidation during 3-4 days after the introduction of particles and a two-fold increase of the free iron content in the liver. Intraperitoneal introduction of ferromagnetic particles with diameter below 1 mu produced rapid death. Intravenous injection of 5-8 mu diameter ferromagnetic particles into rat resulted in their accumulation in the liver, lung and spleen, probably due to the absorption of the iron particles by macrophages. In two weeks these particles were transformed and vanished from the tissues.

[Regularity of changes in magnetic characteristics of Saccharomyces cerevisiae yeast cells at various stages of culture growth]. / Zakonomernosti izmeneniia magnitnykh kharkteristik kletok drozhzhei Saccharomyces cerevisiae na raznykh stadiiakh rosta kultury.

Correlation between cell cycle of the synchronous yeast culture and ESR signal intensity at g 2.2 and 77 K was studied. It was shown that the maximal intensity of ESR signal was reached 10-15 min before the beginning of intensive cell division. The ESR signal with g 2.2 (77 K) is caused by the spin-glass like structure. The "freezing" temperature of these spin-glasses was measured.

[ESR spectra of DNA in the temperature range of 8-300 K]. / Spektry EPR preparatov DNK v intervale 8-300 K.

ESR spectra of DNA from different sources were studied in a wide temperature range. In has been shown that the structures like spin-glass exist in DNA samples and these structures manifest an ESR signal at g approximately 2.2 at room temperature, but after cooling in the magnetic field above 0.4 T they give the ESR signal at g approximately 3.0.

[Electron spin resonance study of the structure of the spin glass type in nodular variant of adenomyosis]. / Issledovanie metodom EPR struktury tipa spinovykh stekol v tkaniakh uzlovatoi formy adenomioza.

Wide ESR signals with g-factor 2.10 (B = 23 mT) and g = 3.0 (B = 80-120 mT) were recorded in the tissues of the nodular form of adenomyosis of women's uterus at the temperature range 20-300 K. The intensity maxima of these signals were at 150 +/- 20 K.

[Distribution and changes in properties of ferromagnetic iron particles administered to the animal body]. / Raspredelenie i izmenenie svoistv ferromagnitnykh chastits zheleza pri vvedenii ikh v organizm zhivotnykh.

The registration of ESR signals of organs and tissues in the wide range of temperatures permits to study properties and distribution of ferromagnetic particles in animal organisms. High dispersed powder (HDP) of iron (particle dimension--50-100 nm) was administered subcutaneously to mice in the doses 2 and 100 mg/kg weight of animals. One week after the administration HDP was accumulated in the animal organs under study. Two weeks after the treatment of mice with HDP in the dose of 2 mg/kg the ESR signals with g = 2.1 appeared in the animal tissues: liver, spleen, kidney, heart and lungs. Six weeks after the treatment the ESR signals of the studied tissues did not differ from those in control animals.

[Transformation of ferromagnetic suspensions in the animal body]. / Prevrashchenie ferromagnitnykh suspenzii v organizme zhivotnykh.

Ferromagnetic suspension (FS) was introduced into rat and mouse organisms by different ways. Transformation of FS into some organs was estimated by ESR-method within the temperature region 80-250 K. It was shown that FS introduced in the animal organism was utilized in it very quickly.

[Spin glass type structures in chloroplasts]. / Struktury tipa spinovykh stekol v khloroplastakh.

The nature of discovered in [2] magnetic anisotropy of ESR signal with g approximately 3 appearing in chloroplasts at helium temperatures has been studied and clarified. It was found that a signal with these properties can arise only when dark adapted chloroplasts are cooled down to temperatures lower than 23 K in an external magnetic field (Ho greater than 2.300 Oe). The results obtained indicate that the system studied belongs to the type of "spin glasses", i. e., of the magnetically diluted structures with cooperative magnetic interaction between centres. These cooperative properties can be most distinctly observed at definite temperature and magnetic field strength values.

[Paramagnetic centers and photochemical reactions of photosystem 2 subchloroplast fragments]. / Paramagnityne tsentry i fotokhimicheskie reaktsii subkhloroplastnykh fragmentov fotosistemy 2.

ESR signals of highly purified subchloroplast fragments containing the Photosystem 2 (PS 2) were obtained. Reversibility of the ESR II signal in darkness in the presence of dichlorophenolindophenol (DCPIP) was observed. Reduction of DCPIP with chloroplast fragments in the absence of exogenous donors takes place in light without simultaneous liberation of O2. Electron transfer may be blocked by the displacement of manganese with Mg2+ and Ba2+ bivalent cations. Cd2+ cations partially suppress the electron transfer catalysed by PS 2. The inhibiting effect of Cd2+ is removed in the presence of exogenous Mn2+ or hydroxylamine.

[Reduction paths of reaction centers of photosystem I of photosynthesis]. / O putiakh vosstanovleniia reaktsionnykh tsentrov fotosistemy I fotosinteza

The behaviour of ESR signal I in blue-green algae and chloroplasts is considered under various illumination and with various previous treatment. It is shown that the main process determining the kinetics of P700 plus reduction is the reaction with reduced components of the electron transfer chain between the two photosystems. If the accumulation of the products is inhibited by any way, then the reduction rate depends on the reactions with reducing agents in the incubation medium accumulated under irradiation before experiment. These exogenic reducing agents may be removed from the chloroplasts with repeated washings, and from algae-by means of the dark incubation for a long time. The relation of cyclic and noncyclic electron transfer is considered with and without artificial cyclic flow mediator.