Binding of Fe3+ ions to halobacterial purple membranes as studied by Mössbauer spectroscopy.

Purple membranes (PM) from Halobacterium were reconstituted with 57Fe ions and investigated by Mössbauer spectroscopy within the temperature range from 5 to 300 K at the Fe/bacteriorhodopsin (BR) ratio 0.6-300. When the Fe/Br ratio was below 2, Fe3+ bonded to PM mostly as hydroxymonomeric particle [FeOH]2+.5H2O, the apparent charge of the iron ion being two. When the Fe/BR ratio exceeded two, the dimeric form [FeOH](2+)4.8H2O along with a cluster form dominated. The temperature dependences of the mean square displacement show that the mobility of Fe ions changes from the solid-state type to the quasi-diffusional one at temperatures approximately 200 and approximately 230 K for the dimeric or monomeric and cluster iron forms, respectively. The nature of the cation binding sites and their location on the PM surface are discussed. A possible role of the divalent cation binding to PM in the mechanism of BR proton pumping is suggested.

An investigation of the electrochemical cycle of bacteriorhodopsin analogs with the modified ring.

5,6-Epoxy-, 4-methoxy-, 4-hydroxy-, and 3,4-dehydrobacteriorhodopsins can generate delta psi coupled to a photochemical cycle with intermediate M. The kinetics of delta psi comprises three main electrogenic phases: the fast small negative, the microsecond, and the millisecond positive phases. The photocycle efficiency is lower in all the analogs. The photocycle is modified insignificantly only in 3,4-dehydrobacteriorhodopsin. In the other pigments the decay of the flash-induced bleaching in the chromophore main absorption band is slower than the decay of M or long-wave intermediates, especially in the 4-hydroxy analog. In the latter analog, such distinctions, according to delta pH measurements, are partly due to deceleration of the decay of the novel intermediate (P). In 5,6-epoxybacteriorhodopsin, at all wavelengths, the decay of the intermediates takes seconds upon M formation. According to our and literature data, no bacteriorhodopsin analogs are known to have a cycle which preserves the M-intermediate and does not transport a proton.

[Cloning of the gene for ribosomal protein HS4 in the archaebacterium Halobacterium halobium]. / Klonirovanie gena ribosomnogo belka HS4 arkhebakterii Halobacterium halobium.

Using a synthetic oligonucleotide probe, a gene of the ribosomal protein HS4 from an archaebacterium Halobacterium halobium has been cloned and partly sequenced. The translation initiation region contains a sequence complementary to the 3' end of the 16S rRNA.

Purification and properties of serine protease from Halobacterium halobium.

Pure extracellular serine protease was isolated from the culture filtrate of Halobacterium halobium by bacitracin-Sepharose affinity chromatography. The enzyme activity was completely and irreversibly lost if the NaCl concentration fell below 2 M. The protease consists of one polypeptide chain with a molecular weight of 41,000. It is characteristically enriched in Asx and Glx content, whereas the level of basic amino acids in the enzyme molecule is unusually low. The protease shows a preference for leucine in the carboxylic side of the scissile bond of the substrate, cleaving the B-chain of oxidized bovine insulin only at the Leu15-Tyr16 bond and liberating p-nitroaniline from L-pyroglutamyl-L-alanyl-L-alanyl-L-leucine-p-nitroanilide.

[Inhibition of bacteriorhodopsin by formalin and lanthanum]. / Ingibirovanie bakteriorodopsina formalinom i lantanom.

It was shown that the penetrating ions (PCB-) method can be used for quantitative estimation of the proton translocation function. Using this method it was found that the maximal inhibition (up to 5.5-6 times) can be achieved by 1% formalin treatment for 0.5 hrs in 0.5 M phosphate buffer, pH 7.8, 70 degrees. Using three methods, the kinetics of the obtained preparation were compared to those of the previously known inhibitor, La3+. The changes observed were as follows: inhibition of intermediate M412 decay in membrane suspension, decrease in the amplitude of the electric response millisecond phase in the purple membrane--collodium film system and deceleration of the overall photochemical cycle turnover in this system. The latter was registered by the rate of resporation of the electric response amplitude to the second light flash in the presence of uncoupling agents. The possible role of crosslinks during protein treatment with formalin is discussed.

[Investigation of the effects of dehydration on bacterial rhodopsin by laser resonance Raman spectroscopy]. / Issledovanie vliianiia degidratatsii na bakterial'nyi rodopsin metodom rezonansnoi spektroskopii kombinatsionnogo rasseianiia sveta.

Using laser resonance Raman spectroscopy the influence of water on the structure of the chromophore centre in bacteriorhodopsin from Halobacterium halobium has been studied. The absorption band has been found to shift from 568 nm to 506 nm due to local protein changes in the chromophore centre near Schiff base bounding retinal with the lysine residue. These changes are not accompanied by the Schiff base deprotonation. Dehydration decreases essentially the reaction rate of the cis in equilibrium trans isomerization processes. In the dry state the potential barriers of the cis in equilibrium trans transition reaction turns out to be higher than that of the reverse reaction. As a result the equilibrium shifts to the cis-retinal form. Comparison of the Raman spectra of the M412 intermediate in wet and dry states of purple membranes leads to the conclusion that in water suspensions of purple membranes the chromophore state of the M412 intermediate is closer to cis- than to trans-retinal.

[Light-induced changes in quantum yields of the photochemical cycle of conversion of bacteriorhodopsin and transmembrane proton transfer in cells of Halobacterium halobium]. / Fotoindutsirovannye izmeneniia kvantovykh vykhodov fotokhimicheskogo tsikla prevrashchenii bakteriorodopsina i transmembrannogo perenosa protonov v kletkakh Halobacterium halobium.

It was found that the rate of proton efflux from the cells, vH+, and the turnover number of bacteriorhodopsin photoconversion cycle per second, vM, are gradually decreased during continuous illumination of Halobacterium halobium with orange light (550-650 nm, 10(3) W/m2). The steady-state value of vH+ after 3 min of illumination is 3 times lower than vH+ at the 1st second of illumination. At the same time the concentration of the photochemical cycle intermediate, M412, the quantum yield of its formation, phi M, and the quantum efficiency of proton efflux, phi H+, are decreased (phi M and phi H+ up to 3-4-fold), whereas the concentration of the initial form of bacteriorhodopsin, BR570, and lifetime of M412, tau, are increased. These light-induced effects are abolished by uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP) and are enhanced by the phosphorylation inhibitor, N,N'-dicyclohexylcarbodiimide (DCCD). This suggests that the observed changes of M412 and BR570 concentrations, tau, VH+, VM, phi M and phi H+ are due to the light-induced changes in the transmembrane electric potential, delta psi, and in pH inside and outside the cell, which control the photochemical cycle reactions. The decrease of phi M and phi H+ is discussed on the basis of an earlier proposed scheme of the branched photochemical cycle. It is assumed that the back reactions of the cycle (shunts) not coupled with the transmembrane proton translocation, e.g. conversion of L550 (P550) into BR570 without M412 formation, etc., become more probably at high values of the proton electrochemical potential gradient.

[Phases of photoelectrical response of bacteriorhodopsin]. / Fazy fotoélektricheskogo otveta bakteriorodopsina.

The generation of electrical potential difference by bacteriorhodopsin sheets incorporated into collodium film impregnated with a lecithine solution in decane was studied. The electrical response to a short light flash inducing a single turnover of bacteriorhodopsin consists of four phases. Phase I of photoresponse (tau less than 0.2 mks) has a small amplitude and is directed oppositely to the electrical response to continuous light. The negative phase is followed by positive phases, i.e. microsecond (II) and millisecond (III) ones, either of which can be decomposed at least into two exponents. Phase IV of the electrical response is a passive discharge of capacity of the "bacteriorhodopsin sheets--collodium film" system. Although phases II and III correspond to the generation and disappearance of the photochemical cycle intermediate with an absorption maximum at 412 nm, the parameters and activation energy of these two processes do not show complete coincidence. The effects of gramicidin A and the trifluoromethoxycarbonylcyanidephenylhydrazone uncoupler on the photoelectrical response were analyzed. The data obtained are discussed in terms of an equivalent electrical scheme, according to which the bacteriorhodopsin sheets form closed vesicles upon being incorporated into collodium film.

[Some features of photochemical conversions of bacteriorhodopsin at low pH values]. / Osobennosti fotokhimicheskikh prevrashchenii bakteriorodopsina pri nizkikh znacheniiakh pH.

Association of bacteriorhodopsin sheets and proteoliposomes with collodium film impregnated with a lecithine solution in decane was carried out. The generation of electrical potential difference in response to a light flash within the pH range of 0-6 was studied. The amplitude of microsecond (II) and millisecond (III) phases of the photoelectrical response was shown to decline with a decrease in pH as in the case when the inhibition by acid is due to protonation of a group with a pK of 3,7. The photoelectrical response of the "acid" form of bacteriorhodopsin with a maximun at 605 nm is represented by a negative phase having a small amplitude. This phase is coupled with the formation of the photochemical cycle bathointermediate and is sharply increased in an acid medium simultaneously with the generation of bacteriorhodopsin form with an absorption maximum at 565 nm. The effects of KCl, phenyldicarbaundecaborane and fluoride on the spectral and photoelectrical responses of bacteriorhodopsin are described.

[Lipid composition of Halobacterium cells and bacteriorhodopsin isolated from them]. / Lipidnyi sostav kletok galobakterii ibakteriorodopsina, vydelennogo iz nikh.

The phospholipids qualitative and quantitative compositions were determined in halobacteria cells and in bacteriorodopsin isolated from them. The phospholipid composition of the cells and bacteriorodopsin is established to be unstable and to depend on the cells cultivation conditions and the culture strain.

[Spectral transformations in purple membranes of Halobacterium halobium: effect of 560-570 transition and blue light on photochemical processes]. / Spektral'nye prevrashcheniia v purpurnykh membranakh iz Halobacterium halobium: vliianie perekhoda 560-570 i sinego sveta na fotokhimicheskie protsessy.

Effect of reversible photodependent spectral transition 560 in equilibrium 570 of the basic absorption band in purpur membranes (PM) in water on the accumulation of short-living forms 412 and 650 of bacteriorhodopsin (BR) photochemical cycle was studied. It has been shown that photochemical transformations in BR proceed as a minimum by two independent cycles, whose initial spectral forms were represented by BR with retinene in the form 13 cis and BR with retinene in the form of trans. In the course of the transition 560-- greater than 570 a long-living spectral form 370--410--430 sensitive to the blue light is formed. On the basis of our own and literature data a new scheme of photochemical processes in PM was suggested and connection between some stages of these processes and ejection and absorption of PM protones proposed.

[Deuterated membranes of Micrococcus lysodeikticus: production and several biochemical properties]. / Deiterirovannye membrany iz bakterii Micrococcus lysodeikticus: poluchenie i nekotorye biokhimicheskie svoistva.

A technique has been elaborated for preparation of deuterated membranes from deuterated cells of Micrococcus lysodeikticus containing 85--90% of deuterium according to the data of IR and PMR spectroscopy. Normal lysis of the deuterated cells of M. lysodeikticus requires a concentration of lysozyme which is eight times higher than for usual cells (8 mg per 1 g of wet deuterated cells) and an addition of the lytic enzymes E-2 (2 mg per 1 g of wet deuterated cells). Preparations of deuterated membranes purified from ribosomes and proteins can be obtained by treating of the lysate with RNAase and washing in 0.5 M NaCl. The purity of the deuterated membranes was evaluated by the evidence of electron microscopy, IR spectra and enzyme activities. After hydrogen atoms were substituted by deuterium, the secondary structure of the total membrane protein, the ratio between the activities of the respiratory chain enzymes, and the relative content of the lipid and protein components of the deuterated membranes remain at the same level as in the protonated ones.

[Growth of Micrococcus lysodeikticus bacteria on a deuterated medium]. / Vyrashchivanie bakterii Micrococcus lysodeikticus na deiterirovannoi srede.

The object of this work was to prepare deuterated growth media and to adapt Micrococcus lysodeikticus to a medium containing deuterated-substituted organic substances and deuterium oxide instead of water. M. lysodeikticus was grown on a medium prepared from the "deuterated-cells" of Chlorella, and was capable of absorbing selectively protons from such a medium containing high concentrations of deuterium. Its deuterated cells ("monsters") produced structures consisting of several (up to 8) smaller cells, angular in shape and having a thicker (2--3 times) cell wall. Apparently, adaptation to a deuterated medium is accompanied with changes in the cell wall biosynthesis as a result of which the separation of daughter cells is interfered with in the course of cell division, and the cells are more resistant to the action of lysozyme.

Spectral transitions in purple membranes from Halobacterium halobium. I. Effect of preliminary illumination on photochemical processes.

The effect of preliminary illumination of purple membranes by yellow light on the difference spectra of short-lived intermediates has been studied. It has been found that changes of the optical density of two of these intermediates, which have the maxima of the difference spectrum at 412 nm and 650 nm, coincides well with the kinetics off the known reversible transitions of the main band of the purple membrane absorption (560 570), i.e., 13-cis-trans transitions. The changes at 412 nm and 650 nm are proportional to the concentration changes of the all-trans retinal and 13-cis retinal, respectively. It was concluded that the formation of the short-lived 412 and 650 intermediates occurs in different photochemical cycles. The pH is found to affect the formation of the 650 intermediate.

[Spectral transformations of photoproducts in Halobacterium halobium cells]. / Spektral'nye prevrashcheniia fotoproduktov v kletkakh Halobacterium halobium.

Long-living spectral products observed earlier in purple membranes (PM) are discovered in the cells of Halobacterium halobium (Hh) under the action of yellow light. Blue spectral region of the light has been found to be optimal for PM arising in the cells cultivated under light. On the basis of the changes discovered in the optical spectra of Hh we may suppose that PM arising in the cells, is the dark processes, occur due to a hypothetic enzyme, which appears under light. The rate of PM arising is maximal during the periods of thermogenesis maximum.