Photosynthetic electrogenic events in native membranes ofChloroflexus aurantiacus. Flash-induced charge displacements within the reaction center-cytochromec 554 complex

The thermophilic phototrophChloroflexus aurantiacus possesses a photosynthetic reaction center (RC) containing a pair of menaquinones as primary (Q_A) and secondary (Q_B) electron acceptors and a bacteriochlorophyll dimer (P) as a primary donor. A tetraheme cytochromec ₅₅₄ with two high(H)- and two low(L)-potential hemes operates as an immediate electron donor for P. The following equilibrium E_m,7 values were determined by ESR for the hemes in whole membrane preparations: 280 mV (H1), 150 mV (H2), 95 mV (L1) and 0 mV (L2) (Van Vliet et al. (1991) Eur. J. Biochem. 199: 317–323). Partial electrogenic reactions induced by a laser flash inChl. aurantiacus chromatophores adsorbed to a phospholipid-impregnated collodion film were studied electrometrically at pH 8.3. The photoelectric response included a fast phase of generation ( < 10 ns, phase A). It was ascribed to the charge separation between P⁺ and Q_A ^– as its amplitude decreased both at high and low E_h values (E_m,high=360±10 mV, estimated E_m,low\s-160 mV) in good agreement with E_m values for P/P⁺ and Q_A/Q_A ^– redox couples. A slower kinetic component appeared upon reduction of the cytochromec ₅₅₄ hemes (phase C). With H1 reduced before the flash the amplitude of phase C was equal to 15–20% of that of phase A and its rise time was 1.2–1.3 s: we attribute this phase to the electrogenic electron transfer from H1 to P⁺. Pre-reduction of H2 decreased the value to about 700–800 ns and increased the amplitude of phase C to 30–35% of that of phase A. Pre-reduction of L1 further accelerated phase C (up to of 500 ns) and induced a reverse electrogenic phase with of 12 s and amplitude equal to 10% of phase A. Upon pre-reduction of L2 the rise time of phase C was decreased to about 300 ns and its amplitude decreased by 30%. The acceleration in the onset of phase C is explained by the acceleration of the rate-limiting H1 P electrogenic reaction after reduction of the other hemes due to their electrostatic influence; a P-H1-(L1-L2)-H2 alignment of redox centers with an approximately rhombic arrangement of the cytochromec ₅₅₄ hemes is proposed. The observed reverse phase is ascribed to the post-flash charge redistribution between the hemes. Redox titration of the amplitude of phase C yielded the E_m,8.3 values of H1, H2 and L2 hemes: 340±10 mV for H1, 160±20 mV for H2 and –40±40 mV for L2.     

Lipid-impregnated filters as a tool for studying the electric current-generating proteins.

Porous filters and collodion film impregnated with decane solution of phospholipids, were used for measurements of electric potential differences generated by bacteriorhodopsin, chromatophore redox chain, H⁺-ATPase, pyrophosphatase, and mitochondrial respiratory chain. It was shown that reconstituted proteoliposomes, containing e.g., bacteriorhodopsin or natural coupling membrane vesicles, such as Rhodospirillum rubrum chromatophores, can be attached to a filter surface by means of Ca²⁺ or Mg²⁺ ions. Addition of the respective energy source was found to result in electric potential difference being generated across the filter. This effect was measured directly by $\text{Ag}\text{AgCl}$ electrodes immersed into electrolyte solutions on both sides of the filter. Using a phospholipid-impregnated collodion film one can measure electric responses as fast as 300 nsec. The phospholipid-impregnated filters turned out to be sensitive and reliable electrodes for measuring the concentration of synthetic penetrating ions, such as phenyldicarbaundecaborane, tetraphenylborate, tetrapentylammonium, and tetraphenylphosphonium. By measuring changes in the concentration of these ions in the suspension of proteoliposomes, chromatophores, mitochondria, or bacterial cells, one can follow the formation and dissipation of transmembrane potential differences in these systems. It is shown that the phospholipid-impregnated filters are much more reliable and handy than planar phospholipid membranes previously used for studying electrogenic activity of electric current-producing membrane proteins.     

Macroscopic Aggregation of Tobacco Mosaic Virus Coat Protein

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry melting curves, TMV CP in the form of a trimer–pentamer mixture (4S-protein) has very low thermal stability, with a transition temperature at about 40°C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial -helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.     

Generation of electric current by chromatophores of Rhodospirillum rubrum and reconstitution of electrogenic function in subchromatophore pigment-protein complexes.

Lipoprotein complexes, containing (1) bacteriochlorophyll reaction centers, (2) bacteriochlorophyll light-harvesting antenna or (3) both reaction centers and antenna, have been isolated from chromatophores of non-sulphur purple bacteria Rhodospirillum rubrum by detergent treatments. The method of reconstituting the proteoliposomes containing these complexes is described. Being associtated with planas azolectin membrane, ptoteoliposomes as well as intact chromatophores were found to generate a light-dependent transmembrane electric potential difference measured by Ag/AgC1 electrodes and voltmeter. The direction of the electric field inproteoliposomes can be regulated by the addition of antenna complexes to the reconstitution mixture. The reaction center complex proteoliposomes generate an electric field of a direction opposite to that in chromatophores, whereas proteoliposomes containing reaction center complexes and a sufficient amount of antenna complexes produce a potential difference as in chromatophores. ATP and inorganic pyrophosphate, besides light, were shown to be usable as energy sources for electric generation in chromatophores associated with planar membrane.     

Similarity of Vibrio alginolyticus, V. cholerae and other Vibrio species with respect to the structure of their flagellar apparatus and ribosomal 5S-RNA

Electron microscopic analysis of basal bodies of the flagella Vibrio alginolyticus revealed a structure composed of four discs. The diameters of two discs localized in the cytoplasmic membrane appeared to be twice as little as those of the other two discs. In this respect the basal body of V. alginolyticus resembles that of V. cholerae. The 5S sequence of ribosomal RNA from V. alginolyticus appeared to be similar to those of V. cholerae, V. harveyi and some other vibrios. Comparison of 5S-RNA sequence culminated in a dendrogram of evolutionary relationships of various bacterial species, suggesting that V. alginolyticus is a typical representative of the Vibrionacea family. The data obtained are discussed in terms of the role of Na+ energy metabolism in living cells.     

The sodium cycle. III. Vibrio alginolyticus resembles Vibrio cholerae and some other vibriones by flagellar motor and ribosomal 5S-RNA structures

An electron microscopic study of the basal bodies of the Vibrio albinolyticus flagellum revealed a four-disc structure. The diameters of the two discs localized closer to the cytoplasmic membrane proved to be about 2-fold shorter than those of the two others. In this respect the basal body of V. alginolyticus resembles very much that of V. cholerae described by Ferris and co-workers. The sequence of the V. alginolyticus ribosomal 5S-RNA showed that it is similar to those of V. cholerae, V. harveyi and some other vibriones. On the basis of the 5S-RNA sequences, a dendrogram of prokaryotes is presented. It confirmed the suggestion that V. alginolyticus is a typical representative of Vibrionaceae rather than a ‘monster’ greatly differing from other vibriones. Possible evolutionary relation of various bacterial species possessing the primary Na⁺ pumps is discussed.     

Reconstitution of Biological Molecular generators of electric current. Bacteriorhodopsin.

1. Photoinduced generation of electric current by bacteriorhodopsin, incorporated into the planar phospholipid membrane, has been directly measured with conventional electrometer techniques. 2. Two methods for bacteriorhodopsin incorporation have been developed: (a) formation of planar membrane from a mixture of decane solution of phospholipids and of the fraction of violet fragments of the Halobacterium halobium membrane (bacteriorhodopsin sheets), and (b) adhesion of bacteriorhodopsin-containing reconstituted spherical membranes (proteoliposomes) to the planar membrane in the presence of Ca2+ or some other cations. In both cases, illumination was found to induce electric current generation directed across the planar membrane, an effect which was measured by macroelectrodes immersed into electrolyte solutions on both sides of the membrane. 3. The maximal values of the transmembrane electric potential were of about 150 mV at a current of about 10(-11) A. The electromotive force measured by means of counterbalancing the photoeffect by an external battery, was found to reach the value of 300 mV. 4. The action spectrum of the photoeffect coincides with the bacteriorhodopsin absorption spectrum (maximum about 570 nm). 5. Both components of the electrochemical potential of H+ ions (electric potential and delta pH) across the planar membrane affect the bacteriorhodopsin photoelectric response in a fashion which could be expected if bacteriorhodopsin were a light-dependent electrogenic proton pump. 6. La3+ ions were shown to inhibit operation of those bacteriorhodopsin which pump out H+ ions from the La3+-containing compartment. 7. The photoeffect, mediated by proteoliposomes associated with thick planar membrane, is decreased by gramicidin A at concentrations which do not influence the planar membrane resistance in the light. On the contrary, a protonophorous uncoupler, trichlorocarbonylcyanidephenylhydrazone, decreases the photoeffect only if it is added at a concentration lowering the light resistance. The dark resistance is shown to be higher than the light one, and decreases to the light level by gramicidin. 8. A simple equivalent electric scheme consistent with the above results has been proposed.     

The effect of the laser microirradiation of the cell center on neutrophil motility

The cell center of human neutrophils spread on polylysine-coated coverslips was irradiated with an argon laser microbeam. After the cells were pretreated with acridine orange, the irradiation of the cell center in a dose of over 0.1 J completely and irreversibly suppressed the motility of neutrophils (both random migration and chemotaxis), even though the cells retained their polarization. The same dose, applied to the cell nucleus and the forward and backward edges of the cytoplasm, resulted in little, if any, effect on cell motility, and did not inhibit their movement toward the target. Electron microscopy of the cells with the irradiated center showed the microtubules to persist for no less than 30 minutes; no visible destruction was caused in the cell center structure. Consequently, the cell center directly controls (not through polymerization of microtubules) the motility of neutrophils.     

Orientation of reaction center complexes fromRhodobacter sphaeroides in proteoliposomes and the effect ofo-phenanthroline on electrogenesis during primary photochemical reaction

The orientation ofRhodobacter sphaeroides reaction center complexes (RC complexes) in proteoliposomal membranes was investigated by a direct electrometric method. Conditions were found that allow monitoring of only that RC complex fraction that is oriented with its donor side to the inner part of the proteoliposome. It is shown thato-phenanthroline, an inhibitor of electron transfer between primary (Q_A) and secondary (Q_B) quinone acceptors, can also inhibit the photoinduced Q_A reduction. The efficiency of this inhibition depends on the concentration of added ubiquinone. It is assumed that the laser flash-inducedo-phenanthroline inhibition of primary dipole (P-870⁺ · Q _A ^– ) formation is of a competitive nature.