Lel A. Drachev

Time resolution of the intermediate steps in the bacteriorhodopsin-linked electrogenesis

Bacteriorhodopsin, the retinal-containing protein from Halobacterium halobium, was discovered by Oesterhelt and Stoeckenius in 197 1 [l] . The study of this compound revealed that it functions as an electrogenic light-dependent proton pump [2-41. Electric potential and current generation mediated by bacteriorhodopsin was directly measured in this group [5-91. Another line of investigation demonstrated several intermediate spectral forms of bacteriorhodopsin, participating in light-induced H’ translocation [IO-131 . The objective of this work was to obtain the time resolution of the early events in bacteriorhodopsinmediated electrogenesis. Electric potential generation accompanying a single turnover of bacteriorhodopsin was estimated by electrodes immersed into solutions on both sides of a phospholipid-impregnated collodion film with bacteriorhodopsin proteoliposomes or membrane fragments attached to one of its surfaces. Potential generation (plus on the bacteriorhodopsin-free side of the film) was found to be composed of two phases, one correlating with the 570 nm + 412 nm spectral transition (r = 25-30 ~.ts) and the other with the reversal to the 570 nm state (7 = 6-l 2 ms). The latter phase was specifically inhibited by La3’. An early photopotential of the opposite direction was also revealed (7 < 0.3 ps). Both direct phases were decelerated by D,O and abolished at pH < 2, while the amplitude of the opposite phase increased. The amplitudes of the direct phases decreased when bacteriorhodopsin was kept in the dark (r = lo-20 min).

Correlation of photochemical cycle, H+ release and uptake, and electric events in bacteriorhodopsin

The kinetics of 3 photoinduced responses of bacteriorhodopsin have been compared: spectral changes, pH shifts in a suspension of open purple membrane sheets and electric potential generation by the sheets incorporated into a lipid‐impregnated collodion film. In the presence of a pH‐buffer, the H+ release by bacteriorhodops in was shown to correlate with the formation of the M412 intermediate and the microsecond phase of the potential generation. The H+/M412 ratio is equal to 0.7±0.1 if the ionic strength of the solution is high. In the absence of the buffer, the H+ release proved to be much slower than spectral and electric responses. The kinetics of H+ uptake by bacteriorhodopsin is close to M412 decay and to the electrogeneous millisecond phase in both the presence and absence of the pH buffer. The bacteriorhodopsin‐induced proton release phase accounts for about 20%, and the uptake phase for about 80% of the overall potential. This is compatible with the model assuming that the proton start‐out point ‐ possibly, the protonated Schiff base connecting lysine 216 with retinal ‐ is closer to the outer rather than the inner (cytoplasmic) surface of the bacterial membrane.

Electrogenesis by bacteriorhodopsin incorporated in a planar phospholipid membrane

In 1966, Mitchell [l] put forward the idea of electrogenesis in coupling membrane systems. According to this concept, there are molecular electric generators incorporated in membranes of mitochondria, chloroplasts, photosynthetic and respiring bacteria. These generators were postulated to represent enzyme complexes charging the membranes by electron or proton transport across hydrophobic barriers. According to Mitchell, the transmembrane electric potential and pH differences generated by enzymes of respiratory or photosynthetic redox chains are utilized by a reversible ATPase to form ATP. Several years ago, Tupper and Tedeschi [2] tried to detect membrane potentials in mitochondria, using microelectrodes, but failed. The size of the mitochondrion is apparently too small to allow an electrode to be introduced into the matrix space without a sharp decrease in the very high electric resistance of the mitochondrial membrane [3]. Some light-dependent electric responses of a complex character were demonstrated by Bulychev et al. [4] with microelectrodes in isolated chloroplast. The electric phenomena in coupling membranes were also studied by other methods. To measure the membrane potential in mitochondria, Mitchell and Moyle determined K+ distribution across the membrane of energized mitochondria whose K+ permeability was increased by valinomycin [5] . Our group, has described the antiport of synthetic penetrating anions and cations across the membranes of energized mitochondria [6], submitochondrial particles [7,8], subchloroplast particles [9] , chromatophores of photo-

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.

Spectral, redox and kinetic characteristics of high-potential cytochrome c hemes in Rhodopseudomonas viridis reaction center

Redox, optical and kinetic characteristics of the four‐heme cytochrome c tightly bound to the reaction center complexes of Rhodopseudomonas viridis have been studied. The two high‐potential hemes, previously thought to be identical, are shown to differ in midpoint potentials, absorption spectra and kinetics of photooxidation. One heme is characterized by E m = 380 ± 10 mV, and a split α‐band (a peak at 559 nm and a shoulder at 553 nm) whereas the other has an E m = 310±10 mV and a symmetrical α‐band at 556 nm. Kinetics of laser flash oxidation of the c‐ heme by the photogenerated P‐960+ (τ ~ 0.3 μs) matches closely that of the bacteriochlorophyll reduction and precedes oxidation of the c‐556 heme, the latter occurring with τ ~ 2.5 μs concurrently with heme c‐ re‐reduction. The data point to heme c‐ being an immediate electron donor to P‐960+. Accordingly, this heme is tentatively identified with the iron‐porphyrin group proximal to the bacteriochlorophyll special pair in the three‐dimensional model of Rps. viridis reaction centers complexes [(1985) Nature 318, 618‐624]. Thus, the following reaction sequence is assumed: c‐556 → c‐559 → P‐960+.

Generation of electric potential by reaction center complexes from Rhodospirillum rubrum

Several lines of indirect evidence indicate that the electrochemical H’ potential is produced by the lightdependent cyclic electron transfer in chromatophores and intact bacterial cells [l-4] , as was originally postulated by Mitchell [ 51. In this paper, data on the direct measurement of electric current generation by bacteriochlorophyll reaction center complexes, isolated from R. rubrum chromatophores, is reported. A method of reconstitution of the reaction center complex-containing proteoliposomes and their association with planar phospholipid membranes was elaborated. Formation of light-induced electric potential difference by the proteoliposomes was demonstrated by the conventional voltmeter techniques as well as by a phenyldicarbaundecaborane (PCB-) probe. The photoelectric effect was shown to increase on addition of TMPD or cytochrome c in combination with CoQ or vitamin Ka , and to decrease on addition of ferricyanide, o-phenanthroline and a protonophorous uncoupler.

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

Abstract 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 Ag 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.

Protonation of a novel intermediate P is involved in the M → bR step of the bacteriorhodopsin photocycle

A novel intermediate (P) of the bacteriorhodopsin (bR) photocycle, appearing between M412 and bR is described. Like bR, intermediate P shows an absorption maximum at 560–570 nm. However, the extinction coefficient of P is somewhat lower than that of bR. Moreover, there are some differences in spectra of bR and P at wavelengths shorter than 450 nm. The P → bR transition correlates with the absorption of H+ from the water medium. The following conditions proved to be favourable for the detection of the new intermediate: a high salt concentration, low light intensity and low temperature (0.5°C). The P → bR transition is strongly decelerated by a small amount of Triton X‐100. Illumination of P does not produce M412 before bR is formed. It is assumed that M412 converts to P when the Schiff base is protonated by a proton transferred from a protein protolytic group which participates in the inward H+‐conductivity pathway. Reprotonation of this group results in the conversion of P to bR. No more than 1 H+ is transported per bR photocycle.

Flash-induced electrogenic events in the photosynthetic reaction center and bc1 complexes of Rhodobacter sphaeroides chromatophores

Electrogenic events in Rb. Sphaeroides chromatophores have been studied (i) electrometrically in the chromatophore/phospholipid-impregnated collodion film system and (ii) spectrophotometrically by measuring the electrochromic spectral shift of carotenoids. Under the conditions when the bc 1 complex and ubiquinone pool were oxidized at pH 7.5, the second flashwas shown to give rise to at least two additional electrogenic phases of τ values approx. 0.2 and approx. 20 ms, which were not induced by the first flash. The fast phase was resistant to the inhibitors of the bc 1 complex, antimycin A and myxothiazol. It seems to be due to the protonation of reduced Q B in the RC complex. The slow phase was partly inhibited by antimycin A and completely by subsequent addition of myxothiazol. The antimycin-sensitive constituent of the slow phase was τ ≈ 40 ms and its rise was non-exponential. The antimycin-insensitive, myxothiazol-sensitive constituent was τ ≈ 7 ms. A comparison of (i) the kinetics of cytochrome b h redox conversion induced by the first and second flashes and (ii) the electrogenic reactions sensitive to the Q-cycle inhibitors suggests that the myxothiazol-sensitive electrogenic phase is associated with the reduction of cytochrome b h ( b -561). The antimycin-sensitive electrogenic phase apparently results from the protonation of reduced Q in the quinone-reducing center of the bct complex. Reduction of ubiquinone to ubisemiquinone by b h seems to be electrically silent, since there is no electrogenic phase to follow the kinetics of this process. Myxothiazol addedin the absence of antimycin A induced a negative electrogenic phase with an opposite polarity (τ ≈ 2.5 ms) after the second flash. This phase, completely abolished by the addition of antimycin A, is assumed to be due to the electrogenic deprotonation of the RC-reduced QH 2 which combines with center C in the bc 1 complex. The data obtained by the electrometric and spectrophotometric methods appear to be very similar, though the electrometric method is more sensitive because of the much higher signal-to-noise ratio.

Electrogenic reduction of the secondary quinone acceptor in chromatophores of Rhodospirillum rubrum: Rapid kinetics measurements

Electron transfer QA → QB has been reconstituted with added Q‐10 in Rhodospirillum rubrum chromatophores associated with a phospholipid‐impregnated collodion film. Rapid kinetics measurements of laser flash‐induced ΔΨ generated in the chromatophores show that whereas electron transfer from Qa − to QB upon the first flash is not electrogenic in dark‐adapted chromatophores, reduction of Q− B to Qbh2 induced by the second flash gives rise to an electrogenic phase with τ = 250 μs at pH 7.5 which contributes about 10% to the total ΔΨ generated upon the flash. The electrogenic phase is ascribed to vectorial protonation of Q2− B.