Kononenko Aa

Evidence for a correlation between the photoinduced electron transfer and dynamic properties of the chromatophore membranes from Rhodospirillum rubrum.

Besides the study of the static structure of biologically important macromolecules, investigation of the dynamic properties has gained much interest in the last few years. X-ray diffraction on myo~obin crystals [l] revealed structural dynamics. Evidence of a large number of confo~ational substates of a molecule was found. Molecules in different conformational substates have the same gross structure but differ in local configurations. To separate the static from the dynamic disorder in the myoglobin crystals, X-ray data and Mossbauer measurements on the heme iron of myoglobin were compared [ 11. It was shown that these Mossbauer experiments themselves gave valuable information on the dynamic properties of the system [2]. The iron labels the motions of the molecule in a specific im~rtant place. In contrast to the study of the dynamic properties on a well-known molecule like myoglobin, the investigations on membrane-bound proteins are much more complicated. Many systems are not described completely even with respect to their biochemical properties. Energy accumulation by photosynthesis demonstrates the importance of these molecules in nature. A large number of membrane-bound proteins involved in the electron transport after light absorption contain iron. The Mijssbauer spectroscopy is, therefore, a valuable tool in the study of such systems, especially with respect to dynamic properties. X-ray ~vestigations, as in [l], cannot be applied because the whole system cannot be crystallized. Despite the large complexity of such systems an interpretation of

Oriented purple-membrane films as a probe for studies of the mechanism of bacteriorhodopsin functioning. II: Photoelectric processes

Abstract Photoelectric processes have been investigated in dry orderly oriented preparations of purple membranes from Halobacterium halobium under both continuous light and flash excitation. An electrophoretic sedimentation on Al, Cu, Fe, Ni, Pt and SnO 2 substrates was used to obtain orderly oriented purple-membrane films. The photoelectric response of the purple-membrane film is the sum of a light-induced ‘displacement’ current and a constant steady-state current, the proportion between the two depending upon the chemical nature of the electrodes and humidity of the film. With a high humidity, the steady-state photocurrent is correlated with the reactivity of the cathode metal (toward H + reduction reaction. A correlation is found to exist between the kinetics of photopotential rise and decay and formation and decay of the K 630 and M 412 intermediates of the bacteriorhodopsin photocycle at temperatures ranging from 293 to 83 K, indicating the electrogenic nature of these intermediates. In purple-membrane films deposited on an SnO 2 substrate, a correlation exists between the ‘dark’ potential and electron work function of the second electrode.

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria.

A method for calculating the rate constant (KA1A2) for the oxidation of the primary electron acceptor (A1) by the secondary one (A2) in the photosynthetic electron transport chain of purple bacteria is proposed. The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of KA1A2 obtained by this method from that measured by the method of Parson ((1969) Biochim, Biophys. Acta 189, 384-396), namely: (4.5 +/- 1.4)-10(3) s-1 and (6.9 +/- 1.2)-10(3) s-1, respectively. The proposed method has also been used for the estimation of the KA1A2 value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P+ at a rate exceeding this for the transfer of electron from A1 to A2. The method of Parson cannot be used in this case. The value of KA1A2 has been found to be (2.7 +/- 0.8)-10(3) s-1. The activation energies for the A1 to A2 electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.

Oriented purple-membrane films as a probe for studies of the mechanism of bacteriorhodopsin functioning. I. The vectorial character of the external electric-field effect on the dark state and the photocycle of bacteriorhodopsin

Abstract Oriented purple-membrane preparations from Halobacterium halobium were obtained by electrophoretic sedimentation of a purple-membrane suspension on a transparent current-conducting surface. Light exposure of orderly oriented purple-membrane films causes the generation of a photopotential amounting to several volts. The effects of external electric field on the dark state and photocycle of bacteriorhodopsin is studied in dry orderly oriented purple-membrane films. In contrast to nonuniformly oriented preparations (Borisevich, G.P., Lakashev, E.P., Kononenko, A.A. and Rubin, A.B. (1979) Biochim. Biophys. Acta 546, 171–174 and Lukashev, E.P., Vozary, E., Kononenko, A.A. and Rubin, A.B. (1980) Biochim. Biophys. Acta 590, 258–266), a specific feature of the field-induced phenomena observed in orderly oriented films is their vectorial character. The field-induced bathochromic shift of the maximum absorbance of bacteriorhodopsin is observed in an electric field, directed from the periplasmatic to cytoplasmatic side of the purple membrane and the field-induced rise of the photo-stationary M412 concentration in a field of opposite sign. This field-induced rise is a result of slowering of M412 decay. The observed effects seem likely to reflect the existence of the potential-dependent regulation of the bacteriorhodopsin photocycle in intact purple membranes.

Probing the fluorescence emission kinetics of the photosynthetic apparatus of Rhodopseudomonas sphaeroides, strain 1760-1, on a picosecond pulse fluorometer

Abstract Using the pulse picosecond fluorometric technique the fluorescence properties of intact cells, isolated chromatophores and photosynthetic reaction centres were studied in bacteria Rhodopseudomonas sphaeroides , strain 1760-1. The fluorescent emission from reduced reaction centres excited by 694.3 nm light has a biphasic character, the lifetimes of the components being τ 1 = 15±8 ps and τ 2 = 250 ps. The faster component, τ 1 , contributes to the integral fluorescence in the long wavelength region. It disappears with oxidation of the reaction centres and is attributed to photoactive bacteriochlorophyll P870. The slow component, τ, is apparently due to both bacteriochlorophyll P800 and bacteriopheophytin. The fluorescence from intact cells exhibits a monophasic pattern and decays with τ = 200 ps. The fluorescence emitted by chromatophores comprises two components with τ 3 = 200 ps and τ 4 = 4200 ps. The duration of fluorescence τ 3 increases to its maximum of 500–550 ps, as P870 is oxidized chemically or photochemically, while τ 4 remains unchanged. The fluorescence with a lifetime of 200 ps was ascribed to the photosystem and the 4200-ps fluorescence to bacteriochlorophyll which had lost its functional links with the photosystem. The rise time of the fluorescence emitted by chromatophores varies from 60 or 70 ps to 350 ps depending on the wavelength of the exciting light and the recorded spectral region. On the basis of our findings the rate for energy migration was estimated to be 10 9 s −1 .

Estimation of the rate of photochemical charge separation in Rhodopseudomonas sphaeroides reaction centers by fluorescence and absorption picosecond spectroscopy

Time‐resolved fluorometry of reaction center (RC) preparations from Rhodopseudomonas sphaeroides, wild strain 1760‐1, shows that the lifetime of the excited state of bacteriochlorophyll P870∗ is τ = 6± 1.5 ps and independent of temperature within the range 293 – 77 K. This value was found to coincide well with the time (7 ± 3 ps) of the RC porphyrin pigment transition into the ion‐radical pair state PF, as measured by picosecond absorption spectroscopy of the same preparations.

4-KETO-BACTERIORHODOPSIN FILMS AS A PROMISING PHOTOCHROMIC AND ELECTROCHROMIC BIOLOGICAL MATERIAL

Photochromic and electrochromic spectral properties of 4-keto-bacteriorhodopsin (4-keto-BR) embedded in a polymer matrix were studied. The light-induced spectral changes were found to be similar to those for 4-keto-BR in suspension, but the duration of the photocycle is substantially longer (up to ten of h). Application of a constant electric field induces a bathochromic shift of the main absorption band, the amplitude of the field-induced spectral changes, showing a quadratic dependence on the field strength. Polymer films containing bacteriorhodopsin analogs show promise as new spectrally-selective photochromic and electrochromic materials.

Modification of protein hydrogen bonds influences the efficiency of picosecond electron transfer in bacterial photosynthetic reaction centers

Picosecond absorption spectroscopy was used to monitor laser‐induced oxidation‐reductions of reaction center (RC) bacteriochlorophyll (P) and bacteriopheophytin (I) in Rhodopseudomonas sphaeroides RC preparations on exposure to different chemicals. The D2O isotope substitution of H2O or partial substitution of water by organic solvents (ethylene glycol, glycerol, propylene glycol, dimethyl sulfoxide) causes the appearance of a fast, nanosecond component of P+ reduction, the result of an increased probability of recombination of the primary ion‐radical products P+I− → PI. The effect is accompanied by a noticeable slowing down of electron transfer from photoreduced bacteriopheophytin to the primary quinone acceptor QA. The effect of the organic solvents, known as cryoprotectors, is correlated with their degree of hydrophobicity, i.e. the ability to penetrate the RC protein and interact with bound water and protein hydrogen bonds. The conclusion drawn from the data is that the dielectric relaxation processes through which the intermediate energy levels of the carriers in the PIQA system are lowered to levels necessary for the stabilization of the photochemically separated charges proceed with the involvement of protons of the nearest water‐protein surrounding of the RC pigments and electron transport cofactors.

Electric field promotion of the bacteriorhodopsin BR570 to BR412 photoconversion in films of Halobacterium halobium purple membranes

The combined action of electric field (105-107 V x m-1) and light (380-580 nm, 80 W x m-2) activating the photoenergetic reaction of bacteriorhodopsin (BR) in dry films of purple membranes from Halobacterium halobium was studied. A new stimulating effect of the field on the BR412 intermediate accumulation in the normal photochromic cycle of BR570 has been observed. The formation of the product BR412 is supposed to be accompanied by specific rearrangements of certain charged, polar and polarizable groups in the BR pigment-protein matrix. Such an intrinsic polarization could be promoted by an external electric field, the displacement vector of those groups being oriented in the direction of the filed. The dielectric polarization properties of the purple membranes have been demonstrated by electret-thermal analysis.

Bacteriorhodopsin (BR570) bathochromic band shift in an external electric field.

In dry films of bacteriorhodopsin-containing purple membranes from Halobacterium halobium the external electric field (10(4) -- 10(5) V . cm-1) induces the appearance of a product spectrally close to the initial intermediate of bacteriorhodopsin (BR) photochromic cycle (bathoform, K). This result and also preliminary data of the electret-thermal analysis of the preparations suggest that the dielectric polarization in chromophore-protein-lipid complexes might be an essential step of the primary stabilization of light energy in photo-bioenergetic processes.