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A method is proposed for spectroscopic probing photo-induced reversible oxidation-reduction changes of high-potential cytochrome in chromatophore films of various humidity. On these preparations of Ect. shaposhnikovii and Chr. minutissium it was found that the characteristic time of cytochrome oxidation, tau, in samples with a high degree of humidity (P/Ps = 0.75) is 2-3 mus, which is close to that seen under physiological conditions (a suspension of intact cells or chromatophores). It was found that under continuous or pulsed illumination the lowering of the relative humidity from 0.75 to 0.15 P/Ps results in a reversible decrease in the amount of cytochrome molecules that can undergo photooxidation. The fraction of cytochrome pool that retains its activity shows a rate of oxidation which remains almost without change. The observed hydration effect and its involvement in the control of the photo-induced oxidation of cytochromes must be taken into account when a model of the molecular mechanism of this process is constructed on the basis of the electron tunneling theory. It is also shown that the dark-reduction kinetics of high-potential cytochrome consist of two components: a fast component with t1/2 = 1-3s which is independent of the sample humidity and a slow component with t1/2 = 5-20 s whose lifetime increases by a factor of 3-5 on reducing the humidity. At a high degree of humidity (P/Ps = 0.75-0.5), the kinetics of cytochrome dark-reduction exhibits only the slow component. The fast component is probably associated with the return of an electron from the primary ferroquinone acceptor and the slow component seems likely to be due to the direct transfer of an electron from exogenous electron donor to the cytochrome.

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Mössbauer spectra were investigated in membranes (chromatophores) of Rhodopseudomonas sphaeroides, enriched in 57Fe, over a temperature range from 4.2 to 300 K. The spectrum of isolated chromatophores is a symmetric doublet characterized by an isomeric shift delta=0.60+/-0.03 mm/s, quadrupole splitting delta=0.54+/-0.03 mm/s and a width gamma delta of 1.42+/-0.04 mm/3 at half maximum. These parameters, which are in fact characteristic of the Mössbauer spectra of bacterial ferredoxins, appeared practically invariable over a wide range of temperatures. The spectrum of dithionite-treated chromatophores, measured immediately after dithionite treatment, exhibits, in addition, a doublet having parameters characteristic of high-spin bivalent iron. The doublet linewidth of the Fe2+ (S=2) iron is equal, at room temperature, to the emission spectrum linewidth. At 4K, some broadening of the spectrum is observed, which is of magnetic origin. The intensity of the Fe2+(S=2) doublet from a dithionite-treated sample shows a pronounced drop after several days of storage, with the intensity of the initial doublet rising. The overall area under the spectra, the linewidth and shape are not changed. Based on experimental data obtained, possible models of the active center composed of most frequently encountered membrane-bound ferredoxins of the photosynthetic bacterium Rhodopseudomonas sphaeroides are discussed.

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A method for isolation of photoactive reaction centers from Rps. sphaeroides (wild type) chromatophores, using lauryldimethylamine oxide (LDAO) as detergent, is described. The preparation obtained is free of a light-harvesting pigment-protein complex and cytochromes. A high degree of purity can be demonstrated from the adsorption indexes of the preparation equal to A280 : A800 = 1.2--1.3; A760 : A800 : A870 = 1 : 2 : 0.9. Data from polyacrylamide gel electrophoresis suggest that the integral protein component of the preparation is made up of three polypeptides with molecular weights of 30 000, 24 000 and 20 000. Under continuous illumination the preparation exhibits an effective electron transfer from the bacteriochlorophyll dimer (BChl)2 to a system of quinone acceptors (X1, X2). The ambient potential Em of (BChl)2 half-recovery was estimated as + 475 mV and pH 7.2. The rate constant for direct electron transfer from X1- to X2 is about 0.5 . 10(3) s-1 at 300 degrees K (0.01 M phosphate buffer; 0.05% LDAO, pH 7.2). The activity of this transfer is exponentially reduced with a decrease in temperature within the range of 300 to 230 degrees K, with an activation energy, Ea, of approximately equal to 8 kcal. The recombination rate constant of light-induced ion-radicals, (BChl)2+ and X1-, was found to be 5 . 10(-2) s-1 at 180 degrees K. The preparations thus obtained can be used for studying mechanisms of primary events in photosynthesis and electron-exchange conformations in the reaction center.

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Using optical differential spectroscopy and EPR, a parallel study of light-induced electron transfer between the primary (X1) and secondary (X2) quinone-like acceptors in the preparations of reaction centers (RC) isolated from bacterial chromatophore membranes with sodium dodecyl sulfate was carried out. The data from direct measurements of the rate constant temperature dependence for the interaction between light-reduced X1 and X2 (KX1X2) are in good agreement with the data calculated from the kinetic analysis of dark reduction of photooxidized bacteriochlorophyll RC on the acceptors X1 and X2 (KX1X2 = 2.10(-1)S at 20 degrees; Ea = 11,8 kcal.mol-1 within the temperature range of 20 degrees-- -20 degrees). This evidence proves the efficiency of the previously used approach /1, 2/ for the evaluation of the X1-X2 interaction. The method proposed was used for a kinetic analysis of a low-temperature electron transfer from X1 to X2 in RC isolated with lauryldimethylaminoxide (KX1X2 = 2,3.10(2) S-1 at 20 degrees; Ea = 5,5 kcal.mol-1 within the temperature range of 10 degrees-- --70 degrees).

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In pigment-protein complexes of photosynthetic reaction centres (RC's), extracted from chromatophore membranes of Rps. sphaeroides with sodium dodecylsulphate, functional activity and intramolecular mobility were studied as a function of temperature and hydration by use of the technique of optical absorbance and ESR spectroscopy. Over the studied temperature range from +20 to -120 degrees C and at a relative humidity (P/Ps) from 0.9 to 0.1, there observed a close interrelationship between reversible kinetic changes of direct and backward redox-reactions of the photo-reduced endogeneous acceptor of quinone nature and the effective parameter of the correlation time of the rotational diffusion of the hydrophobic spin probe as well as of spin labels chemically bound to SH- and COOH-groups of amino acid residues of the RC's protein. The findings support the view that the conformational dynamics in the RC controls the effectiveness of the primary processes of stabilization of photochemically separated charges.

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The work presents the results of the first stage of the study on the valent and structural state and function of the iron atoms in the donor-acceptor environment of the photosynthetic reaction centres of purple bacteria. 57Fe was introduced by cultivating the microorganisms in a medium enriched in this isotope. At 77 K the maxima observed in the Mössbauer spectra in intact freeze-dried cells at a speed of +2 mm/s and --1 mm/s are attributed to doublets 1.11 with the isomer ahifts of 1.3 and 0.5 mm/s respectively and the constants of the quadrupole splitting (Q.S.) of 2 mm/s and 2.2 mm/s. These are presumed to arise from cytochromes type c. The Mössbauer parameters of the intense assymetric quadrupole-split doublet of a more complex nature in the mid of the spectrum with line widths of 0.5 to 7.0 mm/s and 0.5 to 1.5 mm/s fit to these of bacterial ferredoxin. From the analysis of the control and dithionite-treated samples of the temperature dependency of the observable parameters over a temperature range of 77 to 300 K it can be concluded that in cells the iron atoms are present in various valency and spin states and the relation between the redox states of the iron atoms is dependent, in particular, on the age of the culture. The Mössbauer spectra of the cell fragments indicate that most of the intracellular iron, first of all the heme iron, is bound to the fraction of photosynthetic membranes.

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