Sergey K. Chamorovsky

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.

Photoelectric studies of the transmembrane charge transfer reactions in photosystem I pigment-protein complexes

The results of studies of charge transfer in cyanobacterial photosystem I (PS I) using the photoelectric method are reviewed. The electrogenicity in the PS I complex and its interaction with natural donors (plastocyanin, cytochrome c6 ), natural acceptors (ferredoxin, flavodoxin), or artificial acceptors and donors (methyl viologen and other redox dyes) were studied. The operating dielectric constant values in the vicinity of the charge transfer carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment–protein complex (from plastocyanin or cytochrome c6 through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and electron transfer rate constant were discussed.

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.

Electrogenic reduction of the primary electron donor P700 by plastocyanin in photosystem I complexes

An electrometric technique was used to investigate electron transfer between spinach plastocyanin (Pc) and photooxidized primary electron donor P700 in photosystem I (PS I) complexes from the cyanobacterium Synechocystis sp. PCC 6803. In the presence of Pc, the fast unresolvable kinetic phase of membrane potential generation related to electron transfer between P700 and the terminal iron–sulfur acceptor FB was followed by additional electrogenic phases in the microsecond and millisecond time scales, which contribute approximately 20% to the overall electrogenicity. These phases are attributed to the vectorial electron transfer from Pc to the protein‐embedded chlorophyll dimer P700+ within the PsaA/PsaB heterodimer. The observed rate constant of the millisecond kinetic phase exhibited a saturation profile at increasing Pc concentration, suggesting the formation of a transient complex between Pc and PS I with the dissociation constant K d of about 80 μM. A small but detectable fast electrogenic phase was observed at high Pc concentration. The rate constant of this phase was independent of Pc concentration, indicating that it is related to a first‐order process.

The cycle of photochromic reactions of a bacteriorhodopsin analog with 4-keto-retinal

Photochemical reactions in a bacteriorhodopsin analog with 4-keto-retinal (4-keto-BR) were studied by using low-temperature and pulsed laser absorption spectroscopy. A photocycle of the photochemical reactions of 4-keto-BR is proposed, which, unlike the photocycle of native BR, includes several spectrally and kinetically distinguishable M-type and O-type intermediates.

Effects of dehydration and low temperatures on the oxidation of high-potential cytochrome c by photosynthetic reaction centers in Ectothiorhodospira shaposhnikovii

Abstract Effects of temperature and dehydration on the efficiency of electron transfer from membrane-bound high-potential cytochromes ch to the reaction-center bacteriochlorophyll (P-890) in Ectothiorhodospira shaposhnikovii have been studied. A kinetic analysis of the cytochrome oxidation suggests that there are at least two conformational states of the ch-P-890 complex, of which only one allows photoinduced electron transfer from cytochrome to P-890+. Lowering the temperature of dehydration leads to a change in the proportion of the populations in the two conformations. The observed 2-fold deceleration of cytochrome oxidation can be related only to the diminution of the amount of photoactive cytochromes per reaction center. The rate constant for the transfer of an electron from cytochrome ch to bacteriochlorophyll is 2.8 · 105 s−1 and is independent of temperature and dehydration (as estimated within the accuracy of the experiments). The effects produced by low temperature and dehydration are completely reversible. The thermodynamic parameters of the transition of the cytochrome from the nontransfer to electron-transfer conformation were estimated. For room temperature (+ 20°C) in chromatophore preparations, ΔG = −5.4 kJ · M−1, ΔH = 60 kJ · M−1, ΔS = 0.22 kJ · M−1 · K−1. For Triton X-100 subchromatophore preparations, the absolute values of the above parameters are significantly lower: ΔG = −2.8 kJ · M−1, ΔH = 18 kJ · M−1, and ΔS = 0.075 kJ · M−1 · K−1. To a larger extent, the above parameters are diminished for chromatophore preparations in an 80% glycerol solution: ΔG = −1.7 kJ · M−1, ΔH = 6 kJ · M−1, ΔS = 0.025 kJ · M−1 · K−1. The data suggest the hydrophobic character of the forces that maintain the P-890-ch complex in the electron-transfer conformation. The results obtained suggest that electron tunneling within the complex cannot occur until a specific conformational configuration of the complex is formed. The efficiency of cytochrome ch oxidation is determined by the temperature, the degree of dehydration and the environmental conditions, whereas the transfer of an electron itself in the electron-transfer configuration is essentially independent of temperature and hydration.

The oxidation rate of high-potential c-type cytochrome in the photochemical reaction centre is temperature-independent

The temperature dependence of laser-induced (694.3 nm, 30 ns, 10 mJ.cm-2) high-potential cytochrome c (Em=+290 mV) oxidation kinetics was studied in Ectothiorhodospira shaposhnikovii chromatophores. It was shown that the rate constant of this reaction is independent of temperature in the range of 300 K to 120 K.

Electrogenic Reactions Associated with Electron Transfer in Photosystem I

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 I. Structure and Function of PS I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .320 II. Methods of Measurement of Membrane Potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 A. Measurement of the Generation of the Electric Potential Difference at the Heptane/ Water Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .323 B. Measurement of Electric Potential Generated by a Light Gradient in Chloroplast Suspensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 C. Measurement of the Electric Potential Difference in Oriented Multilayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 D. The Direct Electrometrical Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 III. Electrogenic Reactions in PS I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 A. Electrogenic Reactions on the Acceptor Side of PS I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 1. Electrogenicity Accompanies Photoreduction of Iron–Sulfur Clusters FA and FB in Photosystem I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 2. Electron Transfer from the Terminal Cluster FB to External Acceptors is Electrically Silent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 B. Electrogenicity Accompanying Reduction of Photooxidized P700 by Electron Donors . . . . . . . . . . . . . 328 1. Electrogenic Reduction of P700 by Plastocyanin and Cytochrome c6 . . . . . . . . . . . . . . . . . . . . . . . . . . 328 2. Electrogenic Reduction of P700 by Redox Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 IV. Profile of Changes of the Effective Dielectric Constant along the Photosynthetic Electron Transfer Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .329 A. Photosystem I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 B. The Bacterial Reaction Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 C. Photosystem II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 D. Possible Mechanisms Underlying the Correlation Between the Dielectric Constant and the Rate of Charge Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .335 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

Temperature dependence of cytochrome photooxidation and conformational dynamics of Chromatium reaction center complexes

A temperature dependence of multiheme cytochrome c oxidation induced by a laser pulse was studied in photosynthetic reaction center preparations from Chromatium minutissimum. Absorbance changes and kinetic characteristics of the reaction were measured under redox conditions where one or all of the hemes of the cytochrome subunit are chemically reduced (Eh=+300 mV or Eh=−20 to -60 mV respectively). In the first case photooxidation is inhibited at temperatures lower than 190–200 K with the rate constant of the photooxidation reaction being practically independent on temperature over the range of 300 to 190 K (k=2.2×105 s-1). Under reductive conditions (Eh=−20 to -60 mV) lowering the temperature to 190–200 K causes the reaction to slow from k=8.3×105 s-1 to 2.1×104 s-1. Under further cooling down to the liquid nitrogen temperature, the reaction rate changes negligibly. The absorption amplitude decreases by 30–40% on lowering the temperature. A new physical mechanism of the observed critical effects of temperature on the rate and absorption amplitude of the multiheme cytochrome c oxidation reaction is proposed. The mechanism suggests a close interrelation between conformational mobility of the protein and elementary electron tunneling act. The effect of “freezing” conformational motion is described in terms of a local diffusion along a random rough potential.

Fast phases of the generation of the transmembrane electric potential in chromatophores of the photosynthetic bacterium Ectothiorhodospira shaposhnikovii

Abstract Ectothiorhodospira shaposhnikovii chromatophores were associated with a collodion film and kinetics of generation of the transmembrane electric potential (Δψ) were investigated in the ‘chromatophore-collodion film’ system, using an electrometric technique with a high resolution time (over 200 ns). A generation of Δψ (the chromatophore interior positive) following a laser flash was observed, the kinetics consisting of three components of the following half-times: less than 200 ns (phase 1); 2–7 μs (phase 2); and 120 μs (phase 3). A redox titration of the kinetic phases was performed. Computer analysis of the results has shown that the midpoint potentials (Em) of the phase 1 at pH 7.5 are +400 mV and −75 mV, whereas those of the phase 2 are +310 mV and +35 mV. The comparison of the kinetic and potentiometric characteristics of the Δψ generation with analogous characteristics of the electron-transport processes, measured by optical spectroscopy, suggested that phases 1, 2 and 3 are associated with the electron transfer from P-890 to the primary quinone acceptor Qa, from the high-potential cytochrome CH to P-890, and with the reduction of secondary acceptor Qb, respectively. From the amplitude characteristics of the Δψ components, a tentative scheme of the intramembrane localization of the electron carriers is presented.