Fluorescence relaxation in intact cells of photosynthetic bacteria: donor and acceptor side limitations of reopening of the reaction center.

The dark relaxation of the yield of variable BChl fluorescence in the 10(-5)-10 s time range is measured after laser diode (808 nm) excitation of variable duration in intact cells of photosynthetic bacteria Rba. sphaeroides, Rsp. rubrum, and Rvx. gelatinosus under various treatments of redox agents, inhibitors, and temperature. The kinetics of the relaxation is complex and much wider extended than a monoexponential function. The longer is the excitation, the slower is the relaxation which is determined by the redox states, sizes, and accessibility of the pools of cytochrome [Formula: see text] and quinone for donor and acceptor side-limited bacterial strains, respectively. The kinetics of fluorescence decay reflects the opening kinetics of the closed RC. The relaxation is controlled preferentially by the rate of re-reduction of the oxidized dimer by mobile cytochrome [Formula: see text] in Rba. sphaeroides and Rsp. rubrum and by the rate constant of the [Formula: see text] interquinone electron transfer, (350 µs)(-1) and/or the quinol/quinone exchange at the acceptor side in Rvx. gelatinosus. The commonly used acceptor side inhibitors (e.g., terbutryn) demonstrate kinetically limited block of re-oxidation of the primary quinone. The observations are interpreted in frame of a minimum kinetic and energetic model of electron transfer reactions in bacterial RC of intact cells.

Mutational control of bioenergetics of bacterial reaction center probed by delayed fluorescence.

The free energy gap between the metastable charge separated state P(+)QA(-) and the excited bacteriochlorophyll dimer P* was measured by delayed fluorescence of the dimer in mutant reaction center proteins of the photosynthetic bacterium Rhodobacter sphaeroides. The mutations were engineered both at the donor (L131L, M160L, M197F and M202H) and acceptor (M265I and M234E) sides. While the donor side mutations changed systematically the number of H-bonds to P, the acceptor side mutations modified the energetics of QA by altering the van-der-Waals and electronic interactions (M265IT) and H-bond network to the acidic cluster around QB (M234EH, M234EL, M234EA and M234ER). All mutants decreased the free energy gap of the wild type RC (~890meV), i.e. destabilized the P(+)QA(-) charge pair by 60-110meV at pH8. Multiple modifications in the hydrogen bonding pattern to P resulted in systematic changes of the free energy gap. The destabilization showed no pH-dependence (M234 mutants) or slight increase (WT, donor-side mutants and M265IT above pH8) with average slope of 10-15meV/pH unit over the 6-10.5pH range. In wild type and donor-side mutants, the free energy change of the charge separation consisted of mainly enthalpic term but the acceptor side mutants showed increased entropic (even above that of enthalpic) contributions. This could include softening the structure of the iron ligand (M234EH) and the QA binding pocket (M265IT) and/or increase of the multiplicity of the electron transfer of charge separation in the acceptor side upon mutation.

The reaction center is the sensitive target of the mercury(II) ion in intact cells of photosynthetic bacteria.

The sensitivity of intact cells of purple photosynthetic bacterium Rhodobacter sphaeroides wild type to low level (<100 µM) of mercury (Hg²âº) contamination was evaluated by absorption and fluorescence spectroscopies of the bacteriochlorophyll-protein complexes. All assays related to the function of the reaction center (RC) protein (induction of the bacteriochlorophyll fluorescence, delayed fluorescence and light-induced oxidation and reduction of the bacteriochlorophyll dimer and energization of the photosynthetic membrane) showed prompt and later effects of the mercury ions. The damage expressed by decrease of the magnitude and changes of rates of the electron transfer kinetics followed complex (spatial and temporal) pattern according to the different Hg²âº sensitivities of the electron transport (donor/acceptor) sites including the reduced bound and free cytochrome c2 and the primary reduced quinone. In contrast to the RC, the light harvesting system and the bc1 complex demonstrated much higher resistance against the mercury pollution. The 850 and 875 nm components of the peripheral and core complexes were particularly insensitive to the mercury(II) ions. The concentration of the photoactive RCs and the connectivity of the photosynthetic units decreased upon mercury treatment. The degree of inhibition of the photosynthetic apparatus was always higher when the cells were kept in the light than in the dark indicating the importance of metabolism in active transport of the mercury ions from outside to the intracytoplasmic membrane. Any of the tests applied in this study can be used for detection of changes in photosynthetic bacteria at the early stages of the action of toxicants.

Export or recombination of charges in reaction centers in intact cells of photosynthetic bacteria.

The kinetics and thermodynamics of forward and reverse electron transfer around the reaction center of purple bacterium Rhodobacter sphaeroides were studied in vivo by flash-excited delayed fluorescence, prompt fluorescence (induction) and kinetic difference absorption. By protection of the photomultiplier from intense bacteriochlorophyll prompt fluorescence evoked by laser excitation, the time resolution of the fluorometer was reduced typically 10 micros. Two precursor states of the delayed fluorescence were identified: P(+)Q(A)(-) and cyt c(2)(3+)Q(A)(-) whose enthalpy levels were 340 meV and 1020 meV below A, respectively. The free energy of the P(+)Q(A)(-) state relative to A* was -870 meV in whole cells. Similar values were obtained earlier for isolated reaction center and chromatophore. The free energies of cyt c(2)(3+)Q(A)(-) and P(+)Q(A)(-) states showed no or very weak (-6 meV/pH unit) pH-dependence, respectively, supporting the concept of pH-independent redox midpoint potential of Q(A)/Q(A)(-) in intact cells. In accordance with the multiphasic kinetics of delayed fluorescence, the kinetics of re-opening of the closed reaction center is also complex (it extends up to 1 s) as a consequence of acceptor and donor-side reactions. The control of charge export from the reaction center by light regime, redox agents and inhibitors is investigated. The complex kinetics may arise from the distribution of quinones in different redox states on the acceptor side (Q(B) binding site and pool) and from organization of electron transfer components in supercomplexes.

Kinetic bacteriochlorophyll fluorometer.

A pump and probe fluorometer with a laser diode as single light source has been constructed for measurement of fast induction and relaxation of the fluorescence yield in intact cells, chromatophores and isolated reaction centers of photosynthetic bacteria. The time resolution of the fluorometer is limited by the repetition time of the probing flashes to 20 micros. The apparatus offers high sensitivity, excellent performance and can become a versatile device for a range of demanding applications. Some of them are demonstrated here including fast and easy investigation of the (1) organization and redox state of the photosynthetic apparatus of the intact cells of different bacterial strains and mutants and (2) electron transfer reactions on donor and acceptor sides of isolated reaction centers. The compact design of the mechanics, optics, electronics, and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems.

Early detection of mercury contamination by fluorescence induction of photosynthetic bacteria.

The induction (sudden dark-to-light transition) of fluorescence of photosynthetic bacteria has proved to be sensitive tool for early detection of mercury (Hg(2+)) contamination of the culture medium. The major characteristics of the induction (dark, variable and maximum fluorescence levels together with rise time) offer an easier, faster and more informative assay of indication of the contamination than the conventional techniques. The inhibition of Hg(2+) is stronger in the light than in the dark and follows complex kinetics. The fast component (in minutes) reflects the damage of the quinone acceptor pool of the RC and the slow component (in hours) is sensitive to the disintegration of the light harvesting system including the loss of the structural organization and of the pigments. By use of fluorescence induction, the dependence of the diverse pathways and kinetics of the mercury-induced effects on the age and the metabolic state of the bacteria were revealed.

Purple non-sulfur photosynthetic bacteria monitor environmental stresses.

Heavy metal ion pollution and oxygen deficiency are major environmental risks for microorganisms in aqueous habitat. The potential of purple non-sulfur photosynthetic bacteria for biomonitoring and bioremediation was assessed by investigating the photosynthetic capacity in heavy metal contaminated environments. Cultures of bacterial strains Rhodobacter sphaeroides, Rhodospirillum rubrum and Rubrivivax gelatinosus were treated with heavy metal ions in micromolar (Hg(2+)), submillimolar (Cr(6+)) and millimolar (Pb(2+)) concentration ranges. Functional assays (flash-induced absorption changes and bacteriochlorophyll fluorescence induction) and electron micrographs were taken to specify the harmful effects of pollution and to correlate to morphological changes of the membrane. The bacterial strains and functional tests showed differentiated responses to environmental stresses, revealing that diverse mechanisms of tolerance and/or resistance are involved. The microorganisms were vulnerable to the prompt effect of Pb(2+), showed weak tolerance to Hg(2+) and proved to be tolerant to Cr(6+). The reaction center controlled electron transfer in Rvx. gelatinosus demonstrated the highest degree of resistance against heavy metal exposure.