Spectral heterogeneity and time-resolved spectroscopy of excitation energy transfer in membranes of Heliobacillus mobilis at low temperatures.

Transient absorption difference spectra in the Qy absorption band from membranes of Heliobacillus mobilis were recorded at 140 and 20 K upon 200 fs laser pulse excitation at 590 nm. Excitation transfer from short wavelength absorbing forms of bacteriochlorophyll g to long wavelength bacteriochlorophyll g occurred within 1-2 ps at both long wavelength bacteriochlorophyll g occurred within 1-2 ps at both temperatures. In addition, a slower energy transfer process with a time constant of 15 ps was observed at 20 K within the pool of long wavelength-absorbing bacteriochlorophyll g. Energy transfer from long wavelength antenna pigments to the primary electron donor P798 was observed, yielding the primary charge-separated state P798+A0-. The time constant for this process was 30 ps at 140 K and about 70 ps at 20 K. A decay component with smaller amplitude and a lifetime of up to hundreds of picoseconds was observed that was centered around 814 nm at 20 K. Kinetic simulations using simple lattice models reproduce the observed decay kinetics at 295 and 140 K, but not at 20 K. The kinetics of energy redistribution within the spectrally heterogeneous antenna system at low temperature argue against a simple "funnel" model for the organization of the antenna of Heliobacillus mobilis and favor a more random spatial distribution of spectral forms. However, the relatively high rate of energy transfer from long wavelength antenna bacteriochlorophyll g to the primary electron donor P798 at low temperature is difficult to explain with either of these models.

Spectroscopic evidence for the presence of an iron-sulfur center similar to Fx of Photosystem I in Heliobacillus mobilis.

Treatment of membranes of Heliobacillus mobilis with high concentrations of the chaotropic agent urea resulted in the removal of the iron-sulfur centers FA and FB from the reaction center, as indicated by EPR spectra under strongly reducing conditions. In urea-treated membranes, transient absorption measurements upon a laser flash indicated a recombination between the photo-oxidized primary donor P798+ and a reduced acceptor with a time constant of 20 ms at room temperature. Benzylviologen, vitamin K-3 and methylene blue were found to accept electrons from the reduced acceptor efficiently. A differential extinction coefficient of 225-240 mM-1 cm-1 at 798 nm was determined from experiments in the presence of methylene blue. Transient absorption difference spectra between 400 and 500 nm in the presence and absence of artificial acceptors indicated that the electron acceptor involved in the 20 ms recombination has an absorption spectrum similar to that of an iron-sulfur center. This iron-sulfur center was assigned to be analogous to Fx of Photosystem I. Our results provide evidence in support of the presence of Fx in heliobacteria, which was proposed on the basis of the reaction center polypeptide sequence (Liebl et al. (1993) Proc. Natl. Acad. Sci. USA 90: 7124-7128). Implications for the electron transfer pathway in the reaction center of heliobacteria are discussed.

Time-resolved fluorescence and absorption spectroscopy of photosystem I.

Picosecond fluorescence and femtosecond transient absorption spectroscopy have been used to investigate the primary energy transfer and trapping processes in a photosystem II deletion mutant from the cyanobacterium Synechocystis sp. PCC 6803, which contains active photosystem I reaction centers with approximately 100 chlorophylls per P700. In all experiments, low levels of excitation were used which avoid annihilation processes. Following 590-nm excitation, at room temperature, spectral equilibration is observed in both fluorescence and absorption measurements and is characterized by a time constant of 4-6 ps. The shape of the spectra associated with the equilibration process indicates that long wavelength pigments (pigments with absorption maxima at longer wavelength than that of the primary electron donor, P700) are present and functional at physiological temperatures in this preparation. The overall decay of excitations in the antenna is characterized by a time constant of 24-28 ps, in both fluorescence and absorption measurements. The 24-28-ps process results in the appearance of absorption changes associated with only P700+ formation. Absorption changes associated with the reduction of the primary electron acceptor were not resolved under the experimental conditions used here.

Observation of the reduction and reoxidation of the primary electron acceptor in photosystem I.

Femtosecond transient absorption spectroscopy has been used to investigate the primary charge separation in a photosystem II deletion mutant from the cyanobacterium Synechocystis sp. PCC 6803. These cells contain only the photosystem I reaction center and have a pigment content of approximately 100 chlorophylls per P700. Utilizing relatively high excitation intensities, the difference spectrum for the reduction of primary electron acceptor [(A0(-)-A0) difference spectrum] was obtained from experiments performed under both reducing and oxidizing conditions. Both approaches yield very similar results with the (A0(-)-A0) difference spectrum displaying a maximum bleaching at 687 nm. The shape of the difference spectrum suggests that the primary electron acceptor in photosystem I may be a chlorophyll a molecule. The observed rate of primary radical pair formation depends on the overall rate of decay of excitations in the antenna; the radical pair state forms as the antenna decays. The decay of the primary radical pair state is characterized by a 21-ps time constant. Under conditions that avoid annihilation effects, the mean lifetime for excitations in the antenna is 28 ps [Hastings, G., Kleinherenbrink, F.A.M., Lin, S., & Blankenship, R.E. (1994) Biochemistry (preceding paper in this issue)]. This indicates that the reduced acceptor decays faster than it forms. Therefore, only a low concentration of the reduced acceptor will accumulate under most conditions.

Delayed fluorescence from Fe-S type photosynthetic reaction centers at low redox potential.

Fluorescence kinetics were measured in membranes of a photosystem II-deletion mutant of the cyanobacterium Synechocystis sp. PCC 6803 containing photosystem I as the only reaction center and of the anoxygenic photosynthetic bacterium Heliobacillus mobilis. The measurements were performed under conditions where forward electron transfer to secondary acceptors was inhibited by the strong reductant sodium dithionite. Delayed fluorescence due to recombination of the primary radical pair P+A0- in both species was found to consist of several kinetic components. The longest-lived component had a lifetime of 35 ns in photosystem I and 18 ns in H. mobilis, respectively, which corresponds with the lifetime of the primary radical pair. Delayed fluorescence components with lifetimes of about 4 ns, 1 ns, and 200 ps were also observed in both species and were attributed to relaxations within the radical pair. A standard free energy difference of 0.18 eV was calculated for both species between the relaxed primary radical pair and the excited antenna at room temperature. A value of 0.25 eV was estimated for the free energy difference between the relaxed primary radical pair and the excited primary donor. The temperature dependence of the delayed fluorescence between 25 and 2 degrees C indicated that more than half of the free energy difference is due to enthalpy. Our measurements indicate an overall similarity between the primary electron transfer process in the Fe-S type (or low potential) reaction centers and the (bacterio)pheophytin-quinone type (or high potential) reaction centers found in purple photosynthetic bacteria and photosystem II.

Time-resolved spectroscopy of energy and electron transfer processes in the photosynthetic bacterium Heliobacillus mobilis.

The kinetics of excitation energy transfer and electron transfer processes within the membrane of Heliobacillus mobilis were investigated using femtosecond transient absorption difference spectroscopy at room temperature. The kinetics in the 725- to 865-nm region, upon excitation at 590 and 670 nm, were fit using global analysis. The fits returned three kinetic components with lifetimes of 1-2 ps and 27-30 ps, and a component that does not decay within several nanoseconds. The 1- to 2-ps component is attributed to excitation equilibration to form a thermally relaxed excited state. The 27- to 30-ps phase corresponds to the decay of the relaxed excited state to form a charge-separated state. The intrinsic energy and electron transfer rates were estimated using the experimental results and theoretical models for excitation migration and trapping dynamics. Taking into account the number of antenna pigments and their spectral distribution, an upper limit of 1.2 ps for the intrinsic time constant for charge separation in the reaction center is calculated. This upper limit corresponds with the trapping-limited case for excitation migration and trapping. Reduction of the primary electron acceptor A0 was observed in the 640 to 700 nm region using excitation at 780 nm. An instantaneous absorbance increase followed by a decay of about 30 ps was observed over a broad wavelength region due to the excited state absorption and decay of BChl g molecules in the antenna. In addition, a narrow bleaching band centered at 670 nm grows in with an apparent time constant of about 1.0 ps, superimposed on the 30-ps absorbance increase due to excited state absorption. Measurements on a longer time scale showed that besides the 670 nm pigment a BChl g molecule absorbing near 785 nm may be involved in the primary charge separation, and that this pigment may be in equilibrium with the 670 nm pigment. The bleaching bands at 670 nm and 785nm recovered with a time constant of about 600 ps, due to forward electron transport to a secondary electron acceptor. Energy and electron transfer properties of H. mobilis membranes are compared with Photosystem 1, to which the heliobacteria bear an evolutionary relationship.

Lifetimes of bacteriochlorophyll fluorescence in Rhodopseudomonas viridis and Heliobacterium chlorum at low temperatures.

Fluorescence lifetimes of isolated membranes of Rhodopseudomonas viridis were measured in the temperature range of 77 K to 25 K. At room temperature, the main component of the fluorescence decay of bacteriochlorophyll (BChl) b had a time constant of 50 ps. In contrast to other purple bacteria, the emission at low temperature was spectrally homogeneous and showed essentially single lifetimes of 140 ps at 77 K and 180 ps at 25 K, with the primary electron donor in the oxidized state. Taking into account the relative fluorescence yields with open and closed reaction centers, we arrive at numbers of 125 ps and 215 ps, respectively, for open reaction centers. These numbers are significantly smaller than expected on the basis of measurements of the efficiency of charge separation, perhaps suggesting that the excitation decay in the absence of reaction centers is considerably faster at low temperature than at room temperature. At least four different spectral components with different lifetimes were observed at 25 K in the emission of Heliobacterium chlorum, a short-wavelength component of about 30 ps and three longer-wavelength components of about 100 ps, 300 ps, and 900 ps. This indicates a strong heterogeneity in the emitting pigment, BChl g-808. The component with the shortest lifetime does not appear to be affected by the redox state of the reaction center and might reflect energy transfer to BChl g species which are connected to the reaction center.

The fluorescence yield of Rhodopseudomonas viridis in relation to the redox state of the primary electron donor.

The fluorescence yield of bacteriochlorophyll (BChl) b in membranes of Rhodopseudomonas viridis was measured immediately and at a variable time-interval after a saturating laser flash to bring about charge separation. At 4 K a decrease of the yield by 28% was observed immediately after the flash. This yield recovered mono-exponentially with a time constant of 6.3 +/- 0.4 ms to approximately the original level. The same time constant was observed for the re-reduction of the primary electron donor, indicating that the fluorescence quenching can be ascribed to the oxidation of the primary donor. The extent of quenching decreased with increasing temperature and reversed to a fluorescence increase at temperatures above 50 K. These results may be explained by the presence of long-wavelength absorbing BChls b in the antenna which at low temperature transfer their excitation energy more efficiently to the oxidized than to the reduced primary donor, in support of a similar hypothesis used to explain the quenching of fluorescence by 'oxidized' reaction centers in heliobacterium chlorum (Deinum, G., Kramer, H., Aartsma, T.J. Kleinherenbrink, F.A.M. and Amesz, J. (1991) Biochim. Biophys. Acta 1058, 339-344).