Louis N.M. Duysens

On the magnetic field dependence of the yield of the triplet state in reaction centers of photosynthetic bacteria

The yield of the triplet state in reaction centers of Rhodopseudomonas sphaeroides is dependent on the strength of an applied magnetic field. Reaction centers of the wild type that lack a functional iron complexed to the primary acceptor ubiquinone show a dependence similar to that of reaction centers of the mutant R-26 in which the iron-ubiquinone complex is intact. Apparently, the iron of the iron-ubiquinone complex is not essential to the effect, but it does exert an influence on its extent. Inchromatophores, the effect is about 2-fold decreased; the value of the magnetic field at which half the effect is found is about 500 G, in contrast to this value for reaction centers, which is 50--100 G. The magnetodependence of the triplet yield is discussed in terms of the Chemically Induced Dynamic Electron Polarization mechanism (CIDEP).

Picosecond absorbance difference spectroscopy on the primary reactions and the antenna-excited states in Photosystem I particles

Abstract Absorbance difference spectra at various delay times, and kinetics of absorbance changes induced by a 35 ps excitation pulse at 532 nm, were measured of relatively intact Photosystem I particles from spinach containing about 70 chlorophyll a molecules per photoactive primary electron donor P-700. The excitation pulse produced absorbance changes due to the formation of singlet- and triplet-excited antenna chlorophyll a , and, in the case of active reaction centers, also those due to the oxidation of P-700. The formation of excited chlorophyll a was accompanied by the bleaching of the Q y ground state absorption band and by the appearance of a rather flat absorption increase in the region 550–900 nm. The lifetime of singlet-excited chlorophyll a was found to be 40 ± 5 ps. When the iron-sulfur centers were prereduced (photo)chemically, the formation of a radical pair consisting of P-700 + and a chlorophyllous anion was observed. The absorbance-difference spectrum calculated for the reduction of the acceptor was similar to that measured earlier (Shuvalov, V.A., Klevanik, A.V., Sharkov, A.V., Kryukov, P.G. and Ke, B. (1979) FEBS Lett. 107, 313–316), and indicated that the acceptor is a chlorophyll a species absorbing around 693 nm. The lifetime of the radical pair was at least 25 ns. If, however, the acceptor complex was in the oxidized state before the flash, only the oxidation of P-700 was observed. No direct evidence was obtained for the reduction of the chlorophyllous acceptor, implying that if such an anion is formed, it must be reoxidized within 50 ps.

Primary-charge separation and excitation of chlorophyll a in photosystem II particles from spinach as studied by picosecond absorbance-difference spectroscopy

Abstract Photosystem II particles from spinach, containing about 80 chlorophyll a molecules per reaction center, have been investigated with picosecond absorbance-difference spectroscopy. The 35 ps excitation pulse at 532 nm produced absorbance changes due to the formation of singlet excited antenna chlorophyll a and to the primary-charge separation in the reaction centers. The appearance of excited chlorophyll a was accompanied by the bleaching of the ground state Qy absorption band and by the formation of a rather flat absorption band in the region 550–900 nm. At high flash intensity its average lifetime was found to be several tens of picoseconds. In the reaction center charge separation was observed between the primary electron donor P-680 and pheophytin a. Reduction of pheophytin a was accompanied by an absorbance increase between 640 and 675 nm and a bleaching around 685 nm. Electron transfer to a secondary acceptor occurred with a time constant of 250–300 ps. If this secondary acceptor was reduced chemically, the primary radical pair decayed by charge recombination in about 2 ns.

Chlorophyll a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor P-680 Of photosystem II.

1. Changes in the fluorescence yield of aerobic Chlorella vulgaris have been measured in laser flashes of 15 ns, 30 ns and 350 ns half time. The kinetics after the first flash given after a 3 min dark period could be simulated on a computer using the hypothesis that the oxidized acceptor Q and primary donor P+ are fluorescence quenchers, and Q- is a weak quencher, and that the reduction time for P+ is 20-35 ns. 2. The P+ reduction time for at least an appreciable part of the reaction centers was found to be longer after the second and subsequent flashes. In the first 5 flashes an oscillation was observed. Under steady state conditions, with a pulse separation of 3 s, a reduction time for P+ of about 400 ns for all reaction centers gave the best correspondence between computed and experimental fluorescence kinetics.

Function and properties of a soluble c-type cytochrome c-551 in secondary photosynthetic electron transport in whole cells of Chromatium vinosum as studied with flash spectroscopy

Abstract Changes in the absorption spectrum induced by 10-μs flashes and continuous light of various intensities were studied in whole cells of Chromatium vinosum. This paper describes the role and function of a soluble c-type cytochrome, c-551, which was surprisingly found to act in many ways similar to the cytochrome c-420 in Rhodospirillum rubrum, described in a previous paper [1]. After the photooxidation of the membrane bound high potential cytochrome c-555 by a 10-μs flash, (the low potential cytochrome c-552 was kept permanently in the oxidized state) the oxidation of c-551 is observed ( t 1 2 = 0.3 ms ). From a careful analysis of the absorbance difference spectrum and the kinetics it is concluded that there is approximately 0.6–0.7 c-551 per reaction center and that essentially all the c+-555 is reduced via the cytochrome c-551. The oxidized-reduced difference spectrum of c-551 shows peaks at 551 and 421.5 nm. The reduction of c+-551 following the flash-induced oxidation is strongly inhibited by HOQNO, but only slightly by antimycin A. Cytochrome c-551 reduces only the oxidized high potential cytochrome c-555, which is probably located on the outside of the membrane, on the opposite side of the primary acceptor. The low potential cytochrome c-552 does not show any detectable interaction with cytochrome c-551. After the cells have been sonicated, no c-551 is photooxidized and at least part of the cytochrome occurs in the solution. Analysis of the reduction kinetics of c+-551 in the absence and presence of external donors suggests that c+-551 is partly reduced via a cyclic pathway, which is blocked by addition of o-phenanthroline, and partly via a non-cyclic pathway. The non-cyclic reduction rate of c+-551 (k = 6 s−1) is increased approximately 5–10 times upon thiosulphate addition, suggesting a role for c-551 between the final donor pool and the oxidized membrane bound c-type cytochromes.

Carotenoid triplet yields in normal and deuterated Rhodospirillum rubrum

Quantum yields of carotenoid triplet formation in Rhodospirillum rubrum wild type and fully deuterated cells and chromatophores were determined in weak laser flashes for excitation wavelength lambda i = 530 nm (mainly absorbed by the carotenoid spirilloxanthin) and for lambda i = 608 nm (mainly absorbed by bacteriochlorophyll) in the presence and absence of magnetic fields. All experiments were performed at room temperature and in the absence of oxygen. The quantum yield of reaction center bacteriochlorophyll oxidation in wild type preparations, in which all reaction centers are in state PIX, at lambda i = 608 nm is close to unity, whereas the quantum yield of antenna carotenoid triplet formation is low (about 5%); P is the primary electron donor, a bacteriochlorophyll dimer, I the primary acceptor, a bacteriopheophytin, and X the secondary acceptor, an iron-ubiquinone complex. In cells in which the reaction centers are in the state P+IX(-), the antenna carotenoid triplet yield is about 0.2. In contrast, at lambda i = 530 nm, the quantum yield of P+ formation is relatively low (0.3) and the yield of the antenna carotenoid triplet state in state PIX unusually high (0.3). At increasing light intensities of 530 nm only about 3 carotenoids per reaction center of the 15 carotenoids present are efficiently photoconverted into the triplet state, which indicates that there are two different pools of carotenoids, one with a low efficiency for transfer of electronic excitation to bacteriochlorophyll and a high yield for triplet formation, the other with a high transfer efficiency and a low triplet yield. The absorption difference spectrum of the antenna carotenoid triplet, excited by 608 or 530 nm light in the state P+IX(-) does not show the peak at 430 nm, that is present in the difference spectrum of the reaction center carotenoid triplet, mainly observed at lambda i = 608 nm with weak flashes. The yield of the reaction center carotenoid triplet, generated in chromatophores in the state PIX(-), is decreased by about 10% by a magnetic field of 0.6 T. In a magnetic field of 0.6 T the yield of the carotenoid triplet, formed by 530 nm excitation in chromatophores at ambient redox potential, is decreased by about 45%. The high quantum yield of formation and the pronounced magnetic field effect for the carotenoid triplet generated by direct excitation at 530 nm can be explained by assuming that this triplet is not formed by intersystem crossing, but by fission of the singlet excitation into two triplet excitations and subsequent annihilation (triplet pair mechanism), or by charge separation and subsequent recombination (radical pair mechanism). Fully deuterated bacteria give essentially the same triplet yields, both in the reaction center and in the antenna carotenoids and show the same magnetic field effects as non-deuterated samples. This indicates that hyperfine interactions do not play a major role in the dephasing of the spins in the radical pair P+I- nor in the formation of the antenna carotenoid triplet.

Inhibition of the reoxidation of the secondary electron acceptor of Photosystem II by bicarbonate depletion

In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R2- by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q-). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-),)-dimethylurea)- induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR-, but also if added in the state QT2-.

Excited states and primary charge separation in the pigment system of the green photosynthetic bacterium Prosthecochloris aestuarii as studied by picosecond absorbance difference spectroscopy

Abstract Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation flash and kinetics of absorbance changes were measured of the membrane vesicle preparation Complex I from the photosynthetic green sulfur bacterium Prosthecochloris aestuarii . After chemical oxidation of the primary donor the excitation pulse produced singlet and triplet excited states of carotenoid and bacteriochlorophyll a . With active reaction centers present also the flash-induced primary charge separation and subsequent electron transfer were observed. The singlet excited state of the carotenoid, formed by direct excitation at 532 nm, is characterized by an absorbance band peaking at 590 nm. Its average lifetime was calculated to be about 1 ps. Excited singlet states of bacteriochlorophyll a were characterized by a bleaching of their ground state Q y absorption bands. Singlet excited states, localized on the so-called core complex, were produced by energy transfer from excited carotenoid. Their lifetime was about 70 ps. A decay component of about 280 ps was ascribed to singlet excited bacteriochlorophyll a in the bacteriochlorophyll a protein. These singlet excitations were partly converted to the triplet state. With active reaction centers, oxidation of the primary donor, P-840, characterized by the bleaching of its Q y and Q x absorption bands, was observed. This oxidation was accompanied by a bleaching between 650 and 680 nm and an absorbance increase between 680 and 750 nm. These changes, presumably due to reduction of bacteriopheophytin c (Van Bochove, A.C., Swarthoff, T., Kingma, H., Hof, R.M., Van Grondelle, R., Duysens, L.N.M. and Amesz, J. (1984) Biochim. Biophys. Acta 764, 343–346), were attributed to the reduction of the primary electron acceptor. Electron transfer to a secondary acceptor occurred with a time-constant of 550 ± 50 ps. Since no absorbance changes due to reduction of this acceptor were observed in the red or infrared region, we tentatively assume that this acceptor is an iron-sulfur center.

Singlet-singlet annihilation at low temperatures in the antenna of purple bacteria

Abstract Chromatophores of the purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter (Rhodopseudomonas) sphaeroides were excited by means of 35-ps flashes at 532 nm of varying intensities, both at room temperature and at 4 K. With increasing exciting energy densities the integrated yield of fluorescence produced by these flashes was found to decrease considerably due to singlet-singlet annihilation. An analysis of the results showed that in R. rubrum the number of connected antenna molecules between which energy transfer is possible decreases from about 1000 to about 150 when the temperature is lowered from 298 to 4 K. In Rb. sphaeroides the B875 light-harvesting complex appears to contain about 100 connected bacteriochlorophyll (BChl) 875 molecules at 4 K, while the B800–850 complex contains about 45 BChl 850 molecules. The data are explained by a model for the antenna of Rb. sphaeroides in which units of B875, containing about four reaction centres, are separated by an array of B800–850 units that surrounds B875. By applying a random walk model we found that in both species the rate of energy transfer between neighbouring antenna molecules decreased about 10-fold upon lowering the temperature. The rate of energy transfer from antenna molecules to either open or closed reaction centres decreased only 3- to 4-fold in R. rubrum and remained approximately constant in Rb. sphaeroides upon cooling. A blue shift of the emission spectra at 4 K of both species was observed when the excitation energy density was increased to a level where singlet-singlet annihilation plays a significant role. This observation appears to support the notion that an additional long-wave pigment exists in the antenna of these bacteria.

Singlet and triplet excited carotenoid and antenna bacteriochlorophyll of the photosynthetic purple bacterium Rhodospirillum rubrum as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation pulse and kinetics of absorbance changes were measured in chromatophores of the photosynthetic purple bacterium Rhodospirillum rubrum after chemical oxidation of the primary electron donor P-875. Kinetics and spectra were measured of the excited singlet states of carotenoid and bacteriochlorophyll a and also of the triplet state of the carotenoid. The excited singlet state of carotenoid, produced by direct excitation at 532 nm, is characterized by a bleaching of the ground state absorption bands in the region 450–490 nm and by an absorbance increase with a maximum near 570 nm. Its lifetime was calculated to be 0.6 ± 0.1 ps in vitro and less than 1 ps in vivo. The triplet state of carotenoid in vivo is formed within 100 ps after direct carotenoid excitation via a pathway that does not involve excited states of bacteriochlorophyll. Singlet excitation of a bacteriochlorophyll a molecule causes the bleaching of its Qx and Qy absorption bands, and is probably associated with blue shifts of the Qy absorption band of about six neighboring bacteriochlorophyll molecules. Upon increasing the excitation density, the average lifetime of the singlet excitations on bacteriochlorophyll decreased from about 350 ps to about 10 ps or less. The results are in quantitative agreement with the known effect of singlet-singlet annihilation upon the fluorescence yield, and furthermore show that no bacteriochlorophyll or carotenoid triplet formation is associated with this annihilation.