W.A. Arnold's inspiring experiments.

The impact is discussed here of some experiments by W.A. Aronld and coworkers on photosynthetic research, specifically on that at the Leiden Biophysics Department. These experiments involved the following topics: photosynthetic unit and electronic excitation transfer to a reaction center, chlorophyll luminescence and photosynthesis, and unexpected experimental results as a source of discoveries.

Primary electron transfer reactions in modified reaction centers from Rhodopseudomonas sphaeroides.

Absorption spectra were measured by means of an optical multichannel analyzer in Rhodopseudomonas sphaeroides R-26 reaction centers (RCs) modified by treatment with NaBH(4) at various times (>/=1 ps) after the onset of a short excitation flash at 880 nm. Most of these RCs (75-95%) have only one "monomeric" bacteriochlorophyll-800 (B(1)) molecule and are as active as the original RCs. The duration of the excitation and measuring pulses was approximately 33 ps. If the center of the excitation pulse preceded the center of the measuring pulse by 36-40 ps, the formation of a state P(E) (early state), which is converted to the state P(F) (P(+) bacteriopheophytin(-)) in 4 +/- 1 ps (1/e time), was observed. Also the kinetics and the spectrum of the stimulated emission (reflecting the kinetics and the emission spectrum of the excited state P(*)) were determined. The difference spectrum of the state P(E) approximately equals the sum of the spectra of the states P(*) ( approximately 65%) and (1)[P(+)B(1) (-)] ( approximately 35%). This indicates that B(1) (-) is an intermediate in the electron transfer from P(*) to bacteriopheophytin, H(1), transferring this electron with a rate constant of (4 x 0.35 ps)(-1) = 7 x 10(11) s(-1).

Electron donors and acceptors in photosynthetic reaction centers.

A review is given of primary and associated electron transport reactions in various division of photosynthetic bacteria and in the two photosystems of plant photosynthesis. Two types of electron acceptor chains are distinguished: type 'Q', found in purple bacteria, Chloroflexus and system II of oxygenic photosynthesis and type 'F', found in green sulfur bacteria, Heliobacterium and photosystem I. Secondary donor reactions are discussed in relation to plant photosystem II.

Excited states and primary photochemical reactions in the photosynthetic bacterium Heliobacterium chlorum.

The charge separation and excited states of antenna bacteriochlorophyll in membrane fragments of the recently discovered photosynthetic bacterium Heliobacterium chlorum were studied by absorbance-difference spectroscopy. Formation of singlet excited states of bacteriochlorophyll g with a lifetime of 200 ps or less was observed as the disappearance of the ground state absorption bands. From the absorbance-difference spectra, it was concluded that the primary photochemical reaction consists of the transfer of an electron from the primary donor P-798 to a possibly bacteriochlorophyll c-like pigment absorbing at 670 nm. Electron transfer to the secondary acceptor occurred with a time constant of about 500 ps. The midpoint potential of this acceptor (between -450 and -560 mV) and the absence of significant absorbance changes in the near-infrared upon its reduction suggest that this acceptor is an iron-sulfur center. It is concluded that the primary photochemistry of H. chlorum is similar to that of green sulfur bacteria.

Sub-microsecond chlorophyll a delayed fluorescence from photosystem I. Magnetic field-induced increase of the emission yield.

(1) In photosystem I (PS I) particles in the presence of dithionite and intense background illumination at 290 K, an external magnetic field (0-0.22 T) induced an increase, delta F, of the low chlorophyll a emission yield, F (delta F/F approximately or equal to 1-1.5%). Half the effect was obtained at about 35-60 mT and saturation occurred for magnetic fields higher than about 0.15 T. In the absence of dithionite, no field-induced increase was observed. Cooling to 77 K decreased delta F at 685 nm, but not at 735 nm, to zero. Measuring the emission spectra of F and delta F, using continuous excitation light, at 82, 167 and 278 K indicated that the spectra of F and delta F have about the same maximum at about 730, 725 and 700 nm, respectively. However, the spectra of delta F show more long-wavelength emission than the corresponding spectra of F. (2) Only in the presence of dithionite and with (or after) background illumination, was a luminescence (delayed fluorescence) component observed at 735 nm, ater a 15 ns laser flash (530 nm), that decayed in about 0.1 microseconds at room temperature and in approx. 0.2 microseconds at 77 K. A magnetic field of 0.22 T caused an appreciable increase in luminescence intensity after 250 ns, probably mainly caused by an increase in decay time. The emission spectra of the magnetic field-induced increase of luminescence, delta L, at 82, 167 and 278 K coincided within experimental error with those of delta F mentioned above. The temperature dependence of delta F and delta L was found to be nearly the same, both at 685 and at 735 nm. (3) Analogously to the proposal concerning the 0.15 microseconds luminescence in photosystem II (Sonneveld, A., Duysens, L.N.M. and Moerdijk, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5889-5893), we propose that recombination of the oxidized primary donor P-700+ and the reduced acceptor A-, probably A-1, of PS I causes the observed fast luminescence. The effect of an external magnetic field on this emission may be explained by the radical pair mechanism. The field-induced increase of the 0.1-0.2 microseconds luminescence seems to be at least in large part responsible for the observed increase of the total (prompt + delayed) emission measured during continuous illumination in the presence of a magnetic field.

Transfer and trapping of excitation energy in photosystem II as studied by chlorophyll alpha 2 fluorescence quenching by dinitrobenzene and carotenoid triplet. The matrix model.

1. The curves representing the reciprocal fluorescence yield of chlorophyll alpha of Photosystem II (PS II) in Chlorella vulgaris as a function of the concentration of m-dinitrobenzene in the states P Q and P Q-, are found to be straight parallel lines; P is the primary donor and Q the primary acceptor of PS II. In the weakly trapping state P Q- the half-quenching of dinitrobenzene is about 0.2 mM, in vitro it is of the order of 10 mM. The fluorescence yield as a function of the concentration of a quencher is described for three models for the energy transfer between the units, and the matrix model. If it is assumed that the rate constant of quenching by dinitrobenzene is high and thus the number of dinitrobenzene molecules per reaction center low, it can be concluded that the pigment system of PS II in C. vulgaris is a matrix of chlorophyll molecules in which the reaction centers are embedded. Theoretical and experimental evidence is consistent with such an assumption. For Cyanidium caldarium the zero fluorescence yield phi 0 and its quenching by dinitrobenzene were found to be much smaller than the corresponding quantities for C. vulgaris. Nevertheless, our measurements on C. caldarium could be interpreted by the assumption that the essential properties (rate constants, dinitrobenzene quenching) of PS II are the same for these two species belonging to such widely different groups. 2. The measured dinitrobenzene concentrations required for half-quenching in vivo and other observations are explained by (non-rate-limiting) energy transfer between the chlorophyll alpha molecules of PS II and by the assumptions that dinitrobenzene is approximately distributed at random in the membrane and does not diffuse during excitation. 3. The fluorescence kinetics of C. vulgaris during a 350 ns laser flash of variable intensity could be simulated on a computer using the matrix model. From the observed fluorescence quenching by the carotenoid triplet (CT) and the measurement of the the number of CT per reaction center via difference absorption spectroscopy, the rate constant for quenching of CT is calculated to be kT = 3.3 . 10(11)s-1 which is almost equal to the rate constant of trapping by an open reaction center (Duysens, L.N.M. (1979) CIBA Foundation Symposium 61 (New Series), pp. 323--340). 4. The fluorescence quenching by CT in non-treated spinach chloroplasts after a 500 ns laser flash (Breton, J., Geacintov, N.E. and Swenberg, C.E. (1979) Biochim, Biophys. Acta 548, 616--635) could be explained within the framework of the matrix model when the value for kT is used as given in point 3. 5. The observations mentioned under point 1 indicate that the fluorescence yield phi 0 for centers in trapping state P Q is probably for a fraction exceeding 0.8 emitted by PS II.

Magnetic field-induced increase in chlorophyll a delayed fluorescence of photosystem II: A 100- to 200-ns component between 4.2 and 300 K.

At room temperature the delayed fluorescence (luminescence) of spinach chloroplasts, in which the acceptor Q is prereduced, consists of a component with a lifetime of 0.7 mus and a more rapid component, presumably with a lifetime of 100-200 ns and about the same integrated intensity as the 0.7- mus component. Between 4.2 and 200 K only a 100- to 200-ns luminescence component was found, with an integrated intensity appreciably larger than that at room temperature. At 77 K the 150-ns component approached 63% of saturation at roughly the same energy as the variable fluorescence of photosystem II at room temperature. At 77 K the emission spectra of prompt fluorescence but not that of the 150-ns luminescence had a preponderant additional band at about 735 nm. The 150-ns emission also occurred in the photosystem I-lacking mutant FL5 of Chlamydomonas. These experiments indicate that the 150-ns component originates from photosystem II. At room temperature a magnetic field of 0.22 T stimulated the 0.7-mus delayed fluorescence by about 10%. At 77 K the field-induced increase of the 150-ns component amounted to 40-50%, being responsible for the observed approximately 2% increase of the total emission; the magnetic field increased the lifetime about 20%. In order to explain these phenomena a scheme for photosystem II is presented with an intermediary acceptor W between Q and the primary donor chlorophyll P-680; recombination of P-680(+) and W(-) causes the fast luminescence. The magnetic field effect on this emission is discussed in terms of the radical pair mechanism.

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.

On the quenching of the fluorescence yield in photosynthetic systems.

A modified matrix model describing transfer of excitation energy in the photosynthetic pigment system is discussed. In addition to the antenna pigments and reaction centers of the simple matrix model, a coupling complex is postulated mediating energy transfer between antenna and reaction centers. The values of the parameters describing the transfer properties of the coupling complex can be chosen in such a way that a number of recent unexplained measurements of fluorescence properties of various purple bacteria can be described. If such coupling complexes are present in oxygen evolving organisms, some of their properties must be different from those of purple bacteria.

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.

Magnetic field-induced increase of the yield of (bacterio)chlorophyll emission of some photosynthetic bacteria and of Chlorella vulgaris.

In photosynthetic bacteria, in which the iron-ubiquinone complex X is prereduced, a magnetic field induces an increase of the emmission yield, which is correlated with the decrease in reaction center triplet yield reported previously (Hoff, A.J., Rademaker, H., van Grondelle, R. and Duysens, L.N.M. (1977) Biochim. Biophys. Acta 460, 547--554). Our results support the hypothesis that under these conditions charge recombination of the oxidized primary donor and the reduced primary acceptor predominantly generates the excited singlet state of the reaction center bacteriochlorophyll. In Chlorella vulgaris and spinach chloroplasts, at 120 K, the magnetic field has an effect similar to that found in bacteria, which suggests that an intermediary electron acceptor between P-680 and Q is present in Photosystem II also.

Flash-induced photophosphorylation in Rhodospirillum rubrum chromatophores. I. The relationship between cytochrome c-420 content and photophosphorylation.

The content of cytochrome c-420 in Rhodospirillum rubrum chromatophores prepared by grinding with alumina is 5--10% of that in whole cells, and 20--40% in chromatophores by 'French' pressing. Flash-induced phosphorylation of various chromatophores which varied in cytochrome content from 7 to 40% is proportional to the cytochrome content. Extrapolating the cytochrome c-420 content to that observed in whole cells, a ratio ATP/P+X- near 1 is calculated. At low flash intensity the phosphorylation per flash is proportional to flash energy. Photophosphorylation in flashes given after a time of several minutes is only slightly dependent on the number of flashes. If the flashes are spaced from 0.1 to 10 s, relative phosphorylation in the first flash is about 70% and in the second 90+ of that observed in the following flashes. Proton binding is not affected by the cytochrome c-420 content and a ratio of H+/P+x- of 2.3 was found. These results can be explained by a working hypothesis in which charge separation occurring at one reaction centre and the resulting electron transport mediated amongst others by c-420, results in the injection of two protons into an ATPase, this in contrast to a chemiosmotic mechanism, where the protons are released in the chromatophore inner space.

Bacteriochlorophyll fluorescence of purple bacteria at low redox potentials. The relationship between reaction center triplet yield and the emission yield.

This work describes fluorescence yield measurements in suspensions of strains of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides in which the iron . quinone complex (X) was chemically reduced (state [PIX-]; P is the reaction center bacteriochlorophyll dimer, I is the long wavelength bacteriopheophytin), and compares these with the fluorescence observed when all the traps are open (state [PIX]) and with the fluorescence observed when all the traps are closed (state [P+IX]). At 77 K the amplitude and the shape of the fluorescence emission spectrum in [PIX-] are identical to those observed in [PIX]. This is a strong indication that all the extra fluorescence observed at room temperature in [PIX-] is, in fact, caused by an efficient back reaction [P+I-X-] leads to [P*IX-]. Using an equation similar to the original Vredenberg-Duysens relationship (Vredenburg, W.J. and Duysens, L.N.M. (1963) Nature 197, 355-357) but now assuming that a single reaction center has a probability pt of trapping an excitation and (1--pt) of re-emitting it to the surroundings, we are able to calculate pt as a function of the temperature by measuring the fluorescence in [PIX], [PIX-] and [P+IX] as a function of the temperature. The calculated pt values agree reasonably well with triplet yields measured in isolated reaction centers. Finally, we have measured the reaction center triplet yield (PTR) in intact systems and we have shown that the sum of the triplet yield and the remaining loss processes (PL) in the antenna bacteriochlorophyll including the bacteriochlorophyll dimer (such as fluorescence, internal conversion or direct triplet formation) is approximately constant; if we assume that at 77 K the only process which occurs in the reaction center is the formation of a reaction center triplet, than PTR + PL=1. The energy barrier between [P*IX-] and [P+I-X-] was estimated to be 0.11--0.15 eV for a set of preparations.

Flash-induced changes in the in vivo bacteriochlorophyll fluorescence yield at low temperatures and low redox potentials in carotenoid-containing strains of photosynthetic bacteria.

The changes in the in vivo bacteriochlorophyll fluorescence induced by a Xenon flash at low temperatures (77--200 K) with the "primary" acceptor X chemically prereduced have been examined in whole cells of several species of photosynthetic bacteria which contain carotenoids absorbing in the visible part of the absorption spectrum. Two groups of species with different behaviour could be distinguished. In both cases a flash-induced rise of the fluorescence yield was observed with X prereduced at 77 k; as the temperature was increased the ratio of the maximum fluorescence (FM) and the basal fluorescence (F0) decreased and the kinetics of the decay of the high fluorescent state, as observed during the tail of the flash, apparently accelerated. Of the species examined the flash-induced changes in fluorescence-yield kinetics appeared to occur at higher temperatures in the members of one group (Chromatium vinosum, Rhodopseudomonas gelatinosa and Rhodopseudomonas palustris) than in the members of the other (Rhodopseudomonas palustris) than in the members of the other (Rhodopseudomonas sphaeroides and Rhodospirillum rubrum). These effects are interpreted in terms of the light-induced generation of triplet states within the reaction centre. It is suggested that the species-dependent differences may reflect differences in the molecular organisation of the reaction centre. It was found that in all species the reaction centre carotenoid triplet does not act as a fluorescence quencher under these conditions.

Transfer and trapping of excitation energy in photosystem II.

The fluorescence yield of chlorophyll a of system II in spinach chloroplasts as a function of the fraction q- of reaction centres in the weakly trapping state PQ-, with reduced acceptor Q-, and reduced primary donor chlorophyll, P, of the reaction centre, is described by the function phi = a/(1 - pq-), a and p being constants (Van Gorkom et al. 1978); P was estimated to be 0.74. By special treatment and additions it was ascertained that the donor complex (S-states, see below) was in the reduced state. Three models of pigment systems have been considered: separate units; units with a boundary limiting energy transfer; and the matrix or pigment bed model, which was found to describe the experimental data. The following supplementary assumptions were made: ktf greater than kt greater than k't greater than 0. The rate constant ktf is that for electronic excitation transfer from a chlorophyll a molecule (or reaction-centre chlorophyll) to the surrounding chlorophyll molecules; kt and k't are rate constants for trapping at the reaction centres in the state PQ and PQ-, respectively. From this model and additional data such as fluorescence yield in vivo and in vitro, kt was estimated to be 4 X 10(11) S-1 and k't = 7.1 X 10(10) S-1; ktf greater than 10(12) S-1. In dark-adapted Chlorella, a series of curves respresenting changes in fluorescence yield as a function of time in a succession of six 16 microseconds xenon flashes spaced at 3 s crossed at one point. It is concluded from this and other observations that in the states S2 and S3 (with two or three oxidizing equivalents in the donor complex of system II) a certain fraction of the reaction centres occurs in a special conformational state. In this state electron transfer and, possibly, energy transfer to P+ are appreciably decreased.

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).

A one microsecond component of chlorophyll luminescence suggesting a primary acceptor of system II of photosynthesis different from Q.

The kinetics of the luminescence of chlorophyll a in Chlorella vulgaris were studied in the time range from 0.2 mus to 20 mus after a short saturating flash (t 1/2 = 25 ns) under various pretreatment including anaerobiosis, flashes, continuous illumination and various additions. A 1 mus luminescence component probably originating from System II was found of which the relative amplitude was maximum under anaerobic conditions for reaction centers in the state SPQ- before the flash, about one third for centers in the state S+PQ- or SPQ before the flash, and about one tenth for centers in the state S+PQ before the flash. S is the secondary donor complex with zero change; S+ is the secondary donor complex with 1 to 3 positive charges; P, the primary donor, is the photoactive chlorophyll a, P-680, of reaction center 2; Q- is the reduced acceptor of System II, Q. Under aerobic conditions, where an endogenous quencher presumably was active, the luminescence was reduced by a factor two. The 1 mus decay of the luminescence is probably caused by the disappearance of P+ formed in the laser flash according to the reaction ZP+ leads to Z+ in which Z is the molecule which donates an electron to P+ and which is part of S. After addition of hydroxylamine, the 1 mus luminescence component changed with the incubation time exponentially (tau = 27 s) into a 30 mus component; during the same time, the variable fluorescence yield, measured 9 mus after the laser flash, decreased by a factor 2 with the same time constant. Hereafter in a second much slower phase the fluorescence yield decreased as an exponential function of the incubation time to about the dark value; meanwhile the 30 mus luminescence increased about 50% with the same time constant (tau = 7 min). Heat treatment abolished both luminescence components. The 1 mus luminescence component saturated at about the same energy as the System II fluorescence yield 60 mus after the laser flash and as the slower luminescence components. From the observation that the amplutide is maximum if the laser flash is given when the fluorescence yield is high after prolonged anaerobic conditions (state SQ-), we conclude that the 1 mus luminescence is probably caused by the reaction PWQ- + hv leads to P*WQ- leads to P+W-Q- leads to P*WQ- leads to PWQ- + hv in which W is an acceptor different from Q. The presence of S+ reduced the luminescence amplitude to about one third. Two models are discussed, one with W as an intermediate between P and Q and another, which gives the best interpretation, with W on a side path.

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-.