Electron acceptors of photosynthetic bacterial reaction centers. Direct observation of oscillatory behaviour suggesting two closely equivalent ubiquinones.

When reaction centers are illuminated by a series of single turnover flashes ubisemiquinone is formed and destroyed on alternate flashes. This oscillatory behaviour can be observed with both optical and electron spin resonance techniques. The oscillations are dependent upon the presence of excess ubiquinone in a manner which suggests that two molecules may act almost equivalently as metastable primary acceptors forming a two-electron gate between the one-electron primary photoact and a two-electron secondary acceptor pool.     

Secondary electron transfer in reaction centers of Rhodopseudomonas sphaeroides. Out-of-phase periodicity of two for the formation of ubisemiquinone and fully reduced ubiquinone.

Electron transfer between purified reaction centers from Rhodopseudomonas sphaeroides and exogenous ubiquinone has been studied in the presence of electron donors by measurements of light-induced absorbance changes following a sequence of short actinic light flashes. Each odd flash promotes the formation of a molecule of ubisemiquinone; after each even flash the semiquinone disappears and a molecule of the fully reduced quinone appears. We interpret these results by means of a model where a specialized molecule of ubiquinone is reduced by the primary electron acceptor in a one-electron transfer reaction after each flash, and is reoxidized by a molecule of the ubiquinone pool in a two-electron transfer reaction every two flashes.     

Secondary electron transfer in reaction centers of Rhodopseudomonas sphaeroides. Out-of-phase periodicity of two for the formation of ubisemiquinone and fully reduced ubiquinone

Electron transfer between purified reaction centers from Rhodopseudomonas sphaeroides and exogenous ubiquinone has been studied in the presence of electron donors by measurements of light-induced absorbance changes following a sequence of short actinic light flashes. Each odd flash promotes the formation of a molecule of ubisemiquinone; after each even flash the semiquinone disappears and a molecule of the fully reduced quinone appears.We interpret these results by means of a model where a specialized molecule of ubiquinone is reduced by the primary electron acceptor in a one-electron transfer reaction after each flash, and is reoxidized by a molecule of the ubiquinone pool in a two-electron transfer reaction every two flashes.     

Charge accumulation at the reducing side of system 2 of photosynthesis

A study was made of the reactions between the primary and secondary electron acceptors of Photosystem 2 by measurements of the increase of chlorophyll fluorescence induced in darkness by dithionite or by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The experiments were done either with chloroplasts to which hydroxylamine or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) was added, or with chloroplasts treated with tris(hydroxymethyl)aminomethane (Tris) to which phenylenediamine and ascorbate were added as donor system. Under these conditions the fluorescence increase induced by dithionite or DCMU added after illumination with short light flashes was dependent on the flash number with a periodicity of two; it was large after an uneven number of flashes, and small after a long darktime or after an even number of flashes. The results are interpreted in terms of a model which involves a hypothetical electron carrier situated between Q and plastoquinone; this electron carrier is thought to equilibrate with plastoquinone in a two-electron transfer reaction; the results obtained with DCMU are explained by assuming that its midpoint potential is lowered by this inhibitor.     

Modulation of the Primary Electron Transfer Rate in Photosynthetic Reaction Centers by Reduction of a Secondary Acceptor

Photosynthetic application of picosecond spectroscopic techniques to bacterial reaction centers has led to a much greater understanding of the chemical nature of the initial steps of photosynthesis. Within 10 ps after excitation, a charge transfer complex is formed between the primary donor, a “special pair” of bacteriochlorophyll molecules, and a transient acceptor involving bacteriopheophytin. This complex subsequently decays in about 120 ps by donating the electron to a metastable acceptor, a tightly bound quinone.

Recent experiments with conventional optical and ESR techniques have shown that when reaction centers are illuminated by a series of single turnover flashes in the presence of excess electron donors and acceptors, a stable, anionic ubisemiquinone is formed on odd flashes and destroyed on even flashes, suggesting that the acceptor region contains a second quinone that acts as a two-electron gate between the reaction center and subsequent electron transport events involving the quinone pool.

Utilizing standard picosecond techniques, we have examined the decay of the charge transfer complex in reaction centers in the presence of the stable semiquinone, formed by flash illumination with a dye laser 10 s before excitation by a picosecond pulse. In this state the decay rate for the charge transfer complex is considerably slower than when no electron is present in the quinone acceptor region. This indicates fairly strong coupling between constituents of the reaction center-quinone acceptor complex and may provide a probe into the relative positions of the various components.

     

Kinetics of ubisemiquinone redox changes in primary reactions of bacterial photosynthesis

Absorbance changes at 450 nm of the semiquinone form of the secondary electron acceptor were studied in chromatophores of Rhodospirillum rubrum. When chromatophores are illuminated by a series of single turnover flashes ubisemiquinone is formed and destroyed on alternate flashes at ambient redox potential from 100 to 250 mV. A simple kinetic model of the binary oscillations is suggested. On the base of the model it is shown that the rate constant of electron transfer from primary to secondary quinone after the first flash is larger that after the second flash. Cooperativity in electron transfer from primary to secondary quinone can be explained by electrostatic interactions of charged carriers.     

Superoxide flashes in single mitochondria

In quiescent cells, mitochondria are the primary source of reactive oxygen species (ROS), which are generated by leakiness of the electron transport chain (ETC). High levels of ROS can trigger cell death, whereas lower levels drive diverse and important cellular functions. We show here by employing a newly developed mitochondrial matrix-targeted superoxide indicator, that individual mitochondria undergo spontaneous bursts of superoxide generation, termed "superoxide flashes." Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and provide a vital source of superoxide production across many different cell types. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore stimulating superoxide production by the ETC. Furthermore, we observe a flurry of superoxide flash activity during reoxygenation of cardiomyocytes after hypoxia, which is inhibited by the cardioprotective compound adenosine. We propose that superoxide flashes could serve as a valuable biomarker for a wide variety of oxidative stress-related diseases.     

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.     

Kinetic model of the operation of a 2-electron switch in the photosynthetic reaction center of bacteria

A mathematical model, describing the binary oscillation of the concentration of semiquinone form of the secondary acceptor (ubiquinone) in photosynthetic reaction center of purple bacteria is proposed. This model takes into account both the changes of the ubiquinone state when the chromatophores are subjected to short flashes of light, and the successive dark relaxation of the semiquinone form. The model allows to calculate such characteristics as the dependence of the flash number, the stationary level of semiquinone form which is being established, when the flash number increases, the velocity which the concentration of semiquinone form is aspirating towards this stationary level and other characteristics. The model shows that the quantum yield of primary charge separation on the reaction center is higher after odd-number flashes then after even-number flashes.     

Heterogeneity of system II secondary electron acceptors in Tris-washed chloroplasts

Tris-washed chloroplasts were submitted to saturating short flashes, and then rapidly mixed with dichlorophenyldimethylurea (DCMU). The amount of singly reduced secondary acceptor was estimated from the DCMU-induced increase in fluorescence, caused by the reverse electron flow from secondary to primary acceptor. The back-transfer from the singly reduced secondary acceptor to the primary acceptor Q induced by DCMU addition affects only a part (60%) of the variable fluorescence (ΔF_max). As previously shown, the quenchers involved in this phenomenon, ‘B-type’ quenchers, are different from those controlling the complementary part of the fluorescence, the non-B-type. In this report, we show that at pH 8.5 in the B-type systems, there exist two kinds of secondary electron acceptors: B, a two-electron acceptor, the corresponding Q accounting for 40% of the variable fluorescence; B′, a one-electron acceptor, the corresponding Q accounting for 20% of the variable fluorescence. The lifetimes of B^? and B′^? in the absence of DCMU are 40 and 1 s, respectively. The primary acceptors of the B and B′ systems can be considered as corresponding to the Q₁s defined previously (Joliot, P. and Joliot, A. (1981) in Proceedings of the 5th International Congress on Photosynthesis (Akoynoglou, G., ed.), pp. 885–899, Balaban International Science Services, Philadelphia). The B′ centers seems to be equivalent to the Q_β centers as defined by other workers (Van Gorkom, H.J., Thielen, A.P.G.M. and Gorren, A.C.F. (1982) in The Function of Quinones in Energy Conserving Systems (Trumpower, B.L., ed.), Academic Press, New York, in the press).     

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.     

Thermoregulatory physiology of menopausal hot flashes: a review

Hot flashes during the climacteric years have long been a frequent clinical complaint, generally considered within the realm of the internist, gynecologist, or endocrinologist. Yet the underlying mechanism of hot flashes remains unknown. Only within the past 10 years has there been significant research on hot flashes as a disturbance of thermoregulation. This paper focuses on thermoregulatory aspects of hot flashes, reviewing current knowledge of the thermoregulatory physiology and endocrinology of hot flashes and discussing future avenues for research. Hot flashes are compared with fever in terms of thermoregulatory changes and speculated mechanisms. Although several substances in the peripheral circulation are found in increased concentrations during hot flashes, none is a trigger for a hot flash. The pattern of hot flash occurrence is striking in its regularity, and the possibility of endogenous rhythmicity is discussed. Recently, investigators have begun to explore a primate model of menopausal hot flashes. These studies are summarized. Finally, the multiple effects of estrogen on various systems of the body and their interrelationships are discussed. An understanding of the mechanism of hot flashes would not only be of importance to women suffering with hot flashes but would further our knowledge of thermoregulatory function and the interactions between thermoregulatory and reproductive systems.     

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)-1,1-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 QR²⁻.     

Effects of priming flash parameters and dark interval on red-induced afterpotential in Balanus photoreceptors

The sequence (a) priming flash, (b) dark interval, and (c) red light induces a long-lasting afterdepolarization (PDA) in Balanus photoreceptors. The inward flow of membrane current associated with the decay of PDA was independent of red test flashes, provided that PDA had plateaued at a particular intensity. The influence of wavelength and duration of the priming flash and their interaction with the dark interval were investigated. Increasing the duration of the priming flash produced a systematic increase in PDA duration. The dark interval plays a crucial role in PDA induction. The priming flash duration and the dark interval were reciprocally related, i.e, short flashes followed by long dark intervals induced as much PDA as long priming flashes followed by short dark intervals. The action spectrum for the priming flash was found to correspond to that of the primary photopigment (VP537).     

Response of the human circadian system to millisecond flashes of light

Ocular light sensitivity is the primary mechanism by which the central circadian clock, located in the suprachiasmatic nucleus (SCN), remains synchronized with the external geophysical day. This process is dependent on both the intensity and timing of the light exposure. Little is known about the impact of the duration of light exposure on the synchronization process in humans. In vitro and behavioral data, however, indicate the circadian clock in rodents can respond to sequences of millisecond light flashes. In a cross-over design, we tested the capacity of humans (n = 7) to respond to a sequence of 60 2-msec pulses of moderately bright light (473 lux) given over an hour during the night. Compared to a control dark exposure, after which there was a 3.5±7.3 min circadian phase delay, the millisecond light flashes delayed the circadian clock by 45±13 min (p<0.01). These light flashes also concomitantly increased subjective and objective alertness while suppressing delta and sigma activity (p<0.05) in the electroencephalogram (EEG). Our data indicate that phase shifting of the human circadian clock and immediate alerting effects can be observed in response to brief flashes of light. These data are consistent with the hypothesis that the circadian system can temporally integrate extraordinarily brief light exposures.     

Proton translocation in chloroplasts and its relationship to electron transport between the photosystems.

Using dark adapted isolated spinach chloroplasts and sequences of brief saturating flashes the correlation of the uptake and release of protons with electron transport from Photosystem II to Photosystem I were studied. The following observations and conclusions are reported: (1) Flash-induced proton uptake shows a weak, damped binary oscillation, with maxima occurring after the 2nd, 4th, etc. flashes. The damping factor is comparable to that observed in the O2 flash yield oscillation and therefore explained by misses in Photosystem II. (2) On the average and after a steady state is reached, each flash (i.e. each reduction of Q) induces the uptake of 2H+ from outside the chloroplasts. (3) Flash induced proton release inside the chloroplast membrane shows a strong damped binary oscillation with maximum release occurring also after the 2nd, 4th, etc. flashes. (4) This phenomenon is correlated with the earlier reported binary oscillations of electron transport [2] and shows that both electrons and protons are transported in pairs between the photosystems. (5) In two sequential flashes 4H+ from the outside of the thylakoid and 2e- from water are accumulated at a binding site B. Subsequently, the two electrons are transferred to non-protonated acceptors in Photosystem I (probably plastocyanin and cytochrome f) and the 4H+ are released inside the thylakoid. (6) It is concluded that a primary proton transporting site and/or energy conserving step located between the photosystems is being observed.     

A new mechanism-based inhibitor of photosynthetic water oxidation: acetone hydrazone. 2. Kinetic probes

The mechanism of photosynthetic water oxidation in spinach was investigated with a newly developed inhibitor of the water-oxidizing complex, acetone hydrazone (AceH), (CH3)2CNNH2 [Tso, J., Petrouleas, V., & Dismukes, G.C. (1990) Biochemistry (preceding paper in this issue)], by using fluorescence induction and single-turnover flashes to monitor O2 yield and thermoluminescence intensity. AceH binds slowly (1-3 min) in the dark to the S1 (resting) oxidation state of the water-oxidizing complex in thylakoids and PSII membranes. Once bound, it causes a two-flash delay in the pattern of O2 release seen in a train of flashes. This is initiated by reduction of manganese in the S2 oxidation state of the complex in a fast reaction (less than 0.5 s). In thylakoid membranes which have been partially inhibited at low AceH concentrations (less than 2 mM) the inhibition can be reversed by a single flash and a subsequent dark period. This behavior can be explained by two sequential one-electron oxidation steps: S1.AceHhv----S2.AceH in equilibrium S1.AceH+hv----S2.AceH+----S1 + AceH2+ Dissociation of the unobserved radical intermediate, AceH+, from S1 is proposed to account for the recovery from inhibition after one flash. In contrast, recovery from inhibition after a single flash is not observed in detergent-isolated PSII membranes or in intact thylakoid membranes at higher AceH concentrations (greater than 2 mM), where the two-flash delay in O2 release is seen. This suggests either a concerted two-electron process, S2----S0, or tight binding of AceH+ to S1.(ABSTRACT TRUNCATED AT 250 WORDS)     

STIMULATION OF CELLS BY INTENSE FLASHES OF ULTRAVIOLET LIGHT

A study has been made of the effect of sudden intense flashes of ultraviolet light, acting on a wide variety of cells and tissues, with special reference to stimulation. The flashes are obtained by a high voltage condenser discharge through a quartz mercury vapor sterilamp, using the method of Rentschler. The lethal effect of a single such discharge is widespread among unicellular organisms. Medullated nerves and whole muscles are not visibly stimulated, because of absorption by connective tissue. Single muscle fibers undergo immediate contracture in 50 per cent of the experiments. Nitella cells are stimulated, the effect depending on the dosage. Weak ultraviolet flashes slow or stop cyclosis reversibly. Strong flashes stop cyclosis reversibly with the appearance of a local or a propagated action potential. Very strong flashes kill the Nitella cells. The effect of single flashes on myonemes, oscillatory movement, ameboid movement, cilia, flagella, and bioluminescence is described in the text.     

Effects of Flashes of Red or Blue Light on the Composition of Starved Chlorella pyrenoidosa

Autotrophically grown cells of Chlorella pyrenoidosa (211-8b) were starved 3 to 4 days in darkness, flashes of blue light, or flashes of red light. The blue flashes were sufficient to maintain the maximal rate of light-stimulated oxygen uptake during short term experiments. However, after 24 hours, the respiration rate in red flashes was equal to, or greater than, the rate in blue flashes. Starvation in darkness reduced the chlorophyll content by 11%, altered the blue absorbance of the nonsaponifiable material only 1 to 2%, and reduced the dry weight by 13%. Starvation in the presence of blue or red flashes reduced the dry weight by an additional 11 or 12% respectively. Protein per unit cell volume was not changed significantly during 3 to 4 days starvation in darkness or in blue flashes, even though dry weight per unit cell volume decreased 13% in darkness and 23% in blue flashes. In contrast, cells starved under red flashes showed a 20% decrease in protein per unit cell volume and a 24% decrease in dry weight per unit cell volume.     

Light flashes of different durations (0.063-3.33 msec) phase shift the circadian flight activity of a bat

The phase-response curve (PRC) for the circadian rhythm in the flight activity of a cave-dwelling bat, Hipposideros speoris, constructed with 0.063-msec light flashes, reported here is the first of its kind for any circadian system and is unlike any other phase-response curves constructed for other nocturnal animals. The phase responding with maximal advances (90 degrees) and the phase responding with maximal delays (0 degree) of this PRC were exposed to light flashes of systematically varying durations from 0.083 to 3.33-msec. For 0 degree phase, the flashes of 0.063-3.33 msec effected delay phase shifts of comparable magnitude. For 90 degrees phase, the flashes of 0.063-1.0 msec effected advance phase shifts, whereas 3.33-msec flashes effected unmistakable delay phase shifts with advancing transients. Phase shifts evoked with such light flashes are further compared with phase shifts evoked with pulses of longer durations (15 min to 2.8 hr) for H. speoris.