Subsequent Flashes Research Articles

A SHORT‐LIVED INTERMEDIATE IN THE PHOTO‐ENZYMATIC REDUCTION OF PROTOCHLOROPHYLL(IDE) INTO CHLOROPHYLL(IDE) AT A PHYSIOLOGICAL TEMPERATURE

Abstract—The reduction of protochlorophyll(ide) into chlorophyll(ide) has been studied by flash absorption spectroscopy at 21°C, with a time resolution of 0.5 µs. The absorption changes have been recorded in the range 670–720 nm after the first and subsequent flashes given to an extract of etiolated bean leaves.At 695 nm the flash‐induced absorption increase has its maximum value immediately after the flash and then partly decays with a half‐time of about 7–10 µs. A complementary behaviour is observed at 675 nm where the absorption change is very small at time zero and then increases to a stationary value with a half‐time of about 6–9 µs. From measurements at several wavelengths it is concluded that a species with an absorption peak around 695 nm is formed immediately after the flash and is then transformed into a stable species with an absorption peak around 675 nm. Measurements at lower temperatures, down to—50°C, show that the transformation is much slowed down at decreased temperatures. The species absorbing at 695 nm (P695) is attributed to an intermediate in the photoreduction of protochlorophyll(ide) P639,650 into chlorophyll(ide) P676.When the protochlorophyll(ide) is photoreduced before the flash illumination, the newly formed chlorophyll(ide) gets to a triplet state, which decays with a half‐time of 15 µs at 21°C. This result shows that carotenoid molecules do not exert their protective role at this stage of chlorophyll (Chi) formation.

Flash Inactivation of Oxygen Evolution

Brief saturating light flashes were used to probe the mechanism of inactivation of O(2) evolution by Tris in chloroplasts. Maximum inactivation with a single flash and an oscillation with period of four on subsequent flashes was observed. Analyses of the oscillations suggested that only the charge-collecting O(2)-evolving catalyst of photosystem II (S(2)-state) was a target of inactivation by Tris. This conclusion was supported by the following observations: (a) hydroxylamine preequilibration caused a three-flash delay in the inactivation pattern; (b) the lifetimes of the Tris-inactivable and S(2)-states were similar; and (c) reagents accelerating S(2) deactivation decreased the lifetime of the inactivable state. Inactivation proved to be moderated by F, the precursor of Signal II(s), as shown by a one flash delay with chloroplasts having high abundance of F. Evidence was obtained for cooperativity effects in inactivation and NH(3) was shown to be a competitive inhibitor of the Tris-induced inactivation. S(2)-dependent inactivation was inhibited by glutaraldehyde fixation of chloroplasts, possibly suggesting that inactivation proceeds via conformational changes of the S(2)-state.

Airborne and ground‐based studies of thunderstorms in the vicinity of Langmuir Laboratory

AbstractFlights through the central regions of thunderstorms were made over New Mexico on 6 and 15 August 1977 with the ONR/NMIMT Schweitzer aeroplane which carried equipment designed to measure all three components of the electric field, and the charge, Q, and diameter, d, of individual precipitation elements. On the earlier day, information was also obtained with: a rain‐gauge network surrounding Langmuir Laboratory; a 3 cm radar; an acoustic system for locating lightning channels; a ground‐based field‐change meter.The first cell on 6 August produced precipitation at the ground but no lightning. Vertical fields, Ex, of up to about 50kVm−1 and precipitation charge densities ρ of up to −0.5 C km−3 were recorded within the cloud. The second cell, which grew as the first one decayed, produced 7 lightning strokes in 9 minutes during which time the radar revealed vigorous vertical growth in a narrow zone containing precipitation.Thunder reconstructions showed the acoustic sources for the first flash of this cell to be very near the top of the cloud at an altitude of 10 km a.s.1. The subsequent flashes produced acoustic signals from progressively lower in the cloud. When the radar echo reached its maximum height lightning activity ceased. Ex values of up to about 50kVm−1 and pp values of down to −1 Ckm−3 were measured. ρp was consistently negative, individual charges being less than ±40 pC. Q values were within the inductive limit for a thundercloud at breakdown but no systematic relation between Q and d was found.Six penetrations were made through the thundercloud of 15 August, which produced only two lightning strokes. The Ex records were indicative of a (±) dipole located near the cloud top, at around –13°C. Fields of up to about 100kVm−1 and ρp values (positive and negative) of around 5Ckm−3 were measured. Q values of up to ±250 pC were recorded, with charges around ±50 pC being commonly found. No systematic Q‐d relation was revealed, and smaller precipitation particles frequently carried charges (positive or negative) in excess of the inductive limit.On both days estimated precipitation rates were of order 10mmh−1 and on most occasions the pilot reported precipitation particles to be either ‘ice’ or ‘mixed liquid water and ice’.

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 factors limiting the synthesis of ATP by chromatophores exposed to short flash excitation

ATP synthesis was measured after chromatophores from Rhodopseudomonas capsulata had been subjected to illumination by single turnover flashes fired at variable frequencies. Three processes were examined, which under different conditions can limit the net yield of ATP. (1) A process with an apparent relaxation time of 10–20 ms. This reaction probably limits the rate of ATP synthesis in continuous illumination. It has a similar time dependence to the stimulation of the carotenoid shift decay by ADP after a single flash. (2) An active state of the ATPase only persists when the chromatophores are excited more often than once in 10 s. This state decays with similar kinetics to the entire carotenoid shift decay. Full activation is achieved after two flashes. (1) and (2) are not significantly affected by concentrations of antimycin A sufficient to block electron flow through the cytochrome b c 2 oxidoreductase and abolish phase III in the generation of the carotenoid shift. (3) In the presence of antimycin A, after the third, fourth and subsequent flashes ATP synthesis is limited by the quantity of reducing equivalents transported through the reaction centre rather than by the level of the electrochemical proton gradient.

Effects of pH on reactions on the donor side of Photosystem II

The effects of pH on the increase of fluorescene yield measured in the microsecond range, and on the microsecond delayed fluorescence have been studied in dark adapted chloroplasts as a function of flash number. 1. (1) At pH 7, the amplitude of the fast-phase of the microsecond fluorescence yield rise oscillated as a function of flash number with period 4 and with maxima on flashes 1 and 5, and minima on flashes 3 and 7. The damped oscillations were apparent over the range between 6 and 8, although the absolute amplitude of the fast phase was diminished at the lower end of the range. At pH 4, there was no fast phase in the rise and, at pH 9, an enhanced fast-phase occurred only for the first flash. 2. (2) The decay of microsecond delayed fluorescence was described by the sum of exponentials with half-times of 10–15 μs and 40–50 μs. Over the pH range 6– <8, the extrapolated initial amplitude and the proportion of the change due to the faster component showed oscillations which were opposite in phase to those observed for the prompt fluorescence yield rise; the slower component showed weaker oscillations of the same phase. At pH 4, there were no oscillations and the slow phase predominated. At pH 9, the delayed fluorescence intensity was diminished on the first flash, and high on subsequent flashes. 3. (3) The results are interpreted in terms of a model in which protons are released during all transitions of the S-states with the exception of S 1 → S 2, and in which there are two sites of inhibition on the donor side of the photosystem at extreme pH values. At pH 4, electron donation to P + occurs with a half-time approx. 135 μs, either by a back reaction from Q −, or from D; electron transport is interrupted between Z 1 and P. At pH 9, electron transport is inhibited between Z 1 and Z 2; rapid re-reduction of P + by Z 1 occurs after 1 flash, and on subsequent flashes electrons from D, an alternative donor reduce P +. The location of the positive charge on states S 2 and S 3 is discussed.

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited. In chloroplasts pre-treated with Tris, the primary donor of Photosystem II ( P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680 + decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl 2 or reduced phenylenediamine) have no direct influence on the kinetics of P-680 +. In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine. These results are interpreted in terms of multiple pathways for the reduction of P-680 +: a rapid reduction (<1 μs) by the physiological donor D 1; a slower reduction (6 and 22 μs) by donor D′ 1, operative when O 2 evolution is inhibited; a back-reaction (130 μs) when D′ 1 is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680 + has the capacity to deliver only one electron. The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors ( P-680, D 1, D′ 1) are located at the internal side of the thylakoid membrane.

Absorption changes of P-700 reversible in milliseconds at low temperature in Triton-solubilized Photosystem I particles

Triton-solubilized Photosystem I particles from spinach chloroplasts exhibit largely reversible P-700 absorption changes over the temperature range from 4.2 K to room temperature. For anaerobic samples treated with dithionite and neutral red at pH 10 and illuminated during cooling, a brief (1 μs) saturating flash produces absorption changes in the long wavelength region that decay in 0.95 ± 0.2 ms from 4.2 to 50 K. Above 80 K a faster (100 ± 30 μs) component dominates in the decay process, but this disappears again above about 180 K. The major decay at temperatures above 200 K occurs in about 1 ms. The difference spectrum of these absorption changes between 500 and 900 nm closely resembles that of P-700. Using ascorbate and 2,6-dichlorophenolindophenol as the reducing system with a sample of Photosystem I particles cooled in darkness to 4.2 K, a fully reversible signal is seen upon both the first and subsequent flashes. The decay time in this case is 0.9 ± 0.3 ms.

Rapid photopigment conversions in blowfly visual sense cells consequences for receptor potential and pupillary response

Combined optical and electrophysiological experiments on the kinetics of visual pigment conversions in blowfly and the resulting pupillary response and late receptor potential are described. The photometrically detectable conversions of rhodopsin and metarhodopsin in the living wild type fly are completed within 0.5 ms. Prolonged pupillary responses and receptor potentials occur upon intense blue flashes. Subsequent intense red flashes abolish the prolonged responses in the case of both membrane potential and the pupil. The interrelation of potential and pupil is discussed.

Electron acceptors associated with P-700 in Triton solubilized Photosystem I particles from spinach chloroplasts

Flash-induced absorption changes of Triton-solubilized Photosystem I particles from spinach were studied under reducing and/or illumination conditions that serve to alter the state of bound electron acceptors. By monitoring the decay of P-700 following each of a train of flashes, we found that P-430 or components resembling it can hold 2 equivalents of electrons transferred upon successive illuminations. This requires the presence of a good electron donor, reduced phenazine methosulfate or neutral red, otherwise the back reaction of P-700 + with P-430 occurs in about 30 ms. If the two P-430 sites, designated Centers A and B, are first reduced by preilluminating flashes or chemically by dithionite under anaerobic conditions, then subsequent laser flashes generate a 250 μs back reaction of P-700 +, which we associate with a more primary electron acceptor A 2. In turn, when A 2 is reduced by background (continuous) illumination in presence of neutral red and under strongly reducing conditions, laser flashes then produce a much faster (3 μs) back reaction at wavelengths characteristic of P-700. We associate this with another more primary electron acceptor, A 1, which functions very close to P-700. The organization of these components probably corresponds to the sequence P-700- A 1- A 2-P-430[ A B ] . The relation of the optical components to acceptor species detected by EPR, by electron-spin polarization or in terms of peptide components of Photosystem I is discussed. Preliminary experiments with broken chloroplasts suggest that an analogous situation occurs there, as well.

Protolytic Reactions in Photosystem II: a New Model for the Release of Protons Accompanying the Photooxidation of Water

Using pH indicator dye techniques we have investigated the pH changes in dark-adapted chloro- plasts following excitation by short flashes. Two types of pH indicator, cresol red and neutral red, were used, to follow the pH changes either inside or outside the thylakoids, or the net change when the membrane was made permeable to protons by uncoupling agents. (1)With cresol red which showed the net pH changes inside and outside the thylakoids, an oscillation of the flash yield of H+ occurred with a periodicity of 4 (minima on the first and fifth flashes, the yield on the third being not significantly different from the yields on the second and fourth flashes). The pH changes did not occur in synchrony with O2-evolution. (2)The net flash yields without addition of electron acceptor were similar to those with benzyl- viologen. The results were comparable with those obtained with the glass electrode technique by Fowler and Kok (C. F. Fowler and B. Kok, Biochim. Biophys. Acta 357, 299 - 307 [1974]). (3)The net flash yields with ferricyanide as electron acceptor of photosystem I were higher than those in the absence of acceptor, or with benzylviologen. On the first and fifth flashes a net acidification was always observed. (4)In the presence of 3- (3,4-dichlorphenyl) -1,1-dimethylurea (DCMU) a rapid acidification also occured on the first flash, while the pH changes induced by subsequent flashes were inhibited. (5)The uncoupler methylamine did not inhibit the proton uptake outside the thylakoids. (6)With neutral red as indicator for the net pH change inside and outside the thylakoids, the same oscillation of the flash yield occured as with cresol red. (7)With neutral red in the precense of an external buffer, as a pH indicator for the internal aqueous phase alone, an oscillation of the flash yield with a periodicity of 4 also occured. The first and second flash yields were higher compared with the third than the equivalent yields of oxygen. (8)We discuss the results with respect to a model for the release of protons in the water- splitting enzyme reactions, in which protons are not released in synchrony with O2 , but in the transitions of all the states of the watersplitting enzyme with the exception of S1 → S2 . Our results are consistent with this model when account is taken of the release of protons inside the thylakoids with a periodicity of 2, associated with electron transfer from reduced plastoquinone.

Short-term reactions of phytochrome in Mougeotia?

Phytochrome controlled chloroplast movement in Mougeotia is induced by flashes of polarized red light. Two subsequent flashes, separated by a dark interval of a few seconds, are much more effective than two simultaneous flashes; a maximal cumulative effect is reached if the duration of the dark interval is 30 ms or longer. We propose two light reactions in series, separated by a very fast dark reaction. Preliminary evidence is given that the energy requirement for these light reactions is different. It is suggested that the two reactions are related in some way to free and bound phytochrome.

Effects of intracellular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors.

The calcium sequestering agent, EGTA, was injected into Limulus ventral photoreceptors. Before injection, the inward membrane current induced by a long stimulus had a large initial transient which declined to a smaller plateau. Iontophoretic injection of EGTA tended to prevent the decline from transient to plateau. Before injection the plateau response was a nonlinear function of light intensity. After EGTA injection the response-intensity curves tended to become linear. Before injection, bright lights lowered the sensitivity as determined with subsequent test flashes. EGTA injection decreased the light-induced changes in sensitivity. Ca-EGTA buffers having different levels of free calcium were pressure-injected into ventral photoreceptors; the higher the level of free calcium, the lower the sensitivity measured after injection. The effects of inotophoretic injection of EGTA were not mimicked by injection or similar amounts of sulfate and the effects of pressure injection of EGTA buffer solutions were not mimicked by injection of similar volumes of pH buffer or mannitol. The data are consistent with the hypothesis that light adaptation is mediated by a rise of the intracellular free calcium concentration.

Responses of retinal rods of the frog to light

To study the effect of the intensity, duration, spectral composition, and diameter of the light spot on the amplitude and shape of the response of single rods of the frog retina, potentials were recorded intracellularly. The rods tested could be divided into two groups on the basis of their responses to light spots of different spectral composition: those with maxima of sensitivity at 507 ± 8 nm and 442 ± 8 nm. With an increase in the intensity of light the response amplitude rose gradually and the time for the response to rise to its maximum was shortened. A bright flash temporarily inhibited the sensitivity of the cell to subsequent test flashes. If light spots of larger diameter (1000–1500 µ) were presented a delayed depolarization wave, due to illumination of the distant surroundings of the receptor, was observed in the course of recovery of the photic response; this effect was maximal for stimulation with red light and it was evidently induced by horizontal cell activity. The possible functional role of the depolarizing effect of illumination of the distant surroundings of the receptor is discussed.

Photosynthetic reaction center transients, P 435 and P 424, in Chromatium D

1. The correlation of kinetic, saturation and potentiometric evidence shows that P 435 is a spectral component of the same reaction center as P883 in Chromatium D. 2. There is only one cytochrome oxidizing reaction center P883/P 435, since the spectrum of P 435 is the same independent of which cytochrome (C 553 or C 555) is oxidized and the P 435 reduction kinetics correlate with the oxidation kinetics of either cytochrome, depending upon which is being oxidized. 3. The oxidation half-time of P883/P 435 is less than 50 nsec. 4. The E m of P883/P 435 is +486 mV and that of its primary electron acceptor is −134 mV. 5. P 424 is observable only when the primary electron acceptor, X, is in the reduced state prior to the flash: (A) at room temperature in chromatophores between −318 and −145 mV and (B) at 77°K over the same potential range (first laser flash) or at higher potentials (subsequent laser flashes) under conditions in which the first laser flash permanently reduces X. 6. P424, P883/P 435, cytochrome C 553 and cytochrome C 555 are all associated with Thornber's subchromatophore Fraction A, but not Fraction B. 7. The rise half-time of P 424 in chromatophores is 50 nsec at room temperature and 200 nsec at 77°K. Its recovery half-time is 2.2 to 2.4 μsec at both temperatures in chromatophores or 16 msec in whole cells at 77°K. 8. P 424 might represent either an oxidation of bacteriochlorophyll spectrally modified by the presence of reduced primary electron acceptor or a light-induced reduction state of bacteriochlorophyll. 9. Thermodynamically, it is possible that this new species might be a link in the direct reduction of NAD +.

Activation kinetics of photosynthetic oxygen evolution under 20-40 nanosecond laser flashes.

Abstract— Chlorella samples, incubated for varying periods in darkness, were exposed to a series of 20–40 nsec flashes, spaced 15 sec apart, from a Q‐switched ruby laser. A stationary Teflon‐covered platinum electrode measured the microjet of oxygen produced by each flash. After a dark preincubation exceeding 3 min, at 23°C, little or no oxygen is evolved until the third flash in a sequence. The yields from subsequent flashes increase monotonically until a constant value is reached. If low levels of background light are supplied, or if the interval between series of flashes is decreased, oxygen is evolved on the second or even the first flash. Very similar results were obtained from analogous experiments with tailless 28 μsec flashes from a xenon flashtube. In particular, very little oxygen was evolved from the second flash following a long dark period whatever the spacing between flashes. This means that there are no systematic differences between the effects of saturating 20 nsec and 28 μsec light flashes on the activation processes during the first few flashes following a long dark period. No oscillations of flash yield with successive flashes were observed because of the long interval between flashes. These results are consistent with the idea that the reaction center of Photosystem II must undergo a dark process lasting considerably longer than 28 μsec before it can absorb a second photon.