Identification of the 120 mus phase in the decay of delayed fluorescence in spinach chloroplasts and subchloroplast particles as the intrinsic back reaction. The dependence of the level of this phase on the thylakoids internal pH.

After a 500 mus laser flash a 120 mus phase in the decay of delayed fluorescence is visible under a variety of circumstances in spinach chloroplasts and subchloroplast particles enriched in Photosystem II prepared by means of digitonin. The level of this phase is high in the case of inhibition of oxygen evolution at the donor side of Photosystem II. Comparison with the results of Babcock and Sauer (1975) Biochim. Bio-phys. Acta 376, 329-344, indicates that their EPR signal IIf which they suppose to be due to Z+, the oxidized first secondary donor of Photosystem II, is well correlated with a large amplitude of our 120 mus phase. We explain our 120 mus phase by the intrinsic back reaction of the excited reaction center in the presence of Z+, as predicted by Van Gorkom and Donze (1973) Photochem. Photobiol. 17, 333-342. The redox state of Z+ is dependent on the internal pH of the thylakoids. The results on the effect of pH in the mus region are compared with those obtained in the ms region.     

Correlation between flash-induced oxygen evolution and fluorescence yield kinetics in the 0 to 16 mus range in Chlorella pyyrenoidosa during incubation with hydroxylamine.

Following flash excitation, oxygen pulses and fluorescence kinetics in the time range 0-16 mus were studied in the alga Chlorella pyrenoidosa during incubation with various concentrations of hydroxylamine. The obtained results could be explained considering four effects of hydroxylamine. 1. Hydroxylamine removes (reduces) oxidizing equivalents, generated in the water-splitting system by flash excitation. This process does not markedly affect the fluorescence yield kinetics between 0 and 16 mus following the ignition of a flash and reaches a constant rate within a few minutes, but possibly within a few seconds, after addition of hydroxylamine. In a sequence of flashes separated by dark time td, the steady-state oxygen yield in the flashes is exp(-ktd), the yield at td=0 being taken equal to 1, where k=(0.1 + beta[NH2OH])s-1, with [NH2OH] in mM and beta=0.6 mM-1, provided [NH2OH]greater than or equal to 0.5 mM. 2. An inhibition between Z, the physiological donor and the oxidized reaction center pigment P+ occurs, proceeding as exp (-kiti)where ti is the incubation time with hydroxylamine and ki=(alpha[NH2OH]) min-1, with [NH2OH] in mM and alpha=0.14 mM-1. This process not only inhibits oxygen evolution capability, but also decreases the amplitude of the fluorescence yield difference deltaphi=phi(16 mus)-phi(2 mus) induced by a flesh in the steady state. In a fraction of the reaction centers this inhibition occurs "immediately" after the addition of hydroxylamine. These observations, combined with the conslusion of Cheniae and Martin (1971, Plant Physiol. 47, 568-575) that the inhibition of the Hill reaction is related to the extraction of bound manganese indicate that the reaction between Z and P+ requires bound manganese. 3. In the inhibited centers a second donor for P+, D, connected to an entry site for the artificial electron donor hydroxylamine becomes apparent. 4. A flash-induced oxygen uptake signal was observed in the presence of hydroxylamine, which was shown to be caused by a system II reaction. The effects under (1) and (4) were reversed in the dark if hydroxylamine was removed by washing. The effects under (2) and (3) were reversed during illumination of a washed sample.     

Delayed fluorescence from Rhodopseudomonas sphaeroides following single flashes.

Delayed fluorescence from Rhodopseudomonas sphaeroides chromatophores was studied with the use of short flashes for excitation. Although the delayed fluorescence probably arises from a back-reaction between the oxidized reaction center bacteriochlorophyll complex (P+) and the reduced electron acceptor (X-), the decay of delayed fluorescence after a flash is much faster (tau1/2 approximately 120 mus) than the decay of P+X-. The rapid decay of delayed fluorescence is not due to the uptake of a proton from the solution, nor to a change in membrane potential. It correlates with small optical absorbance changes at 450 and 770 nm which could reflect a change in the state of X-. The intensity of the delayed fluorescence is 11-18-fold greater if the excitation flashes are spaced 2 s apart than it is if they are 30 s apart. The enhancement of delayed fluorescence at high flash repetition rates occurs only at redox potentials which are low enough (less than +240 mV) so that electron donors are available to reduce P+X- to PX- in part of the reaction center population. The enhancement decays between flashes as PX- is reoxidized to PX, as measured by the recovery of photochemical activity. Evidently, the reduction of P+X- to PX- leads to the storage of free energy that can be used on a subsequent flash to promote delayed fluorescence. The reduction of P+X- also is associated with a carotenoid spectral shift which decays as PX- is reoxidized to PX. Although this suggests that the free energy which supports the delayed fluorescence might be stored as a membrane potential, the ionophore gramicidin D only partially inhibits the enhancement of delayed fluorescence. With widely separated flashes, gramicidin has no effect on delayed fluorescence. At redox potentials low enough to keep X fully reduced, delayed fluorescence of the type described above does not occur, but one can detect weak luminescence which probably is due to phosphorescence of a protoporphyrin.     

Time-resolved monitoring of flash-induced changes of fluorescence quantum yield and decay of delayed light emission in oxygen-evolving photosynthetic organisms

The present contribution describes a new experimental setup that permits time-resolved monitoring of the rise kinetics of the relative fluorescence yield, Phi(rel)(t), and simultaneously of the decay of delayed light emission, L(t), induced by strong actinic laser flashes. The results obtained by excitation of dark-adapted samples with a train of eight flashes reveal (a) in suspensions of spinach thylakoids, Phi(rel)(t) exhibits a typical period four oscillation that is characteristic for a dependence on the redox states S(i)() of the water oxidizing complex (WOC), (b) the relative extent of the unresolved "instantaneous" rise to the level (100 ns) at 100 ns and the maximum values of Phi(rel)(t) attained at about 45 s after each actinic flash, (45 s) synchronously oscillate and exhibit the largest values at flash nos. 1 and 5 and minima after flash nos. 2 and 3, (c) opposite effects are observed for the normalized extent of the rise kinetics in the 100 ns to 5 s time domain of relative fluorescence yield, Phi(rel)(5 s) - Phi(rel)(100 ns), i.e., both parameters attain minimum and maximum values after the first/fifth and second/third flash, respectively, and (d) analogous features for the "fast" and "slow" ns-kinetics of the fluorescence rise were observed in suspensions of Chlamydomas reinhardtii cells. A slight phase shift by one flash is ascribed to physiological differences. The applicability of this noninvasive technique to study reactions of photosystem II, especially the reduction kinetics of P680(*)(+) and their dependence on the redox state S(i)() of the WOC, is discussed.     

The rise in chlorophyll a fluorescence yield and decay in delayed light emission in Tris-washed chloroplasts in the 6–100 μs time range after an excitation flash

Parallel measurements of the rise in chlorophyll a fluorescence yield and delayed light emission decay, after a 10 ns saturating excitation flash, have been made in tris(hydroxymethyl)aminomethane-washed chloroplasts. Various electron donor systems (Mn²⁺; ascorbate; reduced phenylenediamine and benzidine) were used in conjuction with different preillumination regimes to alter [P⁺-680], the oxidized form of the Photosystem II reaction center chlorophyll a. Conditions giving rise to high [P⁺-680] resulted in only a small rise in fluorescence yield, an inhibition of a 6 μs delayed light component, and an enhancement of a 60 μs component of delayed light emission. These results confirm the hypothesis that P⁺-680 acts as a quencher of fluorescence and that delayed light emission in the microsecond time range is due to the back reaction of P⁺-680 and Q^?. (Q is the first “stable” electron acceptor of Photosystem II.) Two preillumination flashes are required before the full effect of Tris washing is observed in the delayed light emission decay and fluorescence yield rise; this suggests that a capacity to hold two charges exists between the Tris block and P⁺-680. Tris washing has no direct effect on the movement of electrons from Z (the first electron donor to P⁺-680) to P⁺-680. Finally, Mn²⁺ donates electrons to P⁺-680 via Z.     

The mechanism of reduction of the ubiquinone pool in photosynthetic bacteria at different redox potentials.

(1) A flash number dependency of flash-induced absorbance changes was observed with whole cells of Rhodospirillum rubrum and chromatophores of R. rubrum and Rhodopseudomonas sphaeroides wild type and the G1C mutant. The oscillatory behavior was dependent on the redox potential; it was observed under oxidizing conditions only. Absorbance difference spectra measured after each flash in the 275--500 nm wavelength region showed that a molecule of ubiquinone, R, is reduced to the semiquinone (R-) after odd-numbered flashes and reoxidized after even-numbered flashes. The amount of R reduced was approximately one molecule per reaction center. (2) The flash number dependency of the electrochromic shift of the carotenoid spectrum was studied with chromatophores of Rps. sphaeroides wild type and the G1C mutant. At higher values of the ambient redox potential a relatively slow phase with a rise time of 30 ms was observed after even-numbered flashes, in addition to the fast phase (completed within 0.2 ms) occurring after each flash. Evidence was obtained that the slow phase represents the formation of an additional membrane potential during a dark reaction that occurs after flashes with an even number. This reaction is inhibited by antimycin A, whereas the oscillations of the R/R- absorbance changes remain unaffected. At low potentials (E = 100 mV) no oscillations of the carotenoid shift were observed: a fast phase was followed by a slow phase (antimycin-sensitive) with a half-time of 3 ms after each flash. (3) The results are discussed in terms of a model for the cyclic electron flow as described by Prince and Dutton (Prince, R.C. and Dutton, P.L. (1976) Bacterial Photosynthesis Conference, Brussels, Belgium, September 6--9, Abstr. TB4) employing the so-called Q-cycle.     

P680(+)* reduction kinetics and redox transition probability of the water oxidizing complex as a function of pH and H/D isotope exchange in spinach thylakoids.

The rise of fluorescence as an indicator for P680(+)* reduction by YZ and the period-four oscillation of oxygen yield induced by a train of saturating flashes were measured in dark-adapted thylakoids as a function of pH in the absence of exogenous electron acceptors. The results reveal that: (i) the average amplitude of the nanosecond kinetics and the average of the maximum fluorescence attained at 100 micros after the flash in the acidic range decrease with decreasing pH; (ii) the oxygen yield exhibits a pronounced period-four oscillation at pH 6.5 and higher damping at both pH 5.0 and pH 8.0; (iii) the probability of misses in the Si-state transitions of the water oxidizing complex is affected characteristically when exchangeable protons are replaced by deuterons [at pH <6.5, the ratio alpha(D)/alpha(H) is larger than 1 whereas at pH >7.0 values of <1 are observed]. The results are discussed within the framework of a combined mechanism for P680(+)* reduction where the nanosecond kinetics reflect an electron transfer coupled with a "rocket-type" proton shift within a hydrogen bridge from YZ to a nearby basic group, X [Eckert, H.-J., and Renger, G. (1988) FEBS Lett. 236, 425-431], and subsequent relaxations within a network of hydrogen bonds. It is concluded that in the acidic region the hydrogen bond between YZ and X (most likely His 190 of polypeptide D1) is interrupted either by direct protonation of X or by conformational changes due to acid-induced Ca2+ release. This gives rise to a decreased P680(+)* reduction by nanosecond kinetics and an increase of dissipative P680(+)* recombination at low pH. A different mechanism is responsible for the almost invariant amplitude of nanosecond kinetics and increase of alpha in the alkaline region.     

Precursors to microsecond delayed luminescence in oxygenevolving and inhibited chloroplasts

We have investigated submillisecond delayed luminescence in spinach chloroplasts under a variety of conditions. In Tris-washed chloroplasts, which are inhibited on the oxidizing side of P-680, the delayed light emission in the 7–200 μs time-range decayed with biphasic behavior. In fully dark-adapted samples illuminated by a single saturating laser pulse, the fast phase of delayed luminescence followed a nearly identical pH-dependent time-course as that observed optically and by ESR for P⁺-680 reduction, thus verifying the recombination hypothesis for the origin of delayed light. The observed slower phase of delayed luminescence was also pH dependent, but unlike the fast phase, could not be ascribed to specific electron transfer events of PS II. This phase could be rationalized by a heterogeneity in the population of P-680. While kinetic parameters were found to be insensitive to changes in ionic strength, the overall luminescence intensity was quite sensitive to the electrical parameters, thus indicating the role of ionic strength and local charges in delayed luminescence modulation. A similar series of experiments was performed on untreated chloroplasts. The pH-dependent delayed luminescence behavior in both untreated chloroplasts and Tris-washed chloroplasts was similar despite significantly faster kinetics associated with the reduction of P⁺-680 by the secondary PS II electron donor, Z, in the former preparation (e.g., Van Best, J.A. and Mathis, P. (1978) Biochim. Biophys. Acta 503, 178–188). Thus, it was concluded that, in untreated samples, microsecond delayed luminescence emanates primarily from centers which are not competent in oxygen evolution. The nearly identical delayed luminescence intensity in untreated chloroplasts and in Tris-washed chloroplasts was rationalized by a model which predicts modulations in delayed luminescence yield by the exciton-quenching effect of P⁺-680. Computer simulations demonstrate the feasibility of this model. The previously documented flash oscillations in microsecond delayed luminescence intensity in untreated chloroplasts (Bowes, J.M. and Crofts, A.R. (1979) Biochim. Biophys. Acta 547, 336–346), which we readily observed, were attributed to alterations in delayed luminescence yield (in nonfunctional centers) by variations in charge density stored at the oxygen-evolving complex of functional centers. Taken together, our results emphasize the dependence of delayed luminescence kinetics upon electron-transfer kinetics and the dependence of delayed luminescence amplitude upon the photochemical parameters, the exciton yield and the emission yield.     

Reactions between primary and secondary acceptors of photosystem II in Chlorella pyrenoidosa under anaerobic conditions as studied by chlorophyll fluorescence.

The kinetics of the fluorescence yield phi of chlorophyll a in Chlorella pyrenoidosa were studied under anaerobic conditions in the time range from 50 mus to several minutes after short (t 1/2 = 30 ns or 5 mus) saturating flashes. The fluorescence yield "in the dark" increased from phi = 1 at the beginning to phi approximately 5 in about 3 h when single flashes separated by dark intervals of about 3 min were given. After one saturating flash, phi increased to a maximum value (4-5) at 50 mus, then phi decreased to about 3 with a half time of about 10 ms and to the initial value with a half time of about 2 s. When two flashes separated by 0.2 s were given, the first phase of the decrease after the second flash occurred within 2 ms. After one flash given at high initial fluorescence yield, the 10-ms decay was followed by a 10 s increase to the initial value. After the two flashes 0.2 s apart, the rapid decay was not followed by a slow increase. These and other experiments provided additional evidence for and extend an earlier hypothesis concerning the acceptor complex of Photosystem II (Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250-256; Velthuys, B. R. and Amesz. J. (1974) Biochim. Biophys. Acta 333, 85-94): reaction center 2 contains an acceptor complex QR consisting of an electron-transferring primary acceptor molecule Q, and a secondary electron acceptor R, which can accept two electrons in succession, but transfers two electrons simultaneously to a molecule of the tertiary acceptor pool, containing plastoquinone (A). Furthermore, the kinetics indicate that 2 reactions centers of System I, excited by a short flash, cooperate directly or indirectly in oxidizing a plastohydroquinone molecule (A2-). If initially all components between photoreaction 1 and 2 are in the reduced state the following sequence of reactions occurs after a flash has oxidised A2- via System I: Q-R2- + A leads to Q-R + A2- leads to QR- + A2-. During anaerobiosis two slow reactions manifest themselves: the reduction of R (and A) within 1 s, presumably by an endogenous electron donor D1, and the reduction of Q in about 10 s when R is in the state R- and A in the state A2-. An endogenous electron donor, D2, and Q- complete in reducing the photooxidized donor complex of System II in reactions with half times of the order of 1 s.     

Quenching of fluorescence by triplet excited states in chloroplasts

The fluorescence quantum yield in spinach chloroplasts at room temperature has been studied utilizing a 0.5-4.0 mus duration dye laser flash of varying intensities as an excitation source. The yield (phi) and carotenoid triplet concentration were monitored both during and following the laser flash. The triplet concentration was monitored by transient absorption spectoscopy at 515 nm, while the yield phi following the laser was probed with a low intensity xenon flash. The fluorescence is quenched by factors of up to 10-12, depending on the intensity of the flash and the time interval following the onset of the flash. This quenching is attributed to a quencher Q whose concentration is denoted by Q. The relative instantaneous concentration of Q was calculated from phi utilizing the Stern-Volmer equation, and its buildup and decay kinetics were compared to those of carotenoid triplets. At high flash intensities (greater than 10(16) photon . cm-2) the decay kinetics of Q are slower than those of the carotenoid triplets, while at lower flash intensities they are similar. Q is sensitive to oxygen and it is proposed that Q, at the higher intensities, is a trapped chlorophyll triplet. This hypothesis accounts well for the continuing rise of the carotenoid triplet concentration for 1-2 mus after the cessation of the laser pulse by a slow detrapping mechanism, and the subsequent capture of the triplet energy by carotenoid molecules. At the maximum laser intensities, the carotenoid triplet concentration is about one per 100 chlorophyll molecules. The maximum chlorophyll ion concentration generated by the laser pulses was estimated to be below 0.8 ions/100 chlorophyll molecules. None of the observations described here were altered when a picosecond pulse laser train was substituted for the microsecond pulse. A simple kinetic model describing the generation of singlets and triplets (by intersystem crossing), and their subsequent interaction leading to fluorescence quenching, accounts well for the observations. The two coupled differential equations describing the time dependent evolution of singlet and triplet excited states are solved numerically. Using a single-triplet bimolecular rate constant of gammast = 10(-8) cm3 . s-1, the following observations can be accounted for: (1) the rapid initial drop in phi and its subsequent levelling off with increasing time during the laser pulse, (2) the buildup of the triplets during the pulse, and (3) the integrated yield of triplets per pulse as a function of the energy of the flash.     

Comparison of ATP-induced and DCMU-induced increases of chlorophyll fluorescence

A comparative study of the ATP-induced and the DCMU-induced increases of dark chlorophyll fluorescence after activation of the latent ATPase gave the following results: (1) The ATP-induced fluorescence rise exceeds the DCMU-induced rise by an amount equivalent to the rapid component of the biphasic ATP-induced change. There is complementarity between the slow component and any preceding DCMU-induced fluorescence rise. (2) Up to 10^?4 M DCMU (3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea)), with the slow component being completely suppressed, the rapid ATP-induced phase is unaffected. It becomes eliminated, though, with an I₅₀ of about 3 · 10^?4 M. (3) No binary oscillations in dependence of the number of preilluminating flashes are observed for the rapid ATP-induced fluorescence increase. Under identical conditions such oscillations are found upon DCMU-addition. (4) The amplitude of the rapid ATP-induced fluorescence rise is unaffected by closure of Photosystem II reaction centers in presence of DCMU and NH₂OH by a single saturating flash (removal of about 50% of total quenching). With further flashes and gradual complete removal of quenching, the rapid ATP-induced change is eliminated with a two-step dependency. It is concluded that the rapid phase of the ATP-induced increase in fluorescence reflects reverse electron flow at non-B-type reaction centers, while the slow phase is linked to reverse electron flow at B type centers. On the basis of these results a model is proposed for heterogeneous interactions between the ATPase and B-type and non-B-type electron-transport chains. ‘Direct coupling’ appears to be possible between CF₀-CF₁ and those electron-transport chains which are located in the stroma-exposed margin region of the grana stacks (PS IIβ units with non-B-type properties).     

Photosynthetic dioxygen formation studied by time-resolved delayed fluorescence measurements--method, rationale, and results on the activation energy of dioxygen formation

The analysis of the time-resolved delayed fluorescence (DF) measurements represents an important tool to study quantitatively light-induced electron transfer as well as associated processes, e.g. proton movements, at the donor side of photosystem II (PSII). This method can provide, inter alia, insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The underlying rationale and experimental details of the method are described. The delayed emission of chlorophyll fluorescence of highly active PSII membrane particles was measured in the time domain from 10 mus to 60 ms after each flash of a train of nanosecond laser pulses. Focusing on the oxygen-formation step induced by the third flash, we find that the recently reported formation of an S4-intermediate prior to the onset of O-O bond formation [M. Haumann, P. Liebisch, C. Müller, M. Barra, M. Grabolle, H. Dau, Science 310, 1019-1021, 2006] is a multiphasic process, as anticipated for proton movements from the manganese complex of PSII to the aqueous bulk phase. The S4-formation involves three or more likely sequential steps; a tri-exponential fit yields time constants of 14, 65, and 200 mus (at 20 degrees C, pH 6.4). We determine that S4-formation is characterized by a sizable difference in Gibbs free energy of more than 90 meV (20 degrees C, pH 6.4). In the second part of the study, the temperature dependence (-2.7 to 27.5 degrees C) of the rate constant of dioxygen formation (600/s at 20 degrees C) was investigated by analysis of DF transients. If the activation energy is assumed to be temperature-independent, a value of 230 meV is determined. There are weak indications for a biphasicity in the Arrhenius plot, but clear-cut evidence for a temperature-dependent switch between two activation energies, which would point to the existence of two distinct rate-limiting steps, is not obtained.     

The role of hydrogen bonds for the multiphasic P680(+)* reduction by YZ in photosystem II with intact oxyen evolution capacity. Analysis of kinetic H/D isotope exchange effects

The mechanism of multiphasic P680(+)* reduction by YZ has been analyzed by studying H/D isotope exchange effects on flash-induced changes of 830 nm absorption, DeltaA830(t), and normalized fluorescence yield, F(t)/F0, in dark-adapted thylakoids and PS II membrane fragments from spinach. It was found that (a) the characteristic period four oscillations of the normalized components of DeltaA830(t) relaxation and of F(t)/F0 rise in the nanosecond and microsecond time domain are significantly modified when exchangeable protons are replaced by deuterons; (b) in marked contrast to the normalized steady-state extent of the microsecond kinetics of 830 nm absorption changes which increases only slightly due to H/D exchange (about 10%) the Si state-dependent pattern exhibits marked effects that are most pronounced after the first, fourth, fifth, and eighth flashes; (c) regardless of data evaluation by different fit procedures the results lead to a consistent conclusion, that is, the relative extent of the back reaction between P680(+)*QA-* becomes enhanced in samples suspended in D2O; and (d) this enhancement is dependent on the Si state of the WOC and attains maximum values in S2 and S3, most likely due to a retardation of the "35 micros kinetics" of P680(+)* reduction. In an extension of our previous suggestion on the functional role of hydrogen bonding of YZ by a basic group X (Eckert, H.-J., and Renger, G. (1988) FEBS Lett. 236, 425-431), a model is proposed for the origin of the multiphasic P680(+)* reduction by YZ. Two types of different processes are involved: (a) electron transfer in the nanosecond time domain is determined by strength and geometry of the hydrogen bond between the O-H group of YZ and acceptor X, and (b) the microsecond kinetics reflect relaxation processes of a hydrogen bond network giving rise to a shift of the equilibrium P680(+)*YZ <==> P680YZ(OX) toward the right side. The implications of this model are discussed.     

Induction kinetics of delayed light emission in spinach chloroplasts

Induction curves of the delayed light emission in spinach chloroplasts were studied by measuring the decay kinetics after each flash of light. This study differs from previous measurements of the induction curves where only the intensities at one set time after each flash of light were recorded. From the decay kinetics after each flash of light, the induction curves of the delayed light emission measured 2 ms after a flash of light were separated into two components: one component due to the last flash only and one component due to all previous flashes before the last one. On comparing the delayed light induction curves of the two components with the fluorescence induction curves in chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and in chloroplasts treated with hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the component due to the last flash only is found to be dependent on the concentration of open reaction centers and the component due to all previous flashes except the last is dependent on the concentration of closed reaction centers. This implies that the yield of the fast decaying component of the delayed light emission is dependent on the concentration of open reaction centers and the yield of the slow decaying component is dependent on the concentration of closed reaction centers.     

Modifications of the functioning of Photosystem II induced in the dark by alkaline pH in the presence of cations

Submission of chloroplasts to alkaline pH, in the range pH 7.5–9.5, leads to changes in their oxygen-evolving capacities. These changes are enhanced by the addition of divalent cations and also monovalent cations at high concentrations. (1) Dark incubation of chloroplasts at pH ? 9 gives rise to a time-dependent inactivation of electron transport from water to 2,6-dichlorophenolindophenol measured at neutral pH. The rate of inactivation is increased by adding cations. (2) The variable fluorescence is decreased with a dependence on incubation time and concentration of cations similar to that of the Hill reaction. Addition of the electron donor NH₂OH removes most of the fluorescence quenching, (3) EPR measurements indicate that the inactivations are accompanied by loss of Mn²⁺ and the appearance of signal II fast. (4) At lower pH (7.5) the oscillations of oxygen evolved per flash during a sequence of flashes show an increase in damping when 20 mM MgCl₂ is present instead of 100 mM KCI. These changes are not seen at pH 6. (5) None of these Mg²⁺-induced modifications are prevented by glutaraldehyde fixation. We conclude that the effects of alkaline pH and MgCl₂ do not involve major protein structural changes, and that both act on the manganese-containing protein of the oxygen-evolving site.     

Partial characterization of cyclic electron transport in intact chloroplasts

Turnover of the cyclic electron transfer chain around photosystem I in intact chloroplasts was induced by addition of sodium dithionite after poisoning with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A substantial permeability barrier to dithionite allowed redox poising to a level sufficiently negative to activate, but not overreduce, the cycle. Spectral changes could thus be studied without interference from photosystem II reactions. Illumination by repetitive single-turnover flashes showed the participation in the cycle of cytochromes f and b₅₆₃ with an apparent 1:1 stoichiometry. The rise of the flash-induced electrochromic bandshift (“P518”) showed a fast phase with rise time < 10 μs and a slow phase with rise time variable in the millisecond range. The slow phase had an amplitude equal to that of the fast phase and occurred only when electron transfer between cytochromes b₅₆₃ and f was uninhibited. A kinetic correlation was observed between the rise of the slow phase and the rereduction of cytochrome f, whereas cytochrome b₅₆₃ reoxidation was slower than both. Redox titrations of the appearance of the slow rise in the P518 response showed that it was only observed on repetitive flashes when a component of midpoint potential ～- ?55 mV (pH 8.1), n = 2, was reduced before the flash. A comparison is drawn between this protonmotive electron transfer cycle and that of the purple nonsulfur bacterium Rhodopseudomonas capsulata; possible arrangements of electron carriers in the photosystem I cycle are discussed, and a modified Q cycle is proposed to account for the properties observed.