Reduction kinetics of the photo-oxidized chlorophyll a+II in the nanosecond range: Measurements of the absorption changes at 688 nm under repetitive flash excitation

The 688 nm absorption changes (ΔA₆₈₈), indicating the photochemical turnover of chlorophyll a_II (Chl a_II) have been investigated under repetitive laser flash excitation conditions in spinach chlorplasts. It was found that under steady state conditions about 50–60% of the photo-oxidized primary donor of Photosystem II (PS II), Chl a⁺_II, becomes re-reduced with a biphasic kinetics in the nanosecond time scale with half-life times of about 50 ns and 400 ns. The remaining Chl a⁺_II becomes re-reduced in the microsecond range.     

Studies on the functional organization of Photosystem II. The effect of protein-modifying procedures and of ADRY agents on the reaction pattern of photosystem II in Tris-washed chloroplasts

The role of the protein matrix embedding the functionally active redox components of Photosystem II reaction centers has been studied by investigating the effects of procedures which modify the structure of proteins. In order to reduce the influence of the electron transport involving secondary donor and acceptor components, Triswashed chloroplasts were used which are completely deprived of their oxygen-evolving capacity. The functional activity was detected via absorption changes, reflecting at 334 and 690 or 834 nm the turnover of the primary plastoquinone acceptor, X320, and of the photochemically active chlorophyll a complex, Chl a_II, respectively, and at 520 nm the transient formation of a transmembrane electric potential gradient. Under repetitive flash excitation of Tris-washed chloroplasts it was found that: (a) The relaxation kinetics at 690 nm become significantly accelerated in the presence of external electron donors. (b) Trypsin treatment blocks to a high degree the turnover of Chl a_II and X320 unless exogenous acceptors are present, which directly oxidize X320^?, such as K₃Fe(CN)₆. (c) In the presence of K₃Fe(CN)₆ the recovery kinetics of Chl a_II and X320 are retarded markedly by trypsin, followed by a progressive decline in the extent thereof. (d) 2-(3-Chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to reduce the lifetime of S₂ and S₃ in normal chloroplasts, significantly accelerates the recovery of Chl a_II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D₁ to Chl a⁺_II. ANT 2p simultaneously retards the decay kinetics of X320^? and of the electrochromic absorption changes. (e) The kinetic pattern of the electrochromic absorption changes is also affected by the salt content of the suspension. Under dark-adapted conditions, the 10 μs relaxation kinetics of the 834 nm absorption change due to the first flash are hardly affected by mild trypsinization of 5–10 min duration, whereas the amplitude decreases by approx. 30%. The data obtained in Tris-washed chloroplasts could consistently be interpreted as a modification of the back reaction between X320^? and Chl a⁺_II which is caused solely by a change in the reactivity of X320 due to trypsin-induced degradation of the native X320-B apoprotein. Furthermore, ADRY agents are inferred to stimulate cyclic electron flow, which leads to reduction of D⁺₁ between the flashes. A simplified scheme is discussed which describes the functional organization of the reaction center complex.     

Structural and catalytic properties of the oxygen-evolving complex. Correlation of polypeptide and manganese release with the behavior of Z+ in chloroplasts and a highly resolved preparation of the PS II complex

Treatment of intact thylakoid membranes with Triton X-100 at pH 6 produces a preparation of the PS II complex capable of high rates of O₂ evolution. The preparation contains four managanese, one cytochrome b-559, one Signal II_f and one Signal II_s per 250 chlorophylls. By selective manipulation of the preparation polypeptides of approximate molecular weights of 33, 23 and 17 kDa can be removed from the complex. Release of 23 and 17 kDa polypeptides does not release functional manganese. Under these conditions Z⁺ is not readily and directly accessible to an added donor (benzidine) and it appears as if at least some of the S-state transitions occur. Evidence is presented which indicates that benzidine does have increased access to the oxygen-evolving complex in these polypeptide depleted preparations. Conditions which release the 33 kDa species along with Mn and the 23 and 17 kDa polypeptides generate an alteration in the structure of the oxidizing side of PS II, which becomes freely accessible to benzidine. These findings are examined in relationship to alterations of normal S-state behavior (induced by polypeptide release) and a model is proposed for the organization of functional manganese and polypeptides involved in the oxygen-evolving reaction.     

EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II_f and decreased the EPR signal from state S₂. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II_f, but did not relieve the decrease in the state S₂ signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II_f, Z⁺ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Chlorophyll composition of Photosystems IIα, IIβ and I in tobacco chloroplasts

The antenna composition of the Photosystems II_α, II_β and I was studied in tobacco chloroplasts. Absorbance spectra, recorded at 4 K, were analyzed for the wild type and the mutants Su/su and Su/su var. Aurea, containing higher concentrations of the photosystems. With chloroplasts of Su/su we measured the action spectra of the three photosystems from 625 to 690 nm. Above 675 nm absorption by Photosystem I dominated. This sytem had a maximum at 678 nm and a shoulder at 660 nm. Of the long-wavelength chlorophyll a forms, absorbing at 690, 697 and 705 nm at 4 K, which are generally assigned to Photosystem I, the 697 nm form occurred in an amount of four molecules per reaction center of Photosystem I in each type of chloroplast. The Photosystem II_α spectrum was characterized by maxima at 650 and 672 nm, showing clearly the participation of the chlorophyll a and b containing light-harvesting complex. In the mutants the light-harvesting complex has a chlorophyll a to chlorophyll b ratio of more than 1; the amount of the 672 nm chlorophyll a was normal, whereas the amount of chlorophyll b was markedly decreased in the mutants relative to the wild type. The Photosystem II_β spectrum mainly consisted of a band at 683 nm.     

Movement of a sub-population of the light harvesting complex (LHCII) from grana to stroma lamellae as a consequence of its phosphorylation

Phosphorylation in vitro of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex associated with Photosystem II (LHC_II) resulted in the lateral migration of a subpopulation of LHC_II from the grana to the stroma lamellae. This movement was characterized by a decrease in the chlorophyll $\text{a}\text{b}$ ratio and an increase in the 77 K fluorescence emission at 681 nm in the stroma lamellae following phosphorylation. Polyacrylamide gel electrophoresis indicated that the principal phosphoproteins under these conditions were polypeptides of 26–27 kDa. These polypeptides increased in relative amount in the stroma lamellae and decreased in the grana during phosphorylation. Pulse/chase experiments confirmed that the polypeptides were labelled in the grana and moved to the stroma lamellae in the subsequent chase period. A fraction at the phospho-LHC_II, however, was unable to move and remained associated with the grana fraction. LHC_II which moved out into the stroma lamellae effectively sensitized Photosystem I (PS I), since the ability to excite fluorescence emission at 735 nm (at 77 K) by chlorophyll b was increased following phosphorylation. These data support the ‘mobile antenna’ hypothesis proposed by Kyle, Staehelin and Arntzen (Arch. Biochem. Biophys. (1983) 222, 527–541) which states that the alterations in the excitation-energy distribution induced by LHC_II phosphorylation are, in part, due to the change in absorptive cross-section of PS II and PS I, resulting specifically from the movement of LHC_II antennae chlorophylls from the PS-II-enriched grana to the PS-I-enriched stroma lamellae.     

The reduction of the oxygen-evolving system in chloroplasts by thylakoid components

Using thoroughly dark-adapted thylakoids and an unmodulated Joliot-type oxygen electrode, the following results were obtained. (i) At high flash frequency (4 Hz), the oxygen yield at the fourth flash (Y₄) is lower compared to Y₃ than at lower flash frequency. At 4 Hz, the calculated S₀ concentration after thorough dark adaptation is found to approach zero, whereas at 0.5 Hz the apparent $\text{S}{}_0\text{(}\text{S}{}_0\text{+}\text{S}{}_1\text{)}$ ratio increases to about 0.2. This is explained by a relatively fast donation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.0–1.5}\text{s}$) of one electron by an electron donor to S₂ and S₃ in 15–25% of the Photosystem II reaction chains. The one-electron donor to S₂ and S₃ appears to be rereduced very slowly, and may be identical to the component that, after oxidation, gives rise to ESR signal II_s. (ii) The probability for the fast one-electron donation to S₂ and S₃ has nearly been the same in triazine-resistant and triazine-susceptible thylakoids. However, most of the slow phase of the S₂ decay becomes 10-fold faster ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5–6}\text{s}$) in the triazine-resistant ones. In a small part of the Photosystem II reaction chains, the S₂ decay was extremely slow. The S₃ decay in the triazine-resistant thylakoids was not significantly different from that in triazine-susceptible thylakoids. This supports the hypothesis that S₂ is reduced mainly by Q^?_A, whereas S₃ is not. (iii) In the absence of CO₂/HCO^?_A and in the presence of formate, the fast one-electron donation to S₂ and S₃ does not occur. Addition of HCO^?₃ restores the fast decay of part of S₂ and S₃ to almost the same extent as in control thylakoids. The slow phase of S₂ and S₃ decay is not influenced significantly by CO₂/HCO^?₃. The chlorophyll a fluorescence decay kinetics in the presence of DCMU, however, monitoring the Q^?_A oxidation without interference of Q_B, were 2.3-fold slower in the absence of CO₂/HCO^?₃ than in its presence. (iv) An almost 3-fold decrease in decay rate of S₂ is observed upon lowering the pH from 7.6 to 6.0. The kinetics of chlorophyll a fluorescence decay in the presence of DCMU are slightly accelerated by a pH change from 7.6 to 6.0. This indicates that the equilibrium Q^?_A concentration after one flash is decreased (by about a factor of 4) upon changing the pH from 7.6 to 6.0. When direct or indirect protonation of Q^?_B is responsible for this shift of equilibrium Q^?_A concentration, these data would suggest that the pK_a value for Q^?_B protonation is somewhat higher than 7.6, assuming that the protonated form of Q^?_B cannot reduce Q_A.     

Effects of chloride depletion on electron donation from the water-oxidizing complex to the photosystem II reaction center as measured by the microsecond rise of chlorophyll fluorescence in isolated pea chloroplasts

The role of Cl^? in the electron transfer reactions of the oxidizing side of Photosystem II (PS II) has been studied by measuring the fluorescence yield changes corresponding to the reduction of P⁺-680, the PS II reaction center chlorophyll, by the secondary PS II donor, Z. In Cl^?-depleted chloroplasts, a rapid rise in fluorescence yield was observed following the first and second flashes, but not during the third or subsequent flashes. These results indicate that there exists an additional endogenous electron donor beyond P-680 and Z in Cl^?-depleted systems. In contrast, the terminal endogenous donor on the oxidizing side of PS II in Tris-washed preparations has previously been shown to be Z, the component giving rise to EPR signals II_f and II_vf. The rate of reduction of P⁺-680 in the Cl^?-depleted chloroplasts was as rapid as that measured in uninhibited systems, within the time resolution of our instrument. Again, this is in contrast to Tris-washed preparations in which a dramatic decrease in the rate if this reaction has been previously reported. We have also carried out a preliminary study on the rate of rereduction of Z⁺ in the Cl^?-depleted system. Under steady-state conditions, the reduction half-time of Z⁺ in uninhibited systems was about 450 μs, while in the Cl^?-depleted chloroplasts, the reduction of Z⁺ was biphasic, one phase with a half-time of about 120 ms, and a slower phase with a half-time of several seconds. The appearance of the quenching state due to P⁺-680 observed following the third flash on excitation of Cl^?-depleted chloroplasts was delayed by two flashed when low concentrations of NH₂OH (20–50 μM) were included in the medium. Hydrazine at somewhat higher concentrations showed the same effect. This is taken to indicate that the reactions leading to PS II oxidation of NH₂OH or NH₂NH₂ are uninhibited by Cl^? depletion. Addition of NH₂OH at low concentrations to Tris-washed chloroplasts did not alter the pattern of the fluorescence yield, indicating that the reactions leading to the NH₂OH oxidation present in Cl^?-depleted systems are absent following Tris inhibition. The results are discussed in terms of an inhibition by Cl^? depletion of the reactions of the oxygen-evolving complex. It is suggested that no intermediary redox couple exists between the oxygen-evolving complex and Z, and that Z⁺ is reduced directly by Mn of the complex. In terms of the S-state model, Cl^? depletion appears to inhibit the advancement of the mechanism beyond S₂, but not to inhibit the transitions from S₀ to S₁, or from S₁ to S₂.     

Midpoint potential of signal II (slow) in Tris-washed photosystem-II particles

In Tris-washed Photosystem-II particles we are able to induce an EPR signal in the dark by addition of an iridium salt (K₂IrCl₆). This signal is attributed to signal II_s (slow) (D⁺) and the redox titration gives an E_m value of 760 mV for the couple $\text{D}{}^{\mathrm{+}}\text{D}$. On the basis of our previous studies on the equilibrium between D⁺Z and DZ⁺ (K = 10⁴) (Boussac, A. and Etienne, A.L. (1982) Biochem. Biophys. Res. Commun. 109, 1200–1205), we therefore attribute a value of 1 V for the E_m of the $\text{Z}{}^{\mathrm{+}}\text{Z}$ couple. A second effect of K₂IrCl₆ is to modify the spectral characteristics of signal II. We conclude that K₂IrCl₆ is able to change the environment of the species from which signal II_s and signal II_f originate.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

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 (MnCl₂ 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 D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ 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, D₁, D′₁) are located at the internal side of the thylakoid membrane.     

Relation between microsecond reduction kinetics of photooxidized chlorophyll aII (P-680) and photosynthetic water oxidation

The reduction phases of chlorophyll a⁺_II (P-680⁺) in the microsecond range have been studied in O₂-evolving Photosystem II particles from Synechococcus sp. and in spinach subchloroplasts. (1) In selected Photosystem II preparations only approx. 15% of chlorophyll a⁺_II is reduced under repetitive excitation in the microsecond time-range (approx. 85% are reduced in the nanosecond time-range). (2) The size of the microsecond fraction varies as a function of the flash number given to dark-adapted samples, suggesting a correlation to the oxidation states of the O₂-evolving complex (S-states). The oscillatory pattern closely follows the concentration of S₂ + S₃. (3) The microsecond decay can be deconvoluted into three exponential phases with half-life times of approx. 5, 35 and 200 μs. It is the amplitude of the 35 μs phase which depends on S₂ + S₃. Therefore, the 35 μs phase (approx. 10% under repetitive excitation) is connected with water oxidation. (4) Considerably higher values of the μs fraction (up to 50%) reported in former publications were probably due to Photosystem II centers which were inactive in O₂ evolution.     

Characterization of a resolved oxygen-evolving Photosystem II preparation from spinach thylakoids

An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg²⁺. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS II_α. (5) An E_m = ? 10 mV has been measured at pH 7.8 for the primary electron acceptor Q_α of PS II_α. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$. In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.     

Optical characterization of Photosystem II electron donors

Detailed absorbance difference spectra are reported for the Photosystem II acceptor Q, the secondary donor Z, and the donor involved in photosynthetic oxygen evolution which we call M. The spectra of Z and Q could be resolved by analysis of flash-induced kinetics of prompt and delayed fluorescence, EPR signal II_f and absorbance changes in Tris-washed system II preparations in the presence of ferricyanide and 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The spectrum of Z oxidation consists mainly of positive bands at 260, 300 and 390–450 nm on which a chlorophyll a band shift around 438 nm is superimposed, and is largely pH-independent as is also the case for the spectrum of Q reduction. The re-reduction of Z⁺ occurred in the millisecond time range, and could be explained by a competition between back reaction with Q^? (120 ms at pH 6.0) and reduction by ferrocyanide. When the Tris treatment is omitted the preparations evolve oxygen, and the photoreduction of Q (with DCMU present) is accompanied by the oxidation of M. The Q spectrum being known, the spectrum of the oxidation of M could be determined as well. It consists of a broad, asymmetric increase peaking near 305 nm and of a Chl a band shift, which is about the same as that accompanying Z in Tris-washed system II. Comparison with spectra of model compounds suggests that Z is a bound plastoquinol which is oxidized to the semiquinone cation and that the oxidation of M is an Mn(III) → Mn(IV) transition.