Cation-mediated regulation of excitation energy distribution in chloroplasts lacking organized Photosystem II complexes

Despite the total loss of Photosystem II activity, thylakoids isolated from the green nuclear maize mutant hcf1-3 contain normal amounts of the light-harvesting chlorophyll $\text{a}\text{b}$ pigment-protein complex (LHC). We interpret the spectroscopic and ultrastructural characteristics of these thylakoids to indicate that the LHC present in these membranes is not associated with Photosystem II reaction centers and thus exists in a ‘free’ state within the thylakoid membrane. In contrast, the LHC found in wild-type maize thylakoids shows the usual functional association with Photosystem II reaction centers. Several lines of evidence suggest that the free LHC found in thylakoids isolated from hcf1-3 is able to mediate cation-dependent changes in both thylakoid appression and energy distribution between the photosystems: (1) Thylakoids isolated from hcf1-3 and wild-type seedlings exhibit a similar Mg²⁺-dependent increase in the short/long wavelength fluorescence emission peak ratio at 77 K. This Mg²⁺ effect is lost following incubation of thylakoids isolated from either source with low concentrations of trypsin. Such treatment results in the partial proteolysis of the LHC in both membrane types. (2) Thylakoids isolated from both hcf1-3 and wild-type seedlings show a similar Mg²⁺ dependence for the enhancement of the maximal yield of room temperature fluorescence and light scattering; both Mg²⁺ effects are abolished by brief incubation of the thylakoids with low concentrations of trypsin (3) Mg²⁺ acts to reduce the relative quantum efficiency of Photosystem I-dependent electron transport at limiting 650 nm light in thylakoids isolated from hcf1-3. (4) The pattern of digitonin fractionation of thylakoid membranes, which is dependent upon structural membrane interactions and upon LHC in the thylakoids, is similar in thylakoids isolated from both hcf1-3 and wild-type seedlings. We conclude that the surface-exposed segment of the LHC, but not the LHC-Photosystem II core association, is necessary for the cation-dependent changes in both thylakoid appression and energy distribution between the two photosystems, and that the LHC itself is able to transfer excitation energy directly to Photosystem I in a Mg²⁺-dependent fashion in the absence of Photosystem II reaction centers. The latter phenomenon is equivalent to a cation-induced change in the absorptive cross-section of Photosystem I.     

A seconds range component of the reoxidation of the primary Photosystem II acceptor,Q. Effects of bicarbonate depletion in chloroplasts

Broken chloroplasts depleted of bicarbonate (HCO^?₃) show 30–50% inhibition of the Hill reaction in low-intensity light. Also, photoreactions excited by repetitive flashes measured by oxygen evolution, ESR signal IIvf, and absorption changes at 680 and 334 nm show inhibition of 30–50%. An effect of HCO^?₃ was sought to explain these phenomena. The decay of chlorophyll a fluorescence yield in the millisecond and seconds range, following a single flash, was observed to be multiphasic with a very slow component of 1–2 s half-time. In HCO^?₃ -depleted samples this component is enhanced 2- or 3-fold. Since this occurs even after one flash, it is suggested that HCO^?₃ affects the Q^? B → QB^? reaction. In this work it is shown that 40% inhibition of oxygen flash yield is relieved to a great extent if the excitation flash rate is decreased from 2 to 0.33 Hz. A measurement of 520 nm absorption change in the presence of ferricyanide, which is proportional to Photosystem II charge separation, shows a similar inhibition that is dependent on flash rate. The maximum amplitude of variable fluorescence yield and 520 nm absorption change after a single flash are unaffected by HCO^?₃ depletion. The dark distribution of oxygen-evolution S-states is found to be shifted to a more reduced configuration in depleted samples. It is concluded that normal charge separation occurs in HCO^?₃ -depleted Photosystem II reaction centers but that a large fraction of Q^? decays so slowly that not all Q^? is reoxidized between flashes given at a rate of 1 or 2 Hz. Thus, a portion of the Photosystem II centers would be closed to photochemistry. There is a reversible effect of HCO^?₃ depletion on the oxygen-evolution system that is observed as a shift in the dark distribution of S-states.     

Chloroplast membranes of the green alga Acetabularia mediterranea. I. Isolation of the Photosystem II

1. In the presence of Triton X-100, chloroplast membranes of the green alga Acetabularia mediterranea were disrupted into two subchloroplast fragments which differed in buoyant density. Each of these fractions had distinct and unique complements of polypeptides, indicating an almost complete separation of the two fragments.

2. One of the two subchloroplast fractions was enriched in chlorophyll b. It exhibited Photosystem II activity, was highly fluorescent and was composed of particles of approx. 50 Å diameter.

3. The light-harvesting chlorophyll-protein complex of the Photosystem II-active fraction had a molecular weight of 67 000 and contained two different subunits of 23 000 and 21 500. The molecular ratio of these two subunits was 2:1.     

Isolation of Photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition

Partition in an aqueous Dextran-polyethylene glycol two-phase system has been used for the separation of chloroplast membrane vesicles obtained by press treatment of a grana-enriched fraction after unstacking in a low salt buffer.

The fractions obtained were analysed with respect to chlorophyll, photochemical activities and ultrastructural characteristics. The results reveal that the material partitioning to the Dextran-rich bottom phase consisted of large membrane vesicles possessing mainly Photosystem II properties with very low contribution from Photosystem I. Measurements of the H₂O to phenyl-p-benzoquinone and ascorbate-Cl₂Ind to NADP⁺ electron transport rates indicate a ratio of around six between Photosystem II and I.

The total fractionation procedure could be completed within 2–3 h with high recovery of both the Photosystem II water-splitting activity and the Photosystem I reduction of NADP⁺.

These data demonstrate that press treatment of low-salt destabilized grana membranes yields a population of highly Photosystem-II enriched membrane vesicles which can be discriminated by the phase system. We suggest that such membrane vesicles originate from large regions in the native grana membrane which contain virtually only Photosystem II.     

Metastable proton pools in thylakoids and their importance for the stability of Photosystem II

After isolated chloroplast thylakoids have been transferred to a medium which is more alkaline than their storage medium, they retain considerable amounts of unequilibrated protons for often longer than 10 min. Essentially all of these protons are released upon uncoupler addition when the thylakoids are osmotically swollen, but only a portion of them when they are in a shrunken state. Osmotic swelling also greatly accelerates the inactivation of the water-oxidizing system enzyme of Photosystem II, and its depletion of functional Cl^?, at alkaline pH. Analyses of the mestable proton gradient in terms of stoichiometry, temperature dependence, and effect on fluorescent amine probes, suggest that most of the protons involved are bound and exchange readily with the bulk phases only when the thylakoids are swollen. It is concluded that, in shrunken thylakoids, the water-oxidizing enzymes are buried in special H⁺-sequestering domains which probably are formed by cavities in the inner surface of the thylakoid membrane. An observed cooperative action of alkaline pH and divalent cations during Cl^?-extraction from Photosystem II is interpreted as revealing an involvement of both a negatively charged surface region and positively charged groups in maintaining the functional integrity of the site of water oxidization.     

Inhibition of Photosystem II by uncouplers at alkaline pH and its reversal by artificial electron donors

When chloroplasts are aged for 5 min at pH 9.6, or are exposed to uncouplers at pH 8.5–9.0, electron flow from water to Hill acceptors is inhibited. Both treatments induce rapid millisecond dark decay of delayed light emission. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-sensitive electron transport through Photosystem II can be regenerated in both types of inhibited chloroplasts by the artificial electron donor, 1,5-diphenylcarbohydrazide. Neither treatment inhibits electron flow through Photosystem I. Uncouplers at alkaline pH, when added in the light, are less effective in producing the inhibition than when added in the dark. These results are interpreted as indicating inhibition of the oxygen-evolving apparatus by alkaline intrathylakoid pH.     

Stimulation by manganese and other divalent cations of the electron donation reactions of Photosystem II

Using inside-out thylakoid membranes, it has been shown that the oxidation of water and associated reduction of dichlorophenol indophenol is partially inhibited by low concentrations of cation chelators. This inhibition correlates with a removal of two manganese ions per Photosystem II reaction centre. The chelator-induced inhibition was completely reversed by the addition of low levels of Mn²⁺ ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 20 μ}\text{M}$) and higher levels of Mg²⁺ and Ca²⁺ ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1}\text{mM}$). Other cations were not effective, indicating that the ability to overcome the inhibition did not involve a general electrostatic screening process. The degree of inhibition by chelators was greater at lower light intensities and after treatment with glutaraldehyde. In the presence of glutaraldehyde the stimulatory effect of Mn²⁺ was lost, while pretreatment with Mn²⁺ prevented the glutaraldehyde effect. These results are discussed in terms of conformational changes of the electron donation chains involving cation- (preferentially Mn-) dependent coupling between the oxygen evolving and reaction-centre complexes of Photosystem II.     

Cobalt ions inhibit electron-transport activity of Photosystem II without affecting Photosystem I

Investigations on photosynthesis have greatly benefited by the use of specific inhibitors that affect a specific site of inhibition on the electron-transport chain. We show here for the first time that cobalt (Co²⁺) ions can be used specifically to inactivate electron donation to the reaction centre of Photosystem (PS) II without affecting PS I reactions. This conclusion is based on the following observations: (1) addition of exogenous electron donors such as NH₂OH does not relieve Co²⁺-induced inactivation of photoelectron transport or the lowering of steady-state chlorophyll a fluorescence yield; this suggests that the inhibition is beyond the NH₂OH donation site and before the fluorescence quencher Q, i.e., on the reaction centre complex itself. (2) Washing of Co²⁺-pretreated chloroplasts with isolation buffer to remove Co²⁺ does not relieve Co²⁺-induced inhibition of Hill activity, suggesting that the Co²⁺ effect is irreversible. (3) Co²⁺ did not alter the PS I reactions. Thus, Co²⁺-treated chloroplasts can be used to study PS I functions free from PS II reactions in isolated chloroplasts.     

Lactoperoxidase-catalyzed iodination of chloroplast membranes. II. Evidence for surface localization of Photosystem II reaction centers

The enzyme lactoperoxidase was used to specifically iodinate the surface-exposed proteins of chloroplast lamellae. This treatment had two effects on Photosystem II activity. The first, occurring at low levels of iodination, resulted in a partial loss of the ability to reduce 2,6-dichlorophenolindophenol (DCIP), even in the presence of an electron donor for Photosystem II. There was a parallel loss of Photosystem II mediated variable yield fluorescence which could not be restored by dithionite treatment under anaerobic conditions. The same pattern of inhibition was observed in either glutaraldehyde-fixed or unfixed membranes. Analysis of the lifetime of fluorescence indicated that iodination changes the rate of deactivation of the excited state chlorophyll. We have concluded that iodination results in the introduction of iodine into the Photosystem II reaction center pigment-protein complex and thereby introduces a new quenching. The data indicate that the reaction center II is surface exposed.At higher levels of iodination, an inhibition of the electron transport reactions on the oxidizing side of Photosystem II was observed. That portion of the total rate of photoreduction of DCIP which was inhibited by this action could be restored by addition of an electron donor to Photosystem II. Loss of activity of the oxidizing side enzymes also resulted in a light-induced bleaching of chlorophyll a₆₈₀ and carotenoid pigments and a dampening of the sequence of O₂ evolution observed during flash irradiation of treated chloroplasts. All effects on electron transport on the oxidizing side of Photosystem II could be eliminated by glutaraldehyde fixation of the chloroplast lamellae prior to lactoperoxidase treatment. It is concluded that the electron carriers on the oxidizing side of Photosystem II are not surface localized; the functioning of these components is impaired by structural disorganization of the membrane occurring at high levels of iodination.Our data are in agreement with previously published schemes which suggest that Photosystem II mediated electron transport traverses the membrane.     

Oxygen-evolution patterns from spinach Photosystem II preparations

Patterns of O₂ evolution resulting from sequences of short flashes are reported for Photosystem (PS) II preparations isolated from spinach and containing an active, O₂-evolving system. The results can be interpreted in terms of the S-state model developed to explain the process of photosynthetic water splitting in chloroplasts and algae. The PS II samples display damped, oscillating patterns of O₂ evolution with a period of four flashes. Unlike chloroplasts, the flash yields of the preparations decay with increasing flash number due to the limited plastoquinone acceptor pool on the reducing side of PS II. The optimal pH for O₂ evolution in this system (pH 5.5–6.5) is more acidic than in chloroplasts (pH 6.5–8.0). The O₂-evolution, inactivation half-time of dark-adapted preparations was 91 min (on the rate electrode) at room temperature. Dark-inactivation half-times of 14 h were observed if the samples were aged off the electrode at room temperature. Under our conditions (experimental conditions can influence flash-sequence results), deactivation of S₃ was first order with a half-time of 105 s while that of S₂ was biphasic. The half-times for the first-order rapid phase were 17 s (one preflash) and 23 s (two preflashes). The longer S₂ phase deactivated very slowly (the minimum half-time observed was 265 s). These results indicate that deactivation from S₃ → S₂ → S₁, thought to be the dominant pathway in chloroplasts, is not the case for PS II preparations. Finally, it was demonstrated that the ratio of S₁ to S₀ can be set by previously developed techniques, that S₀ is formed mostly from activated S₃ (S₄), and that both S₀ and S₁ are stable in the dark.     

A backflow of electrons around Photosystem II in Chlorella cells

A study was made with a modulated oxygen electrode of the effect of variations of oxygen concentration on photosynthetic oxygen evolution from algal cells. When Chlorella vulgaris is examined with a modulated 650 nm light at 22°C, both the oxygen yield and the phase lag between the modulated oxygen signal and the light modulations have virtually constant values between 800 and 120 ergs · cm^?1 · s^?1 if the bathing medium is in equilibrium with the air. Similar results are obtained at 32°C between 1600 and 120 ergs · cm^?2 · s^?1. Under anerobic conditions both the oxygen yield and the phase lag decrease if the light intensity is lowered below about 500 ergs · cm^?2 · s^?1 at 22°C or about 1000 ergs · cm^?2 · s^?1 at 32°C. A modulated 706 nm beam also gives rise to these phenomena but only at significantly lower rates of oxygen evolution. The cells of Anacystis nidulans and Porphyridium cruentum appear to react in the same way to anaerobic conditions as C. vulgaris. An examination of possible mechanisms to explain these results was performed using a computer simulation of photosynthetic electron transport. The simulation suggests that a backflow of electrons from a redox pool between the Photosystems to the rate-limiting reaction between Photosystem II and the water-splitting act can cause a decrease in oxygen yield and phase lag. If the pool between the Photosystems is in a very reduced state a significant cyclic flow is expected, whereas if the pool is largely oxidized little or no cyclic flow should occur. It is shown that the effects of 706 nm illumination and removal of oxygen can be interpreted in accordance with these proposals. Since a partial inhibition of oxygen evolution by 3-(3.4-dichlorophenyl)-1,1-dimethylurea (10^?8 M) magnifies the decreases in oxygen yield and phase lag, it is proposed that the pool which cycles back electrons is in front of the site of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition and is probably the initial electron acceptor pool after Photosystem II.     

Determination and modification of the redox state of the secondary acceptor of Photosystem II in the dark

The redox state of the secondary electron acceptor B of Photosystem II was studied using fluorescence measurements. Preillumination of algae or chloroplasts with a variable number of short saturating flashes followed rapidly by the addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea induces oscillations of the initial level of fluorescence. The phase of these oscillations is characteristic of a given $\text{B}\text{B}{}^{\mathrm{?}}$ ratio in the dark-adapted samples.We conclude from our results that about 50% of the secondary electron acceptors are singly reduced in the dark in Chlorella cells, but that more than 70% are fully oxidized in the dark adapted chloroplasts.Benzoquinone treatment modifies this distribution in Chlorella leading to the same situation as in chloroplasts, i.e. more than 70% of the secondary acceptors are oxidized in the dark.The same ratio is observed if these algae are illuminated and then dark-adapted, unless an artificial donor (hydroxylamine) is added before this illumination. In that case about 50% B^? is generated and stabilized in the dark.     

Two electron donation sites for exogenous reductants in chloroplast Photosystem II

Two sites are distinguished for the oxidation of exogenous donors by Photosystem II in non-oxygen evolving chloroplasts. In the presence of lipophilic donors (e.g. phenylenediamine, benzidine, diphenylcarbazide), the rate for Signal IIf rereduction following a flash increases as the concentration of exogenous reductant increases. There is a decrease (20–40%) in Signal IIf magnitude accompanying donor addition at low (< 10^?5M) concentrations, but the extent of the decrease does not change further with increasing donor concentration. Complementary polarographic experiments monitoring donor (phenylenediamine) oxidation show an increase in oxidation rate with increasing donor concentration.In the presence of the hydrophilic donor, Mn²⁺, the Signal IIf decay halftime remains constant with increasing Mn²⁺ concentration. However, the flash-induced Signal IIf magnitude progressively decreases with increasing Mn²⁺ concentration.These results are interpreted in terms of two competing paths for the reduction of P680⁺. In one path P680⁺ reduction is accompanied by the appearance of Signal IIf, and lipophilic donors subsequently rereduce the Signal IIf species in a series reaction. This reduction follows pseudo-first order kinetics as a function of donor concentration. In the second path Mn²⁺ reduces P680⁺ in a parallel reaction that competes with the formation of the Signal IIf species. This results in a decrease in the magnitude of Signal IIf, but no change in its decay time.     

Covalent modification of chloroplast Photosystem II polypeptides by p-nitrothiophenol

Illumination of the chlorophyll $\text{a}\text{b}$ light-harvesting complex in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused quenching of fluorescence emission at 685 nm (77 K) relative to 695 nm and covalent modification of light-harvesting complex polypeptides. Fluorescence quenching saturated with one p-nitrothiophenol bound per light-harvesting complex polypeptide (10–13 chlorophylls); $\text{1}\text{2}$ maximal quenching occurred with one p-nitrothiophenol bound per light-harvesting complex polypeptides (190–247 chlorophylls). This result provides direct evidence for excitation energy transfer between light-harvesting complex subunits which contain 4–6 polypeptides plus 40–78 chlorophylls per complex.Illumination of chloroplasts or Photosystem II (PS II) particles in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused inhibition of PS II activity and labeling of several polypeptides including those of 42–48 kilodaltons previously identified as PS II reaction center polypeptides. In chloroplasts, inhibition of oxygen evolution accelerated p-nitrothiophenol modification reactions; DCMU or donors to PS II decreased p-nitrothiophenol modification. These results are consistent with the hypothesis that accumulation of oxidizing equivalents on the donor side of PS II creates a ‘reactive state’ in which polypeptides of PS II are susceptible to p-nitrothiophenol modification.     

Preparation of O2-evolving Photosystem-II subchloroplasts from spinach

An O₂-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O₂ evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O₂ uptake activity. The preparation showed a chlorophyll $\text{a}\text{b}$ ratio of 1.9 and a $\text{P-700}\text{chlorophyll}$ ratio of $\text{1}\text{2400}$. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O₂-evolving Photosystem II preparation.