Inhbition of photosystem II-dependent phosphorylation in chloroplasts by mercurials.

Photophosphorylation supported by the coupling site associated with Phostosystem II electron transport (coupling site II) is 50 to 60 times less sensitive to the energy transfer inhibitor HgCl₂ than phosphorylation supported by the coupling site associated with Photosystem I electron transport (coupling site I). Coupling site II phosphorylation is only about 2 times less sensitive to the lipophilic mercurial p-hydroxymercuribenzoate (PHMB), however. Both coupling sites are equally sensitive to CF₁ antiserum. These results suggest that a portion of the energy conserving apparatus associated with coupling site II is in a more hydrophobic environment than the corresponding apparatus associated with coupling site I.     

The presence of EC collagen and type IV collagen in bovine Descemet's membranes

The inhibitory effect of antimycin A on the slow rise of the flash-induced electrochromic absorbance change was reinvestigated in intact chloroplasts isolated from pea leaves. It is show that in the absence of nigericin and ⁺K at low repetition rates (<0.5 s^?1) of the excitation flashes not only the slow (～ 10 ms) rise but also the initial (?1 ms) rise generated by photosystem 1 is inhibited by antimycin A.     

The role of chloride ion in Photosystem II I. Effects of chloride ion on Photosystem II electron transport and on hydroxylamine inhibition

1. Chloroplasts washed with Cl^?-free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl^? is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems.2. Strong Cl^?-dependence of Hill activity was observed invariably at all pH values tested (5.5–8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll).3. In the absence of added Cl^? the functionally Cl^?-depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H₂O₂ at rates which are 4–12 times faster than the rate of the residual Hill reaction.4. The Cl^?-concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl^?ag E · Cl^? (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation.5. The initial phase of the development of inhibition of water oxidation in Cl^?-depleted chloroplasts during the dark incubation with NH₂OH ($\text{1}\text{2}$ H₂SO₄) is 5 times slower when the incubation medium contains Cl^? than when the medium contains NH₂OH alone or NH₂OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O₂ evolution.)6. We conclude that the Cl^?-requiring step is one which is specifically associated with the water-splitting reaction, and suggests that Cl^? probably acts as a cofactor (ligand) of the NH₂OH-sensitive, Mn-containing O₂-evolving enzyme.     

Nondestructive evaluation of photosynthesis by delayed luminescence in Arabidopsis in Petri dishes

Nondestructive evaluation of photosynthesis is a valuable tool in the field and laboratory. Delayed luminescence (DL) can reflect charge recombination through the backflow of electrons. However, DL detection has not yet been adapted for whole plants in Petri dishes. To compensate for differences in DL decay between sibling Arabidopsis plants grown under the same conditions, we developed a time-sequential double measurement method. Using this method, we examined the influence of photosynthetic electron flow inhibitors, and differences in the DL decay curves were categorized by considering the initial and late phases of the decay curves, as well as their intermediate slopes. The appearance of concavity and convexity in DL curves in Arabidopsis was different from unicellular algae, suggesting complexity in the photosynthetic machinery of higher plants. This detection method should be invaluable for evaluating photosynthetic defects in higher plants under sterile conditions without interrupting plant culture.     

Effects of alcohol/water mixtures on the structure and reactivity of cytochrome c

The oxidation reaction of ferrocytochrome c (produced in situ by pulse radiolysis) by Fe(CN)^3?₆, was used to probe the effect of alcohol/water mixtures on the reactivity of the protein. Reduced cytochrome c is oxidized in a biphasic process. The relative contribution of each phase depended on: pH, alcohol concentration and temperature. pK_a values were derived from the kinetic data. These pK_a values were identical with the spectroscopic pK_a values determined under similar conditions by monitoring the 695 nm absorption band of the oxidized protein. The two phases of oxidation were therefore related to the oxidation of a relaxed and a nonrelaxed conformer of reduced cytochrome c produced in situ. A shift in the pK_a of ferricytochrome c and a retardation of the redox reactions of both the reduced and the oxidized protein were observed at low alcohol concentrations (up to 5 mol %). These low alcohol concentrations are known to affect the structure of water (Yaacobi, M. and Ben-Naim, A. (1973) J. Sol. Chem. 5, 425?443; Ben-Naim, A. (1967) J. Phys. Chem. 71, 4002?4007 and Ben-Naim, A. and Baer, S. (1964) Trans. Faraday Soc. 60, 1736?1741) but have only minor effects on the protein. Accordingly, the kinetic results are interpreted on the basis of involvement of water molecules in the reaction complex of cytochrome c with its redox substrates.     

Calmodulin antagonists inhibit electron transport in photosystem II of spinach chloroplasts

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca²⁺ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca²⁺-specific ionophore, Ca²⁺ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.     

Fluorescence induction in intact spinach chloroplasts

Under conditions in which the Photosystem II quencher is rapidly reduced upon illumination, either after a preillumination or following treatment with dithionite, the fluorescence-induction curve of intact spinach chloroplasts (class I type) displays a pronounced dip. This dip is probably identical with that observed after prolonged anaerobic incubation of whole algal cells (“I-D dip”). It is inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea and occurs in the presence of dithionite, sufficient to reduce the plastoquinone pool. It is influenced by far red light, methylviologen, anaerobiosis and uncouplers in a manner consistent with the interpretation that it represents a photochemical quenching of fluorescence by an electron transport component situated between the Photosystem II quencher and plastoquinone. Glutaraldehyde inhibition may indicate that protein structural changes are involved.     

EGTA, a calcium chelator, inhibits electron transport in photosystem II of spinach chloroplasts at two different sites

A fifteen minute incubation of spinach chloroplasts with the divalent Ca²⁺ chelator, EGTA, in concentrations 50–250 μM, inhibits electron transport through both photosystems. All photosystem II partial reactions, including indophenol, ferricyanide and the DCMU-insensitive silicomolybdate reduction are inhibited from 70–100%. The photosystem II donor reaction, diphenyl carbazide → indophenol, is also inhibited, indicating that the inhibition site comes after the Mn²⁺ site, and that the first Ca²⁺ effect noted (site II) is not on the water oxidation enzyme, as is commonly assumed, but between the Mn²⁺ site and plastoquinone A pool. The other photosystem II effect of EGTA (Ca²⁺ site I), occurs in the region between plastoquinone A and P700 in the electron transport chain of chloroplasts. About 50% inhibition of the reaction ascorbate + TMPD → methyl viologen is given by incubation with 200 μM EGTA for 15 min. Ca²⁺ site II activity can be restored with 20 mM CaCl₂. Ca²⁺ site I responds to Ca²⁺ and plastocyanin added jointly. More than 90% activity in the ascorbate + TMPD → methylviologen reaction can be restored. Various ways in which Ca²⁺ ions could affect chloroplast structure and function are discussed. Since EGTA is more likely to penetrate chloroplast membranes than EDTA, which is known to remove CF₁, the coupling factor, from chloroplast membranes, and since Mg²⁺ ions are ineffective in restoring activity, it is concluded that Ca²⁺ may function in the electron transport chain of chloroplasts in a hitherto unsuspected manner.     

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.     

Photooxidation of cytochrome b559 and the electron donors in chloroplast Photosystem II

The effect of light on the reaction center of Photosystem II was studied by differential absorption spectroscopy in spinach chloroplasts.

At − 196 °C, continuous illumination results in a parallel reduction of C-550 and oxidation of cytochrome b₅₅₉ high potential. With flash excitation, C-550 is reduced, but only a small fraction of cytochrome b₅₅₉ is oxidized. The specific effect of flash illumination is suppressed if the chloroplasts are preilluminated by one flash at 0 °C.

At − 50 °C, continuous illumination results in the reduction of C-550 but little oxidation of cytochrome b₅₅₉. However, complete oxidation is obtained if the chloroplasts have been preilluminated by one flash at 0 °C. The effect of preillumination is not observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

A model is discussed for the reaction center, with two electron donors, cytochrome b₅₅₉ and Z, acting in competition. Their respective efficiency is dependent on temperature and on their states of oxidation. The specific effect of flash excitation is attributed to a two-photon reaction, possibly based on energy-trapping properties of the oxidized trap chlorophyll.     

Primary reactions of Photosystem II at low pH. 2. Light-induced changes of absorbance and electron spin resonance in spinach chloroplasts

The effects of lowering the pH on Photosystem II have been studied by measuring changes in absorbance and electron spin resonance in spinach chloroplasts.At pH values around 4 a light-induced dark-reversible chlorophyll oxidation by Photosystem II was observed. This chlorophyll is presumably the primary electron donor of system II. At pH values between 5 and 4 steady state illumination induced an ESR signal, similar in shape and amplitude to signal II, which was rapidly reversed in the dark. This may reflect the accumulation of the oxidized secondary donor upon inhibition of oxygen evolution. Near pH 4 the rapidly reversible signal and the stable and slowly decaying components of signal II disappeared irreversibly concomitant with the release of bound manganese.The results are discussed in relation to the effects of low pH on prompt and delayed fluorescence reported earlier (van Gorkom, H. J., Pulles, M. P. J., Haveman, J. and den Haan, G. A. (1976) Biochim. Biophys. Acta 423, 217–226).     

Flash photolysis-electron spin resonance studies of Photosystem I. A fast reduction component of P-700+

A 300 μs decay component of ESR Signal I (P-700⁺) in chloroplasts is observed following a 10 μs actinic xenon flash. This transient is inhibited by treatments which block electron transfer from Photosystem II to Photosystem I (e.g. 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), KCN and HgCl₂). The fast transient reduction of P-700⁺ can be restored in the case of DCMU or DBMIB inhibition by addition of an electron donor couple (2,6-dichlorophenol indophenol (Cl₂Ind)/ascorbate) which supplies electrons to cytochrome f. However, this donor couple is inefficient in restoring electron transport in chloroplasts which have been inhibited with the plastocyanin inactivators, KCN and HgCl₂. Oxidation-reduction measurements reveal that the fast P-700⁺ reduction component reflects electron transfer from a component with E_m = 375±10 mV (pH = 7.5). These data suggest the assignment of the 300-μs decay kinetics to electron transfer from cytochrome f (Fe²⁺) to P-700⁺, thus confirming the recent observations of Haehnel et al. (Z. Naturforsch. 26b, 1171–1174 (1971)).     

Kinetics of the photoreduction of cytochrome b-559 by Photosystem II in chloroplasts

The kinetics of the photoreduction of cytochrome b-559 and plastoquinone were measured using well-coupled spinach chloroplasts. High potential (i.e. hydroquinone reducible) cytochrome b-559 was oxidized with low intensity far-red light in the presence of N-methyl phenazonium methosulfate or after preillumination with high intensity light. Using long flashes of red light, the half-reduction time of cytochrome b-559 was found to be 100±10 ms, compared to 6–10 ms for the photoreduction of the plastoquinone pool. Light saturation of the photoreduction of cytochrome b-559 occurred at a light intensity less than one-third of the intensity necessary for the saturation of ferricyanide reduction under identical illumination conditions. The photoreduction of cytochrome b-559 was accelerated in the presence of dibromothymoquinone with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 25–35}\text{ms}$. The addition of uncouplers, which caused a stimulatory effect on ferricyanide reduction under the same experimental conditions, resulted in a decrease in the rate of cytochrome b-559 reduction. The relatively slow photoreduction rate of cytochrome b-559 compared to the plastoquinone pool implies that electrons can be transferred efficiently from Photosystem II to plastoquinone without the involvement of cytochrome b-559 as an intermediate. These results indicate that it is unlikely that high potential cytochrome b-559 functions as an obligatory redox component in the main electron transport chain joining the two photosystems.     

The 520 nm absorbance changes in Scenedesmus obliquus and its relation to Photosystem I

The kinetics (region of seconds) of the light-induced 520 nm absorbance change and its dark reversal have been studied in detail in the wild type and in some pigment and photosynthetic mutants of Scenedesmus obliquus. The following 5 lines of evidence led us to conclude that the signal is entirely due to the photosystem I reaction modified by electron flow from Photosystem II.Gradual blocking of the electron transport with 3(3,4-dichlorophenyl)-1,1-dimethylurea resulted in diminution and ultimate elimination of the biphasic nature of the signal without reducing the extent of the absorbance change or of the dark kinetics. On the contrary, blocking electron flow at the oxidizing side of plastoquinone with 2, $\text{5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isoprophyl}\text{-p-}\text{benzoquinone}$ or inactivating the plastocyanin with KCN, prolonged the dark reversal of the absorbance change apart from abolishing the biphasic nature of the signal.Action spectra clearly indicate that the main signal (I) is due to electron flow in Photosystem I and that its modification (Signal II) is due to the action of Photosystem II.Signal I is pH independent, whereas Signal II demonstrates a strong pH dependence, parallel to the O₂-evolving capacity of the cells.Chloroplast particles isolated from the wild type Scenedesmus cells demonstrated in the absence of any added artificial electron donor or acceptor and also under non-phosphorylation conditions the 520 nm absorbance change with approximately the same magnitude as whole cells. The dark kinetics of the particles were comparatively slower. Removal of plastocyanin and other electron carriers by washing with Triton X-100 slowed down the kinetics of the dark reversal reaction to a greater extent. A similar positive absorbance change at 520 nm and slow dark reversal was also observed in the Photosystem I particles prepared by the Triton method.Mutant C-6E, which contains neither carotenoids nor chlorophyll $\text{b}$ and lacks Photosystem II activity, demonstrates a normal signal I of the 520 nm absorbance change. This latter result contradicts the postulate that carotenoids are the possible cause of the 520 nm absorbance change.     

Effects of carbonyl cyanide m-chlorophenylhydrazone and hydroxylamine on the Photosystem II electron exchange mechanism in 3-(3,4-dichlorophenyl)-1,1-dimethylurea treated algae and chloroplasts

The effects of NH₂OH and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated algae and chloroplasts were studied. In the presence of DCMU, the photochemically separated charges can only disappear through a recombination back reaction; both substances induce an irreversible reduction of the donor side and after sufficient illumination their action in the presence of DCMU leads to the formation of a permanent fluorescent state.

In the DCMU + CCCP system, a fast fluorescence induction curve is observed. The fluorescence yield is brought to its maximum by two flashes. The luminescence emission is strongly inhibited and most centers reach their permanent fluorescent state after one flash.

In the DCMU + NH₂OH system, a slow fluorescence rise is observed and several saturating flashes are needed for the fluorescence yield to reach its maximum. The exhaustion of the NH₂OH oxidizing capacity and the complete transformation to a permanent fluorescent state also require a large number of flashes.

The reduction pathway catalyzed by CCCP appears to be a good competitor to the back reaction, while NH₂OH seems to be a relatively inefficient donor.

In addition the action of NH₂OH and CCCP on fluorescence suggests that the donor side influences the quenching properties of Photosystem II centers. A possible mechanism is proposed.     

Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO₂ showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.