Light-induced rapid absorption changes during photosynthesis. VII. Some reactions in sonicated chloroplasts

1. Spinach chloroplasts subjected to sonication show light-induced absorption changes at 700 mμ characteristic of the photooxidation of the chlorophyll component P700. The appearance of P700 absorption changes probably resulted from the release of plastocyanin thus interrupting the electron flow between pigment systems 1 and 2. The general features of the absorption-change transients are similar to those observed previously with digitonin-treated chloroplasts. The addition of 2 mM ascorbate or 10 μM 3-(3,4-dichlorophenyl)-1, 1-dimethylurea had practically no effect on either the magnitude or the dark decay of the transient absorption change.

2. Phenazine methosulfate (PMS) (in the presence or in the absence of ascorbate) reduction appeared to be coupled to P700 photooxidation, as shown by the corresponding transients at 430 and 388 mμ. The absorbance changes at these two wavelengths indicate that the amount of PMS photoreduced was equivalent to that of P700 photooxidized. Higher PMS concentrations accelerate the dark decay of the P700 signal. When PMS alone is present, anaerobiosis caused the dark decay to become more rapid than in the presence of ascorbate.

3. Unlike PMS, other redox agents such as 2,6-dichlorophenolindophenol, N,N,N′,N′-tetramethyl-p-phenylenediamine or diaminodurol in the presence of excess ascorbate, did not noticeably affect the kinetics of the dark decay at 430 or 703 mμ, suggesting that these reduced species are not efficiently coupled to photooxidized P700.

4. The onset and decay rates of the P700 transient in the presence of PMS and excess ascorbate was insensitive to temperature between 25° and o°. However, when the chloroplast sample was frozen at temperatures ranging from −5° to −196°, all reactions ceased. When the frozen (−196°) sample was brought back to the room temperature, the reaction was restored completely. Fresh broken chloroplasts behave similarly. Digitonin-treated chloroplasts persisted down to about −25° but with diminishing magnitude and slower decay.     

Photochemical systems in mesophyll and bundle sheath chloroplasts of C4 plants

1. The agranal bundle sheath chloroplasts of Sorghum bicolor possess very low Photosystem II activity compared with the grana-containing mesophyll chloroplasts.

2. Sorghum mesophyll chloroplasts have a chlorophyll (chl) and carotenoid composition similar to that of spinach chloroplasts. In contrast, the sorghum bundle sheath chloroplasts have a higher chl a/chl b ratio; they are enriched in β-carotene and contain relatively less xanthophylls as compared to sorghum mesophyll or spinach chloroplasts.

3. Sorghum mesophyll chloroplasts with 1 cytochrome f, 2 cytochrome b₆ and 2 cytochrome b-559 per 430 chlorophylls have a cytochrome composition similar to spinach chloroplasts. Sorghum bundle sheath chloroplasts contain cytochrome f and cytochrome b₆ in the same molar ratios as for the mesophyll chloroplasts, but cytochrome b-559 is barely detectable.

4. The chl/P700 ratios of mesophyll chloroplasts of S. bicolor and mesophyll and bundle sheath chloroplasts of Atriplex spongiosa are similar to that of spinach chloroplasts suggesting that these chloroplasts possess an identical photosynthetic unit size to that of spinach. The agranal bundle sheath chloroplasts of S. bicolor possess a photosynthetic unit which contains only about half as many chlorophyll molecules per P700 as found in the grana-containing chloroplasts.

5. The similarity of the composition of the bundle sheath chloroplasts of S. bicolor with that of the Photosystem I subchloroplast fragments, together with their smaller photosynthetic unit and low Photosystem II activities suggests that these chloroplasts are highly deficient in the pigment assemblies of Photosystem II.     

Mitochondrial functions under hypoxic conditions: The steady states of cytochrome c reduction and of energy metabolism

1. The effect of low oxygen concentration on the oxidation-reduction states of cytochrome c and of pyridine nucleotide, on Ca²⁺ uptake, on the energy-linked reduction of pyridine nucleotide by succinate, and on the rate of oxygen consumption have been examined under various metabolic conditions, using pigeon heart mitochondria.

2. The oxygen concentration required to provide half-maximal reduction of cytochrome c (p50_c) ranges from 0.27 to 0.03 μM (0.2-0.02 Torr) depending upon the metabolic activity. There is a linear increase of the p50_c value with increasing respiratory rate.

3. The fraction of the normoxic respiration that is observed at p50_c is 70–90% under State 4 conditions, but is 30% under State 3 conditions.

4. The oxygen requirement for half-maximal reduction of pyridine nucleotide (p50_PN) varies less than p50_c, being 0.08 μM in State 3 and 0.06 μM in the uncoupled state.

5. The ability of the mitochondria to exhibit an energy-linked reduction of pyridine nucleotide by succinate disappears at an oxygen concentration of 0.09 μM (0.06 Torr). Below this oxygen concentration, endogenous Ca²⁺ begins to be released from the mitochondria. Thus, the critical oxygen concentration for bioenergetic function of mitochondria corresponds approximately to 50% reduction of pyridine nucleotide (p50_PN).     

Evidence for complexed plastocyanin as the immediate electron donor of P-700

The reduction of P-700 by its electron donors shows two fast phases with half-times of 20 and 200 μs in isolated spinach chloroplasts. We have studied this electron transfer and the oxidation kinetics of cytochrome f.

Incubation of chloroplasts with KCN or HgCl₂ decreased the amplitude of the 20 μs phase. This provides evidence for a function of plastocyanin as the immediate electron donor of P-700.

At low concentrations of salt and sugar the fast phases of P-700⁺ reduction were largely inhibited. Increasing concentrations of MgCl₂, KCl and sorbitol (up to 5, 150 and 200 mM, respectively) were found to increase the relative amplitudes of the fast phases to about one-third of the total P-700 signal. Addition of both 3 mM MgCl₂ and 200 mM sorbitol increased the relative amplitude of the 20 μs phase to 70%. The interaction between P-700 and plastocyanin is concluded to be favoured by a low internal volume of the thylakoids and compensation of surface charges of the membrane.

The half-time of 20 μs was not changed when the amplitude of this phase was altered either by salt and sorbitol, or by inhibition of plastocyanin. This is evidence for the existence of a complex between plastocyanin and P-700 with a lifetime long compared to the measuring time. The 200 μs phase exhibited changes in its half-time that indicated the participation of a more mobile pool of plastocyanin.

Cytochrome f was oxidized with a biphasic time course with half-times of 70–130 μs and 440–860 μs at different salt and sorbitol concentrations. The half-time of the faster phase and a short lag of 30–50 μs in the beginning of the kinetics indicate an oxidation of cytochrome f via the 20 μs electron transfer to P-700. An inhibition of this oxidation by MgCl₂ suggests that the electron transfer from cytochrome f to complexed plastocyanin is not controlled by negative charges in contrast to that from plastocyanin to P-700.     

Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP⁺ reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin.

2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a₂). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a₂ from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a₁). The ratio of the fluorescence yields of chlorophyll a₁ and chlorophyll a₂ is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.

3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP⁺ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP⁺ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.

4. The constancy of the fluorescence yield of chlorophyll a₁ in the small fragments, under conditions at which P 700 is oxidized and NADP⁺ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a₁ is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a₁ to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.

5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.     

Effect of growth irradiance on plastocyanin levels in barley

Plastocyanin levels in barley (Hordeum vulgare cv Boone) were found to be dependent on growth irradiance. An immunochemical assay was developed and used to measure the plastocyanin content of isolated thylakoid membranes. Barley grown under 600 mole photons m^–2s^–1 contained two- to four-fold greater quantities of plastocyanin per unit chlorophyll compared with plants grown under 60 mole photons m^–2s^–1. The plastocyanin/Photosystem I ratio was found to be 2 to 3 under high irradiance compared with 0.5 to 1.5 under low irradiance. The reduced plastocyanin pool size in low light plants contributed to a two-fold reduction in photosynthetic electron transport activity. Plastocyanin levels increased upon transfer of low light plants to high irradiance conditions. In contrast, plastocyanin levels were not affected in plants transferred from high to low irradiance, suggesting that plastocyanin is not involved in the acclimation of photosynthesis to shade.Abbreviations: BSA bovine serum albumin - chl chlorophyll - cyt cytochrome - DCIP 2,6-dichlorophenolindophenol - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I - TBS 20 mM Tris-HCl pH 7.5, 500 mM NaCl - TTBS 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.5% (w/v) polyoxyethylenesorbitan monolaurate (Tween-20)     

Pigment systems and electron transport in chloroplasts I. Quantum requirements for the two light reactions in spinach chloroplasts

From studies of electron-transport reactions of isolated spinach chloroplasts, we observe the following quantum requirements: (A) For the photoreduction of NADP⁺, measured both aerobically and anaerobically, in a 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) poisoned system with ascorbate and reduced 2,6-dichlorophenolindophenol (DCIPH₂) present as electron donors, the quantum requirements are 1.0 ± 0.05 at wavelengths longer than 700 nm of actinic light, and 1.5–2.5 for wavelengths between 620 and 680 nm. (B) For the photoreduction of 2,6-dichlorophenolindophenol (DCIP) with water as the electron donor, the quantum requirements are 1.0 ± 0.05 in the range 630–660 nm. (C) For the photoreduction of NADP⁺ with water as the electron donor, the quantum requirements are 2.0 ± 0.1 in the wavelength range 640–678 nm of actinic light, increasing to 6 or greater at wavelengths beyond 700 nm. These results are shown to be inconsistent with the “separate package” model for the two pigment systems in higher plant photosynthetic electron transport. The evidence is most easily interpreted using a “controlled spillover” model, in which the transfer of electronic excitation energy from one pigment system to the other is under the control of incompletely identified factors in the reaction mixture.

At moderate light intensities the steady state rate of the [ascorbate + DCIPH₂ → NADP⁺] reaction (A) in the presence of DCMU and added ferredoxin can be increased more than 3 times when saturating amounts of plastocyanin and ferredoxin-NADP reductase are added to the chloroplasts. Similarly, the steady-state rate of the [H₂O → DCIP] Hill reaction (B) is increased about 3-fold by added MgCl₂ and plastocyanin, but added ferredoxin or ferredoxin-NADP reductase have no effect on this reaction. Plastocyanin appears to be the electron transport component which couples to DCIP, either in the oxidized or in the reduced form, in the reaction media. The steady-state rate of the [H₂O → NADP⁺] reaction (C) with saturating amounts of ferredoxin can be further increased more than 3-fold when MgCl₂, plastocyanin and ferredoxin-NADP reductase are added.     

Cytochrome oxidase and its derivatives. VIII. Formation and properties of the ‘oxygenated’ from of cytochrome oxidase and its reconversion to ferrous and ferric oxidase

1. The ‘oxygenated’ compound of cytochrome c oxidase used in our experiments is more stable than the compound of previous reports. It is quantitatively reversible to ferrous oxidase.

2. It is best formed with an excess of O₂ after reduction with a minimum amount of dithionite. It can also be formed at low O₂ tension, but then contains some ferric oxidase.

3. Its formation from ferrocyanide-reduced oxidase remains incomplete and subsequent reduction by dithionite is also incomplete.

4. Cyanide does not inhibit its formation from ferrous oxidase. If only ferricytochrome a but no ferricytochrome a₃ is reduced in the presence of cyanide by dithionite, there is no reaction with O₂.

5. The anaerobic reduction of ‘oxygenated’ oxidase by dithionite is monophasic and fast. In contrast, that of ferric oxidase is biphasic, with an initial fast reduction of ferricytochrome a followed by a much slower reduction of ferricytochrome a₃. The rate of cytochrome a, but not that of cytochrome a₃ reduction depends on dithionite concentration.

6. In the presence of dissolved O₂, the ferric oxidase reduction comes to a temporary standstill when one-third of the absorbance increase at 444 mμ has been reached.

7. Ethyl hydrogen peroxide reacting with ferrous oxidase forms a compound similar to the ‘oxygenated’ compound.

8. Hydrogen donors known to react with peroxidase-H₂O₂ complexes, particularly pyrogallol, accelerate the transformation of ‘oxygenated’ to ferric oxidase, though not at a rate comparable to that of cytochrome c.

9. These results strengthen the evidence for cytochromes a and a₃ but indicate that this difference has disappeared in ‘oxygenated’ oxidase.     

Mitochondrial oxygen affinity as a function of redox and phosphate potenitals

1. The conditions under which mitochondria might catalyse a net reversal of oxidative phosphorylation are analysed.

2. Rat-liver mitochondria, incubated under such conditions, show a strongly diminished affinity for oxygen.

3. The velocity of respiration under these conditions is a hyperbolic function of the oxygen concentration.

4. The K_m for oxygen is less than 0.1 μM at low phosphate potential, irrespective of substrate, and 1–3 μM under reversal conditions.

5. The observed kinetics can be accounted for in a simple mechanism for cytochrome oxidase action.     

Temperature dependence of delayed light emission in spinach chloroplasts

1. Delayed light of chlorophyll emitted at 0.1–3.9 ms after cessation of repetitive flash light was studied at temperatures between +40 and −196 °C in isolated spinach chloroplasts.

2. Induction kinetics of delayed light varied depending on temperature. It was found to be composed of two phases; one was an initial rapid rise followed by a rather fast decline to a low steady state level (fast phase), and the other was a slow increase after the initial rapid rise to the maximum followed by an insignificant slow decrease to a high steady state level (slow phase). The fast phase existed between −175 and 40 °C with the maximum at −40 °C, while the slow phase, between 0 and 40 °C with the maximum at 25 °C.

3. The intensity of delayed light at −175 °C was found to be less than one fiftieth that at 0 °C, and no delayed light emission was observed at −196 °C within experimental accuracy. This is in contrast to the results reported by Tollin, G., Fujimori, E. and Calvin, M. ((1958) Proc. Natl. Acad. Sci. U.S. 44, 1035–1047) in which the intensity of delayed light measured at −170 °C was about a half that at 0 °C.

4. The induction of delayed light measured at −96 °C was found to be significantly suppressed by the preillumination at −196 °C. This finding suggests that the primary photochemical event still survives at −196 °C without emission of delayed light.

5. Decay kinetics of delayed light after the flash excitation revealed the presence of at least two decay components. A slow decay component with a half decay time of several tens of milliseconds was observed at temperatures higher than 0 °C. A fast decay component with a half decay time of about 0.2 ms was observed at temperatures between −120 and 25 °C. The decay rate of this component was slightly retarded on cooling.

6. The System II particles derived from spinach chloroplasts with digitonin treatment showed a temperature dependence of delayed light similar to that of the chloroplasts. System I particles, on the other hand, scarcely emitted the delayed light at any temperature between 40 and −196 °C.     

On two photoreactions in System II of plant photosynthesis

Light-induced absorbance changes of cytochrome b₅₅₉ and C₅₅₀ in chloroplasts indicate that noncyclic electron transport from water to ferredoxin (Fd)-NADP⁺ is carried out solely by System II and includes not one but two photoreactions (IIa and IIb) that proceed effectively only in short-wavelength light. (C₅₅₀ is a new chloroplast component identified by spectral evidence and distinct from cytochromes.) The evidence suggests that the two short-wavelength light reactions operate in series, being joined by a System II chain of electron carriers that includes (but is not limited to) C₅₅₀, cytochrome b₅₅₉, and plastocyanin (PC).

H₂O → IIb_hv → C₅₅₀ → cyt. b₅₅₉ → PC → IIa_hv → Fd → NADP⁺

Photoreaction IIb involves an electron transfer from water to C₅₅₀ that does not require plastocyanin and is the first known System II photoreaction resistant to inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and o-phenanthroline. Cytochrome b₅₅₉ is reduced by C₅₅₀ in a reaction that is readily inhibited by DCMU or o-phenanthroline. Thus, the site of DCMU (and o-phenanthroline) inhibition of System II appears to lie between C₅₅₀ and cytochrome b₅₅₉. Photoreaction IIa involves an electron transfer from cytochrome b₅₅₉ and plastocyanin to ferredoxin-NADP⁺.     

Oxygen photoreduction and variable fluorescence during a dark-to-light transition in Chlorella pyrenoidosa

When dark-adapted (5 min in the dark) Chlorella cells were deposited on a bare platinum electrode, treated with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and illuminated, O₂ was consumed after a lag time of about 250 ms. The comparison of the O₂ consumption kinetics with the fluorescence O-I-D-P-S transition (the fast change in chlorophyll fluorescence which occurs after the onset of illumination of dark-adapted algae and is over within 2 s) observed in untreated algae indicates that no O₂ is consumed during the fluorescence rise and that O₂ uptake is initiated approximately when the maximum level of fluorescence P is reached. Mass spectrometry measurements of O₂ exchange (using ¹⁸O₂) were performed during dark to light transition with DCMU-untreated Chlorella cells. Under these conditions, O₂ reduction began after a lag time (about 200–400 ms) and stopped after about 5 s of illumination. The above experiments clearly show that the reduction of O₂ starts nearly at the same time that the fluorescence P-S decline. On the other hand, we show that the reduction of CO₂ does not interfere in the fluorescence O-I-D-P-S transient. We found the same apparent affinity for O₂ (about 57 μM) for both the fluorescence P-S decline and the reduction of O₂. At least three consecutive short (2 μs) saturating flashes were required to affect the fluorescence transient significantly and also to induce a significant uptake of O₂. Moreover, parabenzoquinone, an artificial Photosystem I electron acceptor, inhibited both the fluorescence D-P rise and the 250 ms lag time observed in the reduction of O₂. We conclude from the above results that in the early stages of the illumination of dark-adapted algae, some Photosystem I electron acceptors are in an inactive form. In this form, the electron transport chain is unable to reduce either O₂ or CO₂. This would lead to the accumulation of electrons on the Photosystem II acceptors (principally Q⁻_A and the plastoquinone pool) and therefore explains the fluorescence D-P rise. The light activation, probably achieved through the reduction of at least two electron acceptors, first allows the reduction of O₂, and therefore explains the P-S fluorescence decline. By accepting electrons before the site of regulation and mediating rapid O₂ reduction, parabenzoquinone avoids the accumulation of electrons and therefore inhibits the D-P fluorescence rise.     

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F₁) as the triphosphate, either in the absence or presence of Mg²⁺, label covalently bound is not hydrolysed.

2. In the presence of Mg²⁺ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits.

3. After successive photolabelling of F₁ with 8-azido-ATP (no Mg²⁺) and 8-azido-ADP (with Mg²⁺) 4 mol label is bound to F₁, 2 mol to the and 2 mol to the β subunits.

4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F₁ previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits.

5. F₁ that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F₁.

6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F₁.     

The preparation and properties of the membranal DPNH dehydrogenase from Escherichia coli

1. The membrane bound DPNH oxidase of Escherichia coli can reduce the artificial electron acceptors: ferricyanide, dichlorophenolindophenol (DCIP) and menadione. All three are reduced by the flavoprotein of DPNH oxidase, but at different sites of the enzyme.

2. Freeze-drying of the bacterial membranes causes a selective detachment of DPNH dehydrogenase (DPNH: (acceptor) oxidoreductase, EC 1.6.99.3) from the membranes. This solubilization is accompanied by a decrease of K_m(K₃Fe(CN)₆) from 2.0 to 0.25 mM, while no change is detected in K_m(DPNH). This enzyme is not the DPNH diaphorase found in the bacteria.

3. DPNH dehydrogenase of E. coli is a metalloflavoprotein, containing non-heme iron, labile sulfide, FMN and FAD.

4. Reduction of the enzyme with DPNH in the absence of electron acceptor (ferricyanide or DCIP) causes a rapid and irreversible change to a less active state, Form II. Form II is characterized by a higher K_m(DPNH) and slower v_max., while the K_m(K₃Fe(CN)₆) remains unchanged.

5. The transformation of the enzyme to Form II is accompanied by the reduction of the non-heme iron component. The role of non-heme iron in the enzymic reaction is discussed.     

Photochemical properties of a Photosystem II subchloroplast fragment

1. The Photosystem II fraction (D-10) obtained by incubation of spinach chloroplasts with digitonin was further purified by incubation with Triton X-100. The resulting Photosystem II subchloroplast fragment (DT-10) contained 1 mole of cytochrome b-559 per 170 moles of chlorophyll. It lacked cytochrome f and cytochrome b₆ and its content of P700 was low.

2. The DT-10 fragment showed only traces of photochemical activity with water as electron donor, but it was active in a Photosystem II reaction with 2,6-dichlorophenolindophenol as electron acceptor and diphenyl carbazide as donor. Photoreduction of NADP⁺ with diphenyl carbazide as donor was negligible. There was some photoreduction of NADP⁺ with ascorbate plus 2,6 dichlorophenolindophenol as donor but this activity could be accounted for by contamination with Photosystem I. These results are consistent with the Z-scheme of photosynthesis with Photosystems I and II operating in series for the reduction of NADP⁺ from water. DT-10 subchloroplast fragments showed a light-induced rise in fluorescence yield at 20 °C in the presence of diphenyl carbazide. A light-induced fluorescence increase also was observed at 77 °K.

3. During the preparation of the DT-10 fragment, the high potential form of cytochrome b-559 was largely converted to a form of lower potential and C-550 was converted to the reduced state. A photoreduction of C-550 was observed at liquidnitrogen temperature, provided the C-550 was oxidised with ferricyanide prior to cooling. Some photooxidation of cytochrome b-559 was obtained at 77 °K if the preparation was reduced prior to cooling, but the degree of photooxidation was variable with different preparations. C-550 does not appear to be identical with the primary fluorescence quencher, Q.

4. Photosystem I subchloroplast fragments (D-144) released by the action of digitonin were compared with Photosystem I fragments (DT-144) released from D-10 fragments by Triton X-100. There were no significant differences between D-144 and DT-144 fragments either in chlorophyll a/b ratio or in P700 content.     

Studies on the biochemical basis of susceptibility and resistance of the housefly to fluoroacetate

1. Administration of fluoroacetate to sensitive houseflies in amounts close to the L.D.₅₀ range (0.25–0.3 μg/fly) brought about a prompt elevation of their citrate content. With about 10-fold higher doses of fluoroacetate a concurrent increase of both citrate and pyruvate levels took place in the fly tissues.

2. Incubation of sarcosomes of the sensitive housefly strain in the presence of oxidizable substrates and fluoroacetate resulted in accumulation of citrate, inhibition of respiration and uncoupling of oxidative phosphorylation. The magnitude of the effects varied considerably with the different substrates used, being particularly pronounced with pyruvate and malate and inappreciable with succinate and -glycerophosphate.

3. The respiratory inhibition induced by a brief exposure in the cold of housefly sarcosomes to fluoroacetate, persisted after the sarcosomes had been washed free from fluoroacetate. The toxic effect of fluoroacetate on the respiratory chain could be prevented by an excess of simultaneously added acetate.

4. The susceptibility of the respiratory function of the sarcosomes to fluoroacetate inhibition was abolished by sonication. The unresponsiveness of the sonicated sarcosomes to fluoroacetate was attended by a loss of their respiratory chain phosphorylation activity.

5. Sarcosomes derived from a partially resistant housefly strain, when incubated in the presence of fluoroacetate, failed to accumulate citrate, but displayed the characteristic respiratory-inhibition response. Sarcosomes from a highly resistant strain showed no impairment of their functional capacity by fluoroacetate. However, all the different housefly strains tested proved to be equally sensitive to the deleterious effect of fluorocitrate on sarcosomal respiration.

6. The possible biochemical mechanisms underlying the toxicity of fluoroacetate in the housefly are considered with particular reference to the altered response of the target systems exhibited by the fluoroacetate-resistant strains.     

Electron paramagnetic resonance saturation studies of P-700 reaction center chlorophyll in plant photosynthesis

Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T₁, and spin-spin, T₂, relaxation times of P-700⁺ reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K T 100 K. T₁ was 200 μs at 100 K and increased to 900 μs at 10 K. T₂ was 40 ns at 40 K and increased to 100 ns at 10 K. T₁ for 40 K T 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T₁ deviates from the 1/T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T₁ of P-700⁺ were examined: T₁ of P-700⁺ was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700⁺ relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700⁺ indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700⁺ due to either the iron-sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700⁺ when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of the P-700⁺ signal. The absence of diplolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700⁺ reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power.     

Isolation and properties of chloroplast particles of Scenedesmus obliquus D3 with high photochemical activity

An improved and standardized procedure for isolation of chloroplast particles from the unicellular green alga, Scenedesmus obliquus, D₃, is described. The method is generally applicable to heterotrophically- and autotrophically-grown cells of Scenedesmus as well as to Chlamydomonas reinhardti and Chlorella sorokiniana (7-11-05) cultures. Chloroplast particles with high NADP⁺ photoreducing capacity are obtained from heterotrophic cultures only when the cell types are random and the culture is in the logarithmic growth phase; maximal rates of 240–260 μmoles NADP⁺ reduced/h per mg chlorophyll are achieved. Optimal conditions for separation of such chloroplast particles require the use of Tricine buffer (20 mM, pH 7.5), 50 nM EDTA, 10 mM KCl and 0.5 mM dithiothreitol in the breaking medium; for the maintenance of high photochemical activity it is necessary to store particles in a solution consisting of 0.4 M sucrose, 30 mM KCl and 1% bovine serum albumin.

Optimum reaction conditions were developed and the properties of the isolated particles investigated. Maximal activities are obtained when the sucrose concentration is maintained below 0.4 M; the pH optimum with Tricine buffer is between 7.8–8.1; and at least 30 mM Cl⁻ is required. Red actinic light (wavelength >620 nm) with an intensity of 10⁶ ergs/cm² per s is required for saturation.

Ferrodoxin and ferredoxin-NADP⁺ oxidoreductase are lost from the particles during the preparatory procedures and maximum photochemical activity is attained only when they are added back in balanced amounts. Stimulatory effects of added plastocyanin and cytochrome c-553 are noted only with particles having an initially low photochemical activity.     

Studies on the pathway of cyclic electron flow in mesophyll chloroplasts of a C4 plant

1. Cyclic photophosphorylation driven by white light, as followed by ¹⁴CO₂ fixation by mesophyll chloroplast preparations of the C₄ plant Digitaria sanguinalis, was specifically inhibited by disalicylidenepropanediamine (DSPD), antimycin A, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIb), 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC), and KCN suggesting that ferredoxin, cytochrome b₅₆₃, plastoquinone, cytochrome f, and plastocyanin are obligatory intermediates of cyclic electron flow. It was found that 0.2 μM DCMU and 40 μM o-phenanthroline blocked noncyclic electron flow, stimulated cyclic photophosphorylation, and caused a partial reversal (40–100%) of the inhibition by DBMIB and antimycin A, but not DSPD.

2. Cyclic photophosphorylation could also be activated using only far-red illumination. Under this condition, however, cyclic photophosphorylation was much less sensitive to the inhibitors DBMIB, EDAC and antimycin A, but remained completely sensitive to DSPD and KCN. Inhibition in far-red light was not increased by preincubating the chloroplasts with the various inhibitors for several minutes in white light.

3. The striking correspondence between the effects of photosystem II inhibitors, DCMU and o-phenanthroline, on cyclic photophosphorylation under white light and cyclic photophosphorylation under far-red light (in the absence of photosystem II inhibitors) suggests that electrons flowing from photosystem II may regulate the pathway of cyclic electron flow.     

Evidence for a double hit process in Photosystem II based on fluorescence studies

1. The amplitudes of the fast (0–20 μs) and slow (20 μs–2 ms) fluorescence rise induced by a 2 μs flash have been measured as a function of the energy of the flash in chloroplasts inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea. The saturation curve for the slow rise shows a characteristic lag which is not observed for the fast fluorescence rise. This lag indicates that Photosystem II centers undergo a double hit process which implies that (a), each photocenter includes two acceptors Q₁ and Q₂; (b), after the first hit, oxidized chlorophyll Chl⁺ is reduced by a secondary acceptor Y in a time short compared to the duration of the flash; (c), after the second hit, Chl⁺ is reduced by another secondary donor, D.

2. According to Den Haan et al. ((1974) Biochim. Biophys. Acta 368, 409–421), hydroxylamine destroys the secondary donor responsible for the fast reduction of Chl⁺. In the presence of 3 mM hydroxylamine, only the secondary donor D is functional and a flash induces mainly a single hit process.

3. The saturation curves for the fast and the slow rises have been studied in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea for a second actinic flash given 2.5 s after a first saturating one. The large decrease in the half-saturating energy indicates the existence of efficient energy transfer occuring between photosynthetic units.

4. Two alternate hypotheses are discussed (a) in which D is an auxiliary donor and (b) in which D is included in the main electron transfer chain.