Natural diterpenes from <Emphasis Type="Italic">Croton ciliatoglanduliferus</Emphasis> as photosystem II and photosystem I inhibitors in spinach chloroplasts

In our search for new natural photosynthetic inhibitors that could lead to the development of “green herbicides” less toxic to environment, the diterpene labdane-8α,15-diol (1) and its acetyl derivative (2) were isolated for the first time from Croton ciliatoglanduliferus Ort. They inhibited photophosphorylation, electron transport (basal, phosphorylating and uncoupled) and the partial reactions of both photosystems in spinach thylakoids. Compound 1 inhibits the photosystem II (PS II) partial reaction from water to Na⁺ Silicomolibdate (SiMo) and has no effect on partial reaction from diphenylcarbazide (DPC) to 2,6-dichlorophenol indophenol (DCPIP), therefore 1 inhibits at the water splitting enzyme and also inhibits PS I partial reaction from reduced phenylmetasulfate (PMS) to methylviologen (MV). Thus, it also inhibits in the span of P₇₀₀ to Iron sulfur center X (F_X). Compound 2 inhibits both, the PS II partial reactions from water to SiMo and from DPC to DCPIP; besides this, it inhibits the photosystem I (PS I) partial reaction from reduced PMS to MV. With these results, we concluded that the targets of the natural product 2 are located at the water splitting enzyme, and at P₆₈₀ in PS II and at the span of P₇₀₀ to F_X in PS I. The results of compounds 1 and 2 on PS II were corroborated by chlorophyll a fluorescence.     

Multiple anion effects on photosystem II in chloroplast membranes

We investigated the activity of several anions at various sites on photosystem II, in particular those associated with the Cl^- effect (anion binding-site I) and the HCO₃ ^- effect (anion binding-site II). Chlorophyll a fluorescence changes were used to monitor partial photosystem II reactions either in the oxygen-evolving mechanism or involving endogenous quinone electron acceptors. We find that anions such as NO₃ ^-, HCO₃ ^-, HCO₂ ^-, F^-, NO₂ ^-, and acetate can, depending on conditions, bind to either anion binding-site I, anion binding-site II, or both sites simultaneously. The anions N₃ ^- and Au(CN)₂ ^- are exceptions. In their presence, oxygen-consumption reactions are enhanced. The results demonstrate that an exclusive site or mode of action of an anion on photosystem II cannot be determined by measuring the Hill reaction alone. Anion interactions with photosystem II are shown to be very complex and, therefore, caution is advisable in interpreting related experiments. Carbonic anhydrase associated with photosystem II was also investigated as a possible target for some anion effects. In Cl^--depleted thylakoids, NO₃ ^-, stimulated both electron transport and carbonic anhydrase activity at low concentrations, while higher concentrations inhibited both. However, carbonic anhydrase was more sensitive to inhibition by NO₃ ^- than was electron flow. Possible interpretations are discussed; the electron transport and carbonic anhydrase activity appear not to be functionally linked.Abbreviations ABSI Anion binding-site(s) I associated with the oxygen-evolving mechanism - ABSII Anion binding-site(s) II, which controls quinone-related reactions on the electron-acceptor side of photosystem II - OAc^- Acetate - Chl Chlorophyll - DCMU 3—(3,4-dichlorophenyl)-1,1-dimethyl urea - DCBQ 2,6-dichloro-p-benzoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - Mes 2-[N-morpholino]ethanesulphonic acid - Mops 3-[N-morpholino]propanesulphonic acid - Tes N-Tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid - Tricine N-Tris[hydroxymethyl]methylglycine     

Responses of photosystem I compared with photosystem II to high-light stress in tropical shade and sun leaves

Sun and shade leaves of several plant species from a neotropical forest were exposed to excessive light to evaluate the responses of photosystem I in comparison to those of photosystem II. Potential photosystem I activity was determined by means of the maximum P700 absorbance change around 810 nm (ΔA_810max) in saturating far-red light. Leaf absorbance changes in dependence of increasing far-red light fluence rates were used to calculate a ‘saturation constant’, K_s, representing the far-red irradiance at which half of the maximal absorbance change (ΔA_810max/2) was reached in the steady state. Photosystem II efficiency was assessed by measuring the ratio of variable to maximum chlorophyll fluorescence, F_v/F_m, in dark-adapted leaf samples. Strong illumination caused a high degree of photo-inhibition of photosystem II in all leaves, particularly in shade leaves. Exposure to 1800–2000 μ mol photons m⁻² s⁻¹ for 75 min did not substantially affect the potential activity of photosystem I in all species tested, but caused a more than 40-fold increase of K_s in shade leaves, and a three-fold increase of K_s in sun leaves. The increase in K_s was reversible during recovery under low light, and the recovery process was much faster in sun than in shade leaves. The novel effect of high-light stress on the light saturation of P700 oxidation described here may represent a complex reversible mechanism within photosystem I that regulates light-energy dissipation and thus protects photosystem I from photo-oxidative damage. Moreover, we show that under high-light stress a high proportion of P700 accumulates in the oxidized state, P700⁺. Presumably, conversion of excitation energy to heat by this cation radical may efficiently contribute to photoprotection.     

Surface coordination chemistry of noble-metal electrocatalysts: Oxidative addition and reductive elimination of iodide at iridium,platinum and gold in aqueous solutions

The oxidative addition and reductive elimination of the iodo ligand has been compared at smooth polycrystalline gold, platinum and iridium surfaces in aqueous solutions. On these three metals, the iodo species undergoes spontaneous oxidative chemisorption to form a close-packed monolayer of zero-valent iodine, the saturation coverage of which is limited by the van der Waals radius of the iodine atom; this oxidative addition process is further manifested by evolution of hydrogen gas from proton reduction. Elimination of iodine from these surfaces can be achieved by its reduction back to the anion either by application of sufficiently negative potentials or by exposure to ample amounts of hydrogen gas. On Pt and Ir, the reductive desorption of iodine is coupled with reductive chemisorption of hydrogen; consequently, the reaction is a two-electron, pH-dependent process. A plot of E_1/2, the potential at which the iodine coverage is decreased to half its maximum value, against pH yields information concerning the redox potential of the I_(ads)/I⁻_(ads) couple in the surface-coordinated state. On Au, where dissociative chemisorption of hydrogen does not occur, the iodine-stripping process is a pH-independent, one-electron reaction. The difference in the redox potentials [E^o_I(ads) -E^o_I(aq] for the I_(ads) and I_2(aq)/I⁻_(aq) redox couples was found to be −0.90 V on Au, − 0.76 V on Pt, and −0.72 V on Ir. These values imply that the ratio of the formation constants for surface coordination of the iodine and iodide species (K_f,I/K_f,I−) is 2 × 10²⁸ on Au, 1 × 10²⁶ on Pt, and 2 × 10²⁵ on Ir.     

The high-affinity binding site for manganese on the oxidizing side of Photosystem II

Electron donation to Photosystem II (PS II) by diphenylcarbazide (DPC) is interrupted by the presence of endogenous Mn in PS II particles. Removal of this Mn by Tris treatment greatly stimulates the electron transport with DPC as donor. Binding of low concentration of exogenous Mn(II) to Tris-treated PS II particles inhibits DPC photooxidation competitively with DPC. This phenomenon was used to locate a highly specific Mn(II) binding site on the oxidizing side of Photosystem II with dissociation constant about 0.15 μM. The binding of Mn(II) is electrostatic in nature. Its affinity depends not only on the ionic strength, but also on the anion species of the salt in the medium. The effectiveness in decreasing the affinity follows the order F⁻ > SO²⁻₄ > CH₃COO⁻ > CI⁻ > Br⁻ > NO₃⁻. This observation is interpreted as follows: smaller ions, like F⁻, CH₃COO⁻, and larger ions, like SO²⁻₄, have inhibitory effects on Mn(II) binding, whereas ions with optimal size, like Cl⁻, Br⁻ and NO₃⁻, can stabilize the binding, resembling the anion requirement for reactivation of Cl⁻-depleted chloroplasts. We suggest that the binding site for Mn(II) we observed is the site for the endogenous Mn in the O₂-evolving complex of PS II. This site remains after Tris treatment, which removes all the endogenous Mn as well as the three extrinsic proteins, indicating that it is on the intrinsic component(s) of PS II reaction centers. Furthermore, the Cl⁻ requirement for O₂ evolution may be attributed, at least partly to its stabilizing effect on Mn binding.     

Sites of superoxide and hydrogen peroxide production during fatty acid oxidation in rat skeletal muscle mitochondria

H₂O₂ production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined under two conditions: the absence of respiratory chain inhibitors and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H₂O₂ production were low unless carnitine or malate was added to limit acetyl-CoA accumulation. With palmitoylcarnitine alone, H₂O₂ production was dominated by complex II (44% from site II_F in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site I_F). With added carnitine, H₂O₂ production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site III_Qo) and 25% from site I_F. Thus complex II (site II_F in the forward reaction) is a major source of H₂O₂ production during oxidation of palmitoylcarnitine ± carnitine. Under the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H₂O₂ production were highest in the presence of palmitoylcarnitine ± carnitine and were dominated by complex II (site II_F in the reverse reaction). About half the rest was from site I_F, but a significant portion, ∼40 pmol H₂O₂·min⁻¹·mg protein⁻¹, was not from complex I, II, or III and was attributed to the proteins of β-oxidation (electron-transferring flavoprotein (ETF) and ETF-ubiquinone oxidoreductase). The maximum rate from the ETF system was ∼200 pmol H₂O₂·min⁻¹·mg protein⁻¹ under conditions of compromised antioxidant defense and reduced ubiquinone pool. Thus complex II and the ETF system both contribute to H₂O₂ productionduring fatty acid oxidation under appropriate conditions.     

Cu2+ Inhibits Photosystem II Activities but Enhances Photosystem I Quantum Yield of Microcystis aeruginosa

Responses of photosystem I and II activities of Microcystis aeruginosa to various concentrations of Cu²⁺ were simultaneously examined using a Dual-PAM-100 fluorometer. Cell growth and contents of chlorophyll a were significantly inhibited by Cu²⁺. Photosystem II activity [Y(II)] and electron transport [rETR_max(II)] were significantly altered by Cu²⁺. The quantum yield of photosystem II [Y(II)] decreased by 29 % at 100 μg L^?1 Cu²⁺ compared to control. On the contrary, photosystem I was stable under Cu²⁺ stress and showed an obvious increase of quantum yield [Y(I)] and electron transport [rETR_max(I)] due to activation of cyclic electron flow (CEF). Yield of cyclic electron flow [Y(CEF)] was enhanced by 17 % at 100 μg L^?1 Cu²⁺ compared to control. The contribution of linear electron flow to photosystem I [Y(II)/Y(I)] decreased with increasing Cu²⁺ concentration. Yield of cyclic electron flow [Y(CEF)] was negatively correlated with the maximal photosystem II photochemical efficiency (F _v/F _m). In summary, photosystem II was the major target sites of toxicity of Cu²⁺, while photosystem I activity was enhanced under Cu²⁺ stress.     

Reciprocal interactions of progesterone and 17α-OH-Progesterone as exogenous substrates of rat adrenal 21-Hydroxylase

Due to the small concentration and activity of 17α-hydroxylase present in the rat adrenal, the main corticoids secreated in the rat are DOC, B_k, A_k, 18-OH-DOC and aldosterone, formed directly from progesterone (I). Because of the limited amounts of 17α-OH-progesterone (II) available, the biosynthesis of S_R, F_k and E_k is restricted. Since 21-OH steroid hydroxylase (21-OH-ase) uses both I and II in corticoid biosynthesis in other species, it was considered of interest to study the comparative interactions which could exist between these two precursors and the rat adrenal 21-OH-ase, determining enzymatic constants for I and II (usual and unusual substrates, respectively). Homogenized adrenals from normal rats were incubated with various combinations of concentrations of I-7-³H and/or II-¹⁴C, acting as substrates and/or inhibitors of 21-OH-ase. The results showed that 21-OH-ase uses II almost as efficiently as I. The K_m values were about the same for both I and II (13.9 and 14.2 x 10⁻⁶ M/L), respectively, however, the V_max values were 54.6 and 26.0 x 10⁻⁷ M/L/min for I and II, respectively. The amounts of I required to saturate the 21-OH-ase was double that of II. Further kinetic studies showed that both I and II inhibit the 21-hydroxylation of the other in a reciprocal fashion. While II inhibits the 21-hydroxylation of I by competitive inhibition, I inhibits the 21-hydroxylation of II through a mixed type of inhibition. The results suggest that, rather than the existence of two different specific enzymes (one for I and another for II) as it has been postulated by others, it seems that we are dealing with a 21-hydroxylation system with two active sites. One site used only I and the other site uses I and/or II indistinctively.     

Effect of concentration of iodide on the bound species of I2/I−3 in the amylose-iodine complex

A liquid-liquid distribution method, with heptane as the organic solvent, involving evaluation of the concentration of free 1⁻ by magnetic circular dichroism, has been developed for determining the bound amounts of I₂/I⁻₃ in the amylose-iodine complex in unbuffered aqueous solutions. The effect of I₂ and I⁻ concentrations on the bound species of iodine in the complex was investigated by using this method. We found that the stoichiometric bound species of I₂/I⁻₃ is independent of the concentration of I₂ at a given I⁻ concentration. However, the bound species strongly depends on I⁻ concentration, and varies from I⁻₃ at 10 mM KI to I⁻₁₅ at 0M KI. Moreover, the number of d-glucosyl residues required for including one iodine atom is within the range of 2.7 to 3.0, regardless of I⁻ concentration. It was concluded that the bound species are governed by the distribution of the actual species I₂·I₂ (I₄), (I₄), I₂·I⁻₃ (I⁻₅), and I⁻₃·I⁻₃ (I²⁻₆), which are responsible for the blue color of the complex.     

Effects of high potassium iodate intake on iodine metabolism and antioxidant capacity in rats

BackgroundKIO₃ and KI are the most common salt iodization agents. Coincidentally, iodine exists naturally in high-iodine drinking water in the form of iodide (I⁻) or iodate (IO₃⁻). As an oxidizing substance, IO₃⁻ should be reduced to I⁻ before it can be effectively used by the thyroid. However, there is a lack of systematic studies on the metabolic process of high dose KIO₃ in vivo.MethodsThe iodine metabolism processes in the thyroid and serum of rats after high KIO₃ intake were determined using high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC/ICP–MS) and arsenic cerium catalytic spectrophotometry. The changes of redox activity in the serum, thyroid, liver, and kidneys were observed by detecting total antioxidative activity (TAA).ResultsHigh doses of IO₃⁻ were completely reduced to I⁻ in vivo within 0.5 h. The level of organic bound iodine in the serum was stable, while the organic bound iodine in the thyroid increased to a plateau after intake of high-dose KIO₃. The levels of total iodine and I⁻ in serum and thyroid increased quickly, then all decreased after reaching the maximum absorption peak, and I⁻ had two absorption peaks in serum. The thyroid blocking dose of I⁻ was 0.5 mg/kg in rat. Additionally, high KIO₃ intake did not influence the TAA in serum and other tissues.ConclusionThe body is able to reduce and utilize high doses of KIO₃ ingested through the digestive tract. The metabolism of high KIO₃ in vivo is characterized by two absorption process of I⁻ in serum and the thyroid blocking effect. Moreover, a single intake of high-dose KIO₃ does not affect TAA in vivo. The results suggest that such excess IO₃⁻ may have be reduced in the digestive tract before I⁻ enters the blood.     

Modelling of light-induced chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence rise (O-J-I-P transient) and changes in 820 nm-transmittance signal of photosynthesis

Theoretical modelling is often overlooked in photosynthesis research even if it can significantly help with understanding of explored system. A new model of light-induced photosynthetic reactions occurring in and around thylakoid membrane is introduced here and used for theoretical modelling of not only the light-induced chlorophyll (Chl) a fluorescence rise (FLR; the O-J-I-P transient), reflecting function of photosystem II (PSII), but also of the 820 nmtransmittance signal (I₈₂₀), reflecting function of photosystem I (PSI) and plastocyanin (PC), paralleling the FLR. Correctness of the model was verified by successful simulations of the FLR and I₈₂₀ signal as measured with the control (no treatment) sample but also as measured with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone- (inhibits electron transport in cytochrome b ₆/f) and methylviologen- (accepts electrons from iron-sulphur cluster of PSI) treated samples and with the control sample upon different intensities of excitation light. From the simulations performed for the control sample, contribution of the oxidised donor of PSI, P700, and oxidised PC to the I₈₂₀ signal minimum (reflects maximal accumulations of the two components) was estimated to be 75% and 25%, respectively. Further in silico experiments showed that PC must be reduced in the dark, cyclic electron transport around PSI must be considered in the model and activation of ferredoxin-NADP⁺-oxidoreductase (FNR) also affects the FLR. Correct simulations of the FLR and I₈₂₀ signal demonstrate robustness of the model, confirm that the electron transport reactions occurring beyond PSII affect the shape of the FLR, and show usefulness and perspective of theoretical approach in studying of the light-induced photosynthetic reactions.     

Studies on Effect of Certain Quinones: I. Electron Transport, Photophosphorylation, and CO(2) Fixation in Isolated Chloroplasts

The effect of quinone herbicides and fungicides on photosynthetic reactions in isolated spinach (Spinacia oleracea) chloroplasts was investigated. 2,3-Dichloro-1,4-naphthoquinone (dichlone), 2-amino-3-chloro-1,4-naphthoquinone (06K-quinone), and 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) inhibited ferricyanide reduction as well as ATP formation. Benzoquinone had little or no effect on these reactions. The two reactions showed a differential sensitivity to these inhibitors. Dichlone was a strong inhibitor of both photosystems I and II; photosystem I was more sensitive to 06K-quinone than was photosystem II, whereas the reverse was true of chloranil. Chloranil and 06K-quinone inhibited ferricyanide reduction and the coupled photophosphorylation to the same extent, whereas dichlone affected photophosphorylation to a greater extent than the ferricyanide reduction.     

Low temperature spectral properties of subchloroplast fractions purified from spinach

Spinach (Spinacia oleracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, F_I, F_II, and F_III, corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampholine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at −196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra. The activity of purified F_II (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at −196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms of chlorophyll. The fluorescence emission spectra of F_I, F_II, and F_III at −196 C were also unique with maxima at 734, 685 and 681 nm, respectively. F_I showed negligible emission at wavelengths shorter than 700 nm and the long wavelength tails of F_II and F_III in the 730 nm region were relatively small (approximately 10% of emission of their wavelength maxima). Addition of 0.1% Triton to F_I and F_II caused the longer wavelength absorbing forms of chlorophyll to shift to 670 nm and the fluorescence emission maxima (of both fractions) to shift to 679 nm at −196 C with an increase in the yield of fluorescence especially in the case of F_I.     

Inhibition of chloroplast photochemical reactions with 2-chloromercuri 4,6-dinitrophenol.

2-Chloromercuri 4,6-dinitrophenol inhibited photosystem I mediated photochemical reactions of Euphorbia hirta chloroplasts. The compound inhibited cyclic photophosphorylation and NADP reduction (in the presence of dichlorophenol indophenol and ascorbate couple) at concentrations as low as 10^?6m. At higher concentrations (above 10^?4m), however, it affected all NADP reductions but still showed negligible effect on ferricyanide reduction or noncyclic photophosphorylation. The compound may be used as an inhibitor of cyclic photophosphorylation.     

Photosynthetic control, “energy-dependent” quenching of chlorophyll fluorescence and photophosphorylation under influence of tertiary amines

The effects of the tertiary amines tetracaine, brucine and dibucaine on photophosphorylation and control of photosynthetic electron transport in isolated chloroplasts of Spinacia oleracea were investigated. Tertiary amines inhibited photophosphorylation while the related electron transport decreased to the rates, observed under non-phosphorylating conditions. Light induced quenching of 9-aminoacridine fluorescence and uptake of ¹⁴C-labelled methylamine in the thylakoid lumen declined in parallel with photophosphorylation, indicating a decline of the transthylakoid proton gradient. In the presence of ionophoric uncouplers such as nigericin, no effect of tertiary amines on electron transport was seen in a range of concentration where photophosphorylation was inhibited. Under the influence of the tertiary amines tested, pH-dependent feed-back control of photosystem II, as indicated by energy-dependent quenching of chlorophyll fluorescence, was unaffected or even increased in a range of concentration where 9-aminoacridine fluorescence quenching and photophosphorylation were inhibited. The data are discussed with respect to a possible involvement of localized proton flow pathways in energy coupling and feed-back control of electron transport.Abbreviations 9-AA 9-aminoacridine - J _e flux of photosynthetic electron transport - PC photosynthetic control - pH₁ H⁺ concentration in the thylakoid lumen - pmf proton motive force - _P potential quantum yield of photochemistry of photosystem II (with open reaction centers) - Q _A primary quinone-type electron acceptor of photosystem II - q _Q photochemical quenching of chlorophyll fluorescence - q _E energy-dependent quenching of chlorophyll fluorescence - q _AA light-induced quenching of 9-amino-acridine fluorescence     

p-nitroacetophenoxime N-methylcarbamate,a new electron acceptor in isolated thylakoids

p-Nitroacetophenoxime N-methylcarbamate (MCPNA) is a rather potent inhibitor of the electron transfer in spinach class A chloroplasts. In isolated thylakoids, MCPNA is an electron acceptor at the level of photosystem I (PS I). It inhibits O₂ evolution in the presence of NADP and ferredoxin but not the reduction of ferricyanide. MCPNA is active as an acceptor between 3 μM and 100 μM. At concentrations higher than 300 μM, inhibition of photosystem II (PS II) occurs. MCPNA has no uncoupling effect on photophosphorylation. Reduction of MCPNA by thylakoids in the presence of light is in accordance with the E′_o of this compound (??0.57 V) and is followed by an electron transfer to O₂. This reaction probably explains the inhibitory effect of MCPNA on class A chloroplasts.     

Photoproduction of hydrogen by photosystem I of Scenedesmus

Summary Anaerobically adapted and illuminated Scenedesmus evolves molecular hydrogen from endogenous organic compounds. This photoproduction of H₂ does not require photosystem II, since 5x10^-6 M DCMU, which inhibited normal photosynthesis almost completely, did not significantly inhibit the photoevolution of H₂. The relative efficiencies in far-red light of photosynthesis, photoreduction and H₂ production were determined. Photohydrogen evolution was comparatively the most efficient of these three processes. Three mutants of Scenedesmus (isolated and characterized by Dr. N. I. Bishop) were also tested. Mutant PS-50, which lacks cytochrome 552, did not photoproduce H₂. Mutant No. 11, blocked in photosystem II, showed rates of H₂ production comparable to those of the wild type. Cl-CCP, an uncoupler of photophosphorylation, caused an apparent stimulation of H₂ production by mutant No. 11 and wild-type cells. Mutant No. 8, which is partially blocked in photosystem I, showed a diminished photohydrogen production which was inhibited by Cl-CCP. These results suggest that photoproduction of hydrogen by photosystem I is due either to cyclic photophosphorylation, which supplies energy needed for a dark, H₂-yielding reaction, or to a more direct photooxidation of organic compounds by the photosynthetic electron transfer chain.The following abbreviations were used: Cl-CCP=carbonyl cyanide m-chlorophenylhydrazone; DCMU=3-(3,4-dichlorophenyl)-1,1-dimethylurea.This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission to Professor H. Gaffron.     

Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L

High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of ³²P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.

A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively.

     

Rates and mechanisms of the reactions of octacyanomolybdate(V) anion with L-cysteine,penicillamine and thioglycolic acid in aqueous acidic solutions

The rates and mechanisms of the reactions of octacyanomolybdate(V) anion with L-cysteine, penicillamine and thioglycolic acid have been studied in aqueous acidic solution and constant ionic strength I = 1.00 mol dm⁻³ (NaClO₄) for cysteine and I = 0.10 mol dm⁻³ (NaClO₄) for penicillamine and thioglycolic acid respectively. The reactions show a second order substrate dependence and the rates are found to decrease with increasing hydrogen ion concentration [H⁺]. This is attributed to the deprotonation of the −SH and −CO₂H groups in these thiols prior to electron-transfer. No evidence was found for the formation of an intermediate complex of significant stability. The reactions are interpreted in terms of outer-sphere mechanism.     

Photophosphorylation Associated with Photosystem II: II. Effects of Electron Donors, Catalyst Oxidation, and Electron Transport Inhibitors on Photosystem II Cyclic Photophosphorylation

Incubation of KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts with p-phenylenediamine for 10 minutes in the dark prior to illumination produced rates of photosystem II cyclic photophosphorylation up to 2-fold greater than the rates obtained without incubation. Partial oxidation of p-phenylenediaine with ferricyanide produced a similar stimulation of ATP synthesis; addition of dithiothreitol suppressed the stimulation observed with incubation. Addition of ferricyanide in amounts sufficient to oxidize completely p-phenylenediamine failed to inhibit completely photosystem II cyclic activity. This is due at least in part to the fact that the ferrocyanide produced by oxidation of p-phenylenediamine is itself a catalyst of photosystem II cyclic photophosphorylation. N,N,N′N′-Tetramethyl-p-phenylenediamine catalyzes photosystem II cyclic photophosphorylation at rates approaching those observed with p-phenylenediamine. The activities of both proton/electron and electron donor catalysts of the photosystem II cycle are inhibited by dibromothyoquinone and antimycin A. These findings are interpreted to indicate that photosystem II cyclic photophosphorylation requires the operation of endogenous membrane-bound electron carriers for optimal coupling of ATP synthesis to electron transport.