PHYTOCHROME INTERMEDIATES IN VIVO—IV. KINETICS OF Pfr EMERGENCE*

Abstract— Rate constants for the formation of P_fr, the far-red-absorbing form of phytochrome, from photochemical intermediates, have been measured in vivo in a number of plants. Two first order processes were observed, the slowest of which has been studied in detail. In pea epicotyl hooks, the rate constants proved dependent on temperature as well as on composition of the gas phase. A transition point was observed at about—2°C. Above this temperature the Arrhenius activation energy is 61 ± 6 kJ/mol, below the transition temperature 200 + 17 kJ/mol. The data correspond to half lives of between 0.4 and 0.2 s in an atmosphere of oxygen at 20°C, depending upon the species of plant. In oxygen-free atmospheres, the rates are between 1.7 and 1.9 times as fast. The data prove that, on the time scale relevant to the usual type of photomorphogenetic experiments, the occurrence of phytochrome intermediates generally can be neglected. Exceptions to this generality are formed by experiments involving continuous or very rapidly intermittent exposures to very high light intensities. Under these conditions the steady state fraction of P_fr may be considerably lower than calculated from the spectroscopic data for the pigment and the light sources.     

124-kDa PHYTOCHROME IN MODEL MEMBRANE SYSTEMS: STUDIES OF THE Ii700 INTERMEDIATES WITH THE PROTEIN COVALENTLY BOUND TO PREFORMED LIPOSOMES

Abstract— We have described the covalent binding of 124-kDa oat phytochrome to large unilamellar liposomes composed of either dioleoyl phosphatidylcholine or dipalmkoyl phosphatidylcholine or soybean lecithin, without affecting the photochromic properties of the protein. These phytochrome-liposome systems have now been studied by laser flash photolysis. The liposomes, independent of their membrane rigidity (liquid-crystal vs gel-like phase), do not influence the ratio and reactivity of the two primary photoproducts, Iⁱ₇₀₀- of the red absorbing form of phytochrome, P_l Thus, the lifetimes of the Iⁱ₇₀₀ intermediates and the activation parameters associated with Iⁱ₇₀₀Iⁱ_bl are the same as those measured for nonbound phytochrome in buffer solution. The temperature increase from about 273 K. to 297 K lowers the population of the shorter-lived Iⁱ₇₀₀ intermediate to the same extent both in the liposome-P_l and in nonbound P_l, whereas it does not affect the relative population of the Iⁱ₇₀₀ intermediates from non-bound P_l in the presence of 25% ethylene glycol added to the buffer solution (ionic strength 0.17).     

INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris-I DEMONSTRATION AND QUANTITATIVE CHARACTERIZATION OF THE PHOTOCHROMIC SYSTEM Pr⇔ Ii700USING NANOSECOND-LASER PULSES*,†

Abstract— Spores of Dryopteris paleacea and D. filix-mas are positively photoblastic with an optimum in the action spectrum around 665 nm. Light is perceived by phytochrome and the relationship between germination and mole fraction of the far-red-absorbing form of this pigment, P_fr, was investigated with saturating irradiations between 662 and 747 nm under low-fluence-rate conditions. These control irradiations establish a proportion of the total phytochrome, P,_tot, as P_fr with P_fr/P_tot–φ at equilibrium. These φ -values were calculated according to data for native oat phytochrome (Kelly and Lagarias, 1985, Biochemistry 24, 6003) and the spectral characteristics of the interference filters. With this method a linear relationship could be found between φ and germination from 2 to 70% for D. paleacea and from 2 to 90% for D. filix-mas, if probit germination was plotted vs probit φ This correlation formed the basis of investigating the phytochrome photoconversion by dye-laser pulses of 380 ± 30 ns under high-fluence-rate conditions, and thus to test quantitatively the impact of the photoreversibility of intermediate reactions of the photoconversion and the red-absorbing form of phytochrome, P_fr on the final P_fr-level. Spore germination was initiated by a single-laser pulse in the range from 592 to 700 nm. The most effective wavelengths were 649 and 660 nm in both species, and at saturation maximal germination (ca. 50%) was obtained from 592 to 665 nm for D. paleacea or ca. 60% germination from 592 to 670 nm for D. filix-mas. Both saturation levels correspond to a ø-value between 0.40 and 0.45. This significantly diminished photoconversion is a consequence of the high-fluence-rate conditions during the laser pulse which establishes the photochromic system between P_r and a set of very early intermediates, Iⁱ_700, (= P_r? Iⁱ₇₀₀). This system can be described by the extinction coefficients of P_r and the intermediates Iⁱ₇₀₀, and by the quantum yields, 4,φ for the forward and reverse reactions as φ If φ is calculated, assuming a quantum yield of 1:1 for both reactions and with the extinction coefficients of P_r and Iⁱ_7(l() (= lumi-R) given by Eilfeld and Riidiger (1985, Z. Naturforsch. 40c , 109), significantly higher values are calculated for / as compared to φ found in the control experiments. These results can be explained either: (i) with a quantum yield ratio φ_pr-φ1700: φ_1700φpr=1:1 and an assumed additional dark reaction leading from Iⁱ₇₀₀ or later intermediates back to P_r: or (ii) with a quantum yield ratio φ_{pr φ 1700}: φ_{1700 φpr}=1:2. In this case all Iⁱ₇₀₀ have to relax to P_fr. In this case all Iⁱ₇₀₀ have to relax to P_fr.     

EVIDENCE FOR A SEQUENTIAL PATHWAY FROM Pr TO Pfr OF THE PHOTOTRANSFORMATION OF 124-kDa OAT PHYTOCHROME

Abstract. Phototransformation kinetics of 124-kDa oat phytochrome at 298 K after a red (660-nm) laser flash excitation were recorded at different wavelengths. The kinetics of the dark relaxation processes for lumi-R to P_fr can be satisfactorily described by only 3 rate constants: k = 28000 s^-1 370 s^-1 and 20 s^-1. The first rate constant is due to the decay of lumi-R to meta -Ra. The latter two rate constants correspond to processes establishing the far-red (>700 nm) absorption band. No meta -Rb could be detected. From the wavelength dependency of the amplitudes of these two rates, parallel pathways in the formation of P_fr could be excluded. A unique sequential pathway for the dark relaxation leading to P_fr seems to be an intrinsic property of 124-kDa phytochrome, however. Assuming a sequential pathway, molar extinction coefficients for intermediates have been calculated. These values agree with molar extinction coefficients obtained from low-temperature spectra. The process with a rate constant of 370 s^-1 corresponds to absorbance changes for the formation of meta -Rc from meta -Ra and the rate constant of 20 s^-1 describes the absorbance changes due to the transformation of meta -Rc to P_fr.     

LIGHT-INDUCED FORMATION OF O-2˙ OXYGEN RADICALS IN SYSTEMS CONTAINING CHLOROPHYLL

It has been shown recently that photosystem 1 particles, photosystem 1 lipid vesicles and chlorophyll-a lipid vesicles show identical photochemical reactions in the presence of oxygen e.g. H⁺-and O₂-uptake (Van Ginkel, 1979). Therefore, spin-trapping experiments were done to identify the oxygen radicals formed. The spintrap phenyltertiarybutylnitrone (PBN) failed to yield information about oxygen radicals. With the spintrap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), however, we obtained a mixed spectrum of O^-_2˙ and OH·-adducts generated in chloroplasts, photosystem 1 particles or chlorophyll-a lipid vesicles. These data indicate that chlorophyll-a in an artificial membrane can also catalyze O^-_2˙-formation. Chlorophyll-a lipid vesicles catalyze light-induced formation of the Tiron-semiquinone free radical, which has been proposed as a specific O^-_2˙-probe (Greenstock and Miller, 1975). However, OH· scavengers strongly reduce the formation of this radical, whereas superoxide dismutase does not. Pulse-radiolysis measurements showed that the rate constant for the reaction of Tiron with OH· is 8.2 · 10⁹M^-1 s^-1, which is considerably higher than the published Tiron/O^-_2˙ rate constants. Therefore, Tiron is a better spin probe for OH· than for O^-_2˙. We suggest that light-induced H⁺-and O^-_2˙-uptake in membranes containing chlorophyll-a in the presence of ascorbate is caused mainly by the very rapid reaction of OH· with ascorbate.     

A PHYTOCHROME PHOTOTRANSFORMATION STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: ANALYSIS OF THE DECAY MECHANISM OF I700*

The mechanism of I₇₀₀ decay, representing an early event in the phytochrome P_r→ P_fr phototransformation, was reanalyzed in the microsecond range by conventional laser flash photolysis as well as by two-laser/two-color flash photolysis. Three kinetic models that might describe the I₇₀₀ decay mechanism following P_r excitation were considered: a parallel, a sequential, and an equilibrium model. These models were used to mathematically simulate both the one- and two-laser flash experiments in an effort to select the model best describing the I₇₀₀ decay. The sequential model could be excluded already on the basis of the one-laser flash photolysis results alone. Discussion of the two-laser/two-color flash rcsults in the context of the equilibrium and the parallel models is presented.     

ON THE FORMATION AND REACTIVITY OF DIOXIRANE INTERMEDIATES IN THE REACTION OF PEROXOANIONS WITH ORGANIC SUBSTRATES*

Abstract— Kinetics and ¹⁸O-labeling studies have provided evidence for the involvement of dioxirane intermediates (V) in the ketone-catalysed decomposition of peroxomonosulfate (Caroate) HSO;. Reaction rates depend on ketone structure. In competition with catalysis of peroxide decomposition, the dioxirane intermediate is capable of oxidizing several organic and inorganic substrates. Thus, cis- and rrans-cinnammic acids could be converted stereospecifically into the corresponding epoxides. Also, oxidation of phenylpropiolic acid, a substrate which is representative of weakly nucleophilic alkynes, could be carried out using the Caroatehetone oxidizing system. Under the conditions adopted, no oxidation of the substrates examined was found to occur in the absence of ketone. The possibility that formation of dioxathiirane intermediates (XXII) occurs following a side pathway in the reaction of Caroate with sulfoxides (which produces sulfones in high yield) has been explored. Preliminary experiments using ¹⁸O-labeling of p-tolyl phenylsulfoxide, however. failed to support this hypothesis. pointing out the need for further detailed studies.     

A PHYTOCHROME STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: I700 IS A MANDATORY INTERMEDIATE IN THE PrPfr PHOTOTRANSFORMATION

Abstract— The phototransformation of native (124 kDa)oat phytochrome, P_r P_fr, Has been studied at 10C by two laser/ two-color flash photolysis. the overall P_rP_fr reaction yield did not vary with temperature within the range4–21C. Foloeing the excitation of P_r with a single 15 ns laser flash at 650nm, the formation of P_fr was quantitavely measured in a time-resolved experiment in the presence of a second 8 ns laser flash at 710 nm delayed from the initial flash. the second laser flash causes at 1.0 s after the initial laser flash a depletion of the uintermediate I₇₀₀ as welll as a reduction of the P_fr absorption at 730 nm. The depletion of I₇₀₀ correlates quantitavely with the reduction of P_fr formation. The absorpton spectra of I₇₀₀ and of the following intermendiate, I_bi, were calculated assuming that the amount of P_r, which is photoconverted by a single laser, equals the amount of P_fr formed.     

GENERATION AND PARTICIPATION OF O-2 IN 2-NITROPROPANE DIOXYGENASE REACTION

Abstract— 2-Nitropropane dioxygenase (EC 1. 13. 11) of the yeast Hansenula mrakii catalyzes the oxygenative denitrification of 2-nitropropane as follows:

The enzyme is significantly inhibited by superoxide dismutase and various scavengers for superoxide such as cytochrome c , epinephrine, thiols and polyhydric phenols. The scavengers added to the reaction mixture were oxidized or reduced. The addition of superoxide dismutase and the omission of 2-nitropropane or oxygen prevented the oxidation and the reduction of the scavengers. The enzyme catalyzes the formation of nitrite from 2-nitropropane by KO₂ added anaerobically.
One mole of NADH is bound per mole of the enzyme and predominantly the pro-R hydrogen of bound NADH is transferred to superoxide formed enzymatically or provided externally. The enzyme shows incomplete stereospecificity for hydrogen transfer from NADH.     

DETERMINATION OF THE BINDING CONSTANT OF H14 CO−3 TO THE PHOTOSYSTEM II COMPLEX IN MAIZE CHLOROPLASTS: EFFECTS OF INHIBITORS AND LIGHT

The binding (dissociation) constant for HCO^?₃ to the photosystem II complex in maize chloroplasts is approximately 80 μM. One HCO^?₃ binds per 500–600 chlorophyll molecules. In the dark, formate is a competitive inhibitor of HCO^?₃ binding, while 3-(3′,4′-dichlorophenyl)-1, 1-dimethylurea (DCMU) inhibits HCO^?₃ binding non-competitively. Light decreases HCO^?₃ binding in the presence of formate. Light increases the binding of HCO^?₃ in the presence of DCMU. The high binding constant for HCO, discriminates strongly among the various hypotheses attempting to explain the “bicarbonate-effect” on photosystem II. The proposal by Stemler and Jursinic (Arch. Biochem. Biophys. 221, 227–237 1983), that HCO^?₃ is one of a class of monovalent anionic inhibitors of photosystem II, is favored. These anions compete for a specific binding site on the photosystem II complex.     

EVIDENCE FOR THE FORMATION OF A CHLOROPHYLL a/ZEAXANTHIN COMPLEX IN LECITHIN LIPOSOMES FROM FLUORESCENCE DECAY KINETICS

The interaction of Chi a with zeaxanthin (Zea), which is an analogue of lutein, has been studied in soya bean lecithin liposomes using the fluorescence of Chi as monitor. The fluorescence emission spectrum at 4.2 K of Chi a showed characteristic changes in the presence of Zea: the emission maximum shifted from 688 nm to 680 nm, and a peak at 731 nm appeared. The fluorescence decay kinetics of Chi a alone could be described by the sum of two exponential components (T₁,≅0.8 ns, T₂≅2.5 ns). In the presence of Zea a component with a long lifetime, T≅5 ns, appeared with a large relative amplitude (40%). This indicated the formation of a Chl a /Zea complex, in which Chl a /Chl a interaction is negligible, presumably because of strong interaction between Chl a and Zea. The fluorescence anisotropy decay kinetics supported the hypothesis of the formation of a large Chl a containing complex in the presence of Zea. A rotational correlation time, φ≅14 ns at 4°C and φ≅21 ns at 30°C, was found, which is distinctly larger than for samples containing Chl a only. We interpret these results as further evidence for a strong interaction between Chl a and Zea in the hydrophobic environment of the lecithin liposomes. This interaction may also occur in the Chl-proteins of the Chi alb light-harvesting complex of plant photosynthesis.     

THE INWARD H+ PATHWAY IN BACTERIORHODOPSIN: THE ROLE OF M412 AND P(N)560 INTERMEDIATES*

Abstract— In purple bacteriorhodopsin sheets adsorbed onto the phospholipid-impregnated collodion film, electrogenic stages are identified correlating with decays of the M and N(P)-type intermediates. It is concluded that both M → N and N → bR transitions are electrogenic.
The M decay is shown to be of a complex kinetics. In purple sheets, the lower the light intensity, the higher the rate of "slow M" decay. Such a dependence, which is absent from monomeric bacteriorhodopsin in proteoliposomes and from Triton X-100-solubilized protein, may be explained by the inhibiting effect of a light-induced conformation change in a bacteriorhodopsin molecule upon the M decay in some other bacteriorhodopsin molecules within the same sheet.
The light intensity-independent "slow M" decay in solubilized bacteriorhodopsin is shown to correlate with the decay of the N intermediate and H⁺ uptake after the flash. In contrast to "fast M", "slow M" is pH dependent, closely resembling in this respect the N intermediate. It is suggested that there is a fast light-independent equilibration between M and N so that "slow M" represents the portion of the M pool that monitors the N concentration. The M → N equilibrium is assumed to be involved in the effect of the light-induced electric field on the M decay. No direct effect of light on the equilibrium was found.     

THE ROLE OF O2- IN THE CHEMILUMINESCENCE OF LUMINOL*

Abstract— The chemiluminescence of luminol in buffered aqueous solutions is inhibited by superoxide dismutase. This occurs whether the luminescence is induced by ferricyanide, persulfate, hypochlorite, or by the action of xanthine oxidase on xanthine. Since superoxide dismutase inhibits reactions which involve O₂^-, we conclude that this radical is a constant factor in the chemiluminescence of luminol in aqueous solutions. The kinetics of light production are discussed in terms of hypothetical mechanisms that fit the available data. The strong luminescence of luminol in aprotic solvents or in aqueous systems containing relatively high concentrations of H₂O₂ could not be explored in this way, because superoxide dismutase is inactive under such conditions.     

A CORRELATION OF EPR SPINS WITH P700 IN SPINACH SUBCHLOROPLAST PARTICLES

Abstract— The ratio of the concentrations of P₇₀₀, as measured by oxidized-minus-reduced (also light-minus-dark) spectroscopy, and electron paramagnetic resonance (EPR) signal I, as measured by EPR spectroscopy, were determined on aliquots of photosystem I spinach subchloroplast particles. The results substantiated a 1:1 ratio with a precision of ±25%. This constitutes correlative evidence that P₇₀₀ is the molecular source of the EPR signal I.     

DIFFERENCE SPECTRA OF THE OXIDIZED INTERMEDIATES IN THE PHOTOSYNTHETIC OXYGEN-EVOLVING SYSTEM: EVIDENCE FOR A SMALL S0→S1 ABSORPTION CHANGE

A consensus has emerged in the recent literature on the fact that the UV difference spectrum of the first oxidation step (S₀→ S₁) of the photosynthetic oxygen-evolving complex is significantly different and generally smaller than the spectra of the higher oxidation steps (S₀→ S₁and S₂→ S₃). Discrepancies still persist, however, notably in the 300 nm region where the S₀→ S₁ change was either reported to be markedly smaller than the other changes, or, at variance, to have a similar amplitude. A novel approach is proposed here for estimating the ratio of these changes, requiring no estimate of the Kok model parameters, such as the initial S₀/S₁ratio, or damping coefficients. This was achieved by comparing the absorption difference between two fully deactivated states, differing only in their S₀/S₁, distribution, with the flash-induced changes measured from these states. The results show that, at two wavelengths around 300 nm, the S₀→ S₁ change is at least 4 times, and probably 5–6 times smaller than the S₀→ S₁change.     

原位担载Fe2S3催化剂煤的热解动力学研究

用加压热天平研究了原位担载纳米级Fe2S3催化剂的大柳塔次烟煤的加氢热解动力学,考查了2.0MPa氢气氛或氮气氛下原煤及担载催化剂的煤热解失重过程,计算了活化能E和指前因子A等热解动力学参数。结果表明,煤原位担载Fe2S3催化剂后热失重速率比原煤有较大程度的增加,最大热解速率的特征温度也比原煤降低。特别是煤在表面性剂十六烷基三甲基溴化铵溶液中担载Fe2S3催化剂后的热解反应速率高于水溶液中担载同样催化剂的热解反应速度。原位担载Fe2S3的煤及原煤的热解反应过程符合一级反应动力学。     

STUDIES ON THE KINETICS OF PROPYLENE POLYMERIZATION WITH THE COMPLEXTYPE TiCl_3 CATALYST

The active center concentration C_p, the rate constant k_p, and the activation energy of chain propagation E_p in the polymerization of propylene with complex-type TiCl_3-(C_2H_5)_2AlCl catalyst system were studied. The Mn was corrected by (?) value determined by GPC. The values thus obtained for C_p, k_p, and E_p at 50℃were 3.01 mol/mol Ti, 6.27 1/mol·sec, and 5.10 Kcal/mol respectively.The kinetic parameters were compared with those obtained from conventional TiCl_3·AlCl_2 catalyst, showing that the higher activity of the complex-type catalyst over the conventional catalyst is not only due to the higher C_p of the former, but to a greater extent due to the increase of the k_p value.     

PHOTOPOLYMERIZATION IN THE MICELLAR SYSTEMSⅡ THE CATALYSIS OF PKT FUNCTIONAL MICELLE*

A kind of cationic compounds, having benzophenone end group and various length chain (PKT) (BP-CH_2N+R_2R'·X~-, R&R' different chain iength alkyl group) were used as photosensitizers. Various BP/TEA systems have been used for study, The efficiency of MMA photopolymerization initiated by them shows PKT>BP in homogeneous water solution and PKT> BP/CTAB in micellar water solution. The results obtained indicate that catalytic effects of PKT type functional micelles are far greater than that of common micelle with the enhancement of polymerization rate over 10 times compared with BP in water solution. The catalytic role, reaction character of PKT, effect of counter ions and retarding effect of oxygen have also been discussed.