ELECTROPHORETIC AND SPECTROPHOTOMETRIC STUDIES OF CHLOROPHYLL-PROTEIN COMPLEXES FROM TOBACCO CHLOROPLASTS. ISOLATION OF A LIGHT HARVESTING PIGMENT PROTEIN COMPLEX WITH A MOLECULAR WEIGHT OF 70,000

Abstract— …After a short-term solubilization with sodium dodecyl sulphate, chloroplast membranes of tobacco were separated by polyacrylamide gel electrophoresis into three chlorophyll-protein complexes. In addition to the two major complexes termed I and II_c corresponding respectively to P700 chlorophyll a -protein and light-harvesting chlorophyll a/b -protein described by Thornber (1975), a relatively stable complex termed II_a has been observed. This new complex has an apparent molecular weight of 70,000 daltons and possesses Chl a and b.
Complexes I, II_a and II_C have been isolated and precise spectroscopic analyses have been performed. Fourth derivative analyses of low temperature absorption spectra suggest that complex II_a seems more representative than II_C of chlorophyll a forms present in intact thylakoid membranes.
Moreover, the electrophoretic study reveals that CP_I and CP_II are composed of only one polypeptidic subunit with respective molecular weights of 68,000 and 24,000 daltons.     

THE REACTION OF METHYLENE WITH HYDROGEN

Abstract— An investigation has been made of the reaction between methylene, formed by the photolysis of ketene, and hydrogen. Ethane, ethylene and methane are the major hydrocarbon products, and it has been shown that the formation of these products may be adequately described by the sequence of processes
CH₂CO + hv → CH₂+ CO (1)
CH₂+ H₂→ CH₃+H (2)
2CH₃→ C₂H₆ (3)
CH₃+ H₂+ CH₄+ H (4)
CH₂+ CH₂CO → C₂H₄+ CO (7)
In particular, the relative rates of ethane and methane formation are consistent with the known rate constants for reactions (3) and (4), and it is not therefore necessary to postulate the participation of an 'insertion' process
CH₂+ H₂→ CH₄ (6) to account for the formation of methane.
Decrease of the energy possessed by the methylene, either by increase of the wavelength of ketene photolysis, or by increase of gas pressure, is shown to result in an increase in the reactivity of the methylene towards ketene relative to its reactivity towards hydrogen (i.e. the ratio k₂/k₂ increases).     

EFFECTS OF TRYPTOPHAN OXIDATION ON BACTERIORHODOPSIN STRUCTURE AND FUNCTION

Abstract— N -bromosuccinimide at low molar ratios specifically oxidizes tryptophan residues in purple membranes (bR). Loss of 1 mol tryptophan per mol bR (which corresponds to oxidation of Trp₁₀ or Trp₁₂ or part of both) produces slight changes in the absorption and CD spectra and in the photobleaching kinetics. The efficiency of reconstituting the retinylidene protein after bleaching and heptane extraction of these membranes was comparable to control values(–80%). Photocycling yield (M₄₁₂) with 265 nm or 530 nm excitation (15 ns pulse) was only slightly decreased. Another mol of tryptophan was reacted at higher NBS:bR molar ratio (10:1). Partial oxidation of several residues rather than titration of a specific residue occurred: HPLC indicated no reaction with the chromophore. A considerable loss of extinction at 570 nm including enhanced red and blue shifts of LD_max at low and high pH respectively, was observed. Also the photobleaching rate was faster and functional retinylidene membranes could not be reconstituted from heptane-extracted, apomembranes. Exogenous retinal could still locate the attachment site, based on formation of the fluorescent NaBH₄-reduced retinoyl adduct. Perturbation in the near UV and visible CD infer changes in helical conformation, trimer dissociation and decreased asymmetry of the chromophore. Photocycling efficiency was greatly decreased. The relative decrease was greater for 265 nm rather than 530 nm excitation. These results are consistent with co-operative destabilization of the protein conformation by oxidized tryptophan residues, which leads to a decrease in the hydrophobicity of chromophoric site.     

ABSORPTION OF METHYLBACTERIOPHEOPHORBIDE a SINGLE CRYSTALS: SPECTRAL SHIFTS DUE TO π-π INTERACTIONS

Abstract— Polarized absorption spectra were obtained for a single crystal of methylbacteriophorbide a (MeBPhide a). The Q_y band is red-shifted ∽ 1660 cm^-1 (∽ 110 nm) relative to MeBPhide a in a CH₂Cl₂/benzene solution. This is equivalent to the largest red shifts observed for in vivo bacteriochlorophyll a. The Soret band exhibits a smaller red shift and a significant reduction in intensity, and the Q_x band is not observed. The crystal spectra are qualitatively similar to spectra reported for several other aggregated (bacterio)chlorophyll and bacteriopheophytin systems. Since crystalline MeBPhide a contains no Mg, water or hydrogen bonding (Barkigia etal. , 1981), these results demonstrate that the spectral changes associated with the aggregation of photosynthetic chromophores can arise solely from IT-IT interactions between macrocycles.     

ESTIMATION OF ENERGY DISTRIBUTION AND REDISTRIBUTION AMONG TWO PHOTOSYSTEMS USING PARALLEL MEASUREMENTS OF FLUORESCENCE LIFETIMES AND TRANSIENTS AT 77 K

Abstract— A single-sample method for estimating energy distribution and redistribution among the two photosystems using fluorescence lifetimes and transients at 77 K is presented. In this method,α(the fraction of photons absorbed by photosystem I, PSI) is F_1(α)/(F_1(α)+ (τ_{F 1(M)}/τ_{F 2(M)}).F_2(M)) where, F_1(α) is the fluorescence intensity from PSI excited by photons initially absorbed by the latter, τ_{F 1(M)} and τ_{F 2(M)} are the maximum lifetimes of fluorescence from chlorophyll- a in PSI (1) and II (2), and, F_2(M) is the maximum fluorescence intensity from PSII (P level). Analysis of the intensities and lifetimes of wavelength resolved fluorescence of thylakoids (pH 7.0), with and without cations, leads to the following conclusions: The addition of 10 m M Na⁺ to cation-depleted thylakoids (pH 7.0) increases α by ˜ 10%, while the subsequent addition of 10 m M Mg²⁺ leads to three principal concomitant changes (in the order of importance): a 50% decrease in PSII to PSI energy transfer, a 20% increase in other radiation-less losses, and a 10% decrease in α.     

LASER PHOTOLYSIS OF 3-METHYLLUMIFLAVIN

Abstract— Using high-intensity actinic light, the chlorophyll a fluorescence transient from HCO^-₃-depleted chloroplasts shows a rapid initial rise (O → I) followed by a slow phase (I → P). In the presence of HCO^-₃, the O → I rise is delayed but the I → P phase is much more rapid. Using low-intensity actinic light, the chlorophyll a fluorescence transient from 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU)-treated chloroplasts is delayed in the presence of HCO^-₃. Bicarbonate increases the amount of delayed light emission from chloroplasts given 10 s illumination with weak blue light (0·4 W/m²). DCMU greatly increases the amount of delayed light seen in the presence of HCO^-₃ under these conditions but decreases the amount seen in the absence of HCO^-₃. It is suggested that HCO^-₃ may somehow form or stabilize, in the dark, a number of reaction centers corresponding to the S₁ state in the model of B. Forbush, B. Kok and M. McGloin ( Photochem. Photobiol. 14, 307–321, 1971).     

PRODUCTS AND RELATIVE REACTION RATES OF THE OXIDATION OF TOCOPHEROLS WITH SINGLET MOLECULAR OXYGEN

Abstract— The relative reactivity of singlet molecular oxygen, 0₂(¹Δ_g), α-,β-,Γ-and δ with -tocopherol (vitamin E) was investigated using microwave discharge generation as a uniquely clean source of singlet oxygen and using a hydrocarbon solvent to approximate the membrane environment. The relative efficiencies of the tocopherols for O₂(¹Δg) were found to decrease in the order: D-α-tocopherol > D-β-tocopheroI > D–Γ-tocopherol > D-δ-tocopherol. The reaction products in all cases were found to be mixtures of quinone and quinone epoxides apparently resulting from decomposition of the primary product, the hydroperoxydienone.     

THE EFFECT OF ALIPHATIC ALCOHOLS ON FLUORESCENCE QUENCHING AND FLUORESCENCE POLARIZATION OF LUMINESCENCE PROBES IN HEXADECYLTRIMETHYLAMMONIUM BROMIDE MICELLES

Abstract— The fluorescence quenching of the indole chromophore by NO⁻₂ and the fluorescence depolarization of several luminescence probes in aqueous solutions containing hexadecyltrimethylammonium bromide (HDTBr) were measured as a function of added C₂–C₄ aliphatic alcohol concentration. The fluorescence decay profiles of pyrene in the micellar solutions were also measured to estimate the aggregation number of the micelles. The addition of n -butyl alcohol significantly reduces the fluorescence quenching rate and the aggregation number and increases the extent of fluorescence depolarization in HDTBr micellar systems. The addition of ethyl alcohol shows a similar but smaller effect.     

Synthesis and reactivity of alkynyl niobocene complexes

The synthesis of alkynyl containing niobocene complexes with differing auxiliary ligands is described. The niobium(III) derivatives of the general formula [Nb(η⁵-C₅H₄SiMe₃)₂(CCR)(L)] where L is either carbonyl, phosphine, phosphite, or isocyanide, were prepared by the reaction of the bis(alkynyl)magnesium reagent with the corresponding cloro-niobocene precursor. In a similar manner the niobium(V) imido compounds, of the general formula, [Nb(=NR′)(η⁵-C₅H₄R″)₂(CCR)], were prepared. The characterization of these complexes is discussed. The reactivity of the alkynyl compounds towards oxidation and protonation has been studied.     

ON THE FORMATION OF PHOTOSYNTHETIC MEMBRANES IN BEAN PLANTS*

Abstract— The formation of lamellar chlorophyll-protein complexes I and II, solubilized by sodium dodecyl sulfate, was studied by hydroxylapatite column chromatography during greening of etiolated Phaseohis vulgaris leaves.
The protein moiety of both complexes preexists in the prolamellar body of etiolated tissue. The complex II to complex I protein ratio is of the order of 0.5. During greening in intermittent illumination the 'proto'-chloroplast is agranal, and contains 'primary' thylakoids and chlorophyll a (Chl a ). At this stage the complex II to complex I protein ratio increases only slightly. Further greening of the plant tissue in continuous illumination results in grana, Chi b (chlorophyll b ) and more Chl a formation. The complex II to complex I protein ratio in unfractionated thylakoids is now of the order of 2.5, while in grana it is of the order of 4.0.
The binding of chlorophyll formed during greening to the protein moiety of the two complexes is found to be selective. The Chi a selectively formed under intermittent illumination is more strongly bound to the complex I protein. The Chi b and Chl a formed in continuous illunination are found bound to both complex I and complex II proteins.
Analysis by hydroxylapatite column chromatography of subchloroplast fractions obtained by different fractionation procedures have shown that these two chlorophyll-protein complexes are most probably derived from the PSI (photosystem I) and PSII (photosystem II) particles of the photosynthetic membrane. These findings suggest that PSI units are assembled ahead of PSII units. Moreover, they indicate that the complex I protein is the main protein component in the prolamellar body membranes, the 'primary' thylakoids. and the stroma lamellae, while in the grana membranes the major protein is the complex II protein. Finally our results show that formation of the photosynthetic membranes is a multi-step process.     

Aggregation of the Light-Harvesting Complex in Intact Leaves of Tobacco Plants Stressed by CO2 Deficit

Pronounced aggregation of the photosystem II light-harvesting complex (LHC II) was observed in low-lightgrown tobacco plants stressed with a strong CO₂ deficit for 2–3 days. The LHC II aggregates showed a typical band at 697–700 nm (F₆₉₉) in low-temperature emission spectra. Its excitation spectrum corresponded to that of detergent-solubilized LHC II. Formation of F₆₉₉ in stressed plants was not reversed in the dark and leaves did not contain any zeaxanthin showing that neither a light-induced transthylakoid pH gradient nor zeaxanthin was required for LHC II aggregation. The CO₂-stressed plants showed clear signs of photodamage: depression of the potential yield of photosystem II photochemistry (F,/F_M) by 50–70% and a decline in chlorophyll content by 10–15%. Therefore, we propose that the photodamage to the photosynthetic apparatus is the cause of the LHC II aggregation in plants. The F₆₉₉ exhibited a reversible decrease of its intensity upon irradiation of leaves with intensive light. There was no or only slight decrease around 700 nm in unstressed plants. The nonphotochemical quenching of chlorophyll fluorescence showed the opposite relation, being higher before than after the strong CO₂ deficit. This discrepancy was likely related to the different LHC II aggregation state in control and stressed plants.     

RIBOFLAVIN-PHOTOSENSITIZED OXIDATIVE DEGRADATION OF A VARIETY OF LIGNIN MODEL COMPOUNDS

Oxidation of several lignin model compounds with alkylated paraphenolic groups by photosensitizing riboflavin (RF). rose bengal (RB) and methylene blue (MB) was examined. Photosensitizing RF cleaved l–(3'-4'-diethoxyphenyl)-1.3 dihydroxy-2-(4-methoxyphenyl)propane (I). 4-ethoxy-3-methoxyphenylglycerol-(3-guaiacyl ether (II) and l-(4'-ethoxy-3'-methoxyphenyI)-1,3 dihydroxypropane (IV) at their respective C_α-C_β bonds. Riboflavin also oxidized 3.4-diethoxy-benzaldehyde (VI) to the corresponding acid, and hydroxylated the conjugated olefin l-(4'-ethoxy-3'-methoxyphenyl)1.2 propene (III) to yield the initial product IV. In contrast, MB and RB hydroxylated III but had no effect on I, II, IV or VI under identical conditions. This suggested RF effected transformations via a hydrogen radical abstraction (Type I) rather than a ¹O₂ mediated reaction.
To confirm this, the effects of deuterium oxide (D_:0) and oxygen pressure on the photosensitizing dye reactions were examined. The effect of D₂0 on tryptophan (Trp) oxidation and hydroxylation of III by MB and RB was significant, indicating involvement of ¹O₂. D₂O had no significant effect on cleavage of the diarylpropane (I) and the olefin (III) by RF, indicating these reactions did not proceed by a Type II mechanism. While O₂ pressure effect on Trp oxidation by MB was insignificant, it had a large negative effect on cleavage of I by RF. These results, coupled with the relatively slow oxidation rate of Trp by RF. indicate that photosensitizing RF produces ¹O₂ inefficiently and is not effecting oxidation of these lignin model compounds via a Type II mechanism.     

PHYSICAL AND PHOTOCHEMICAL PROPERTIES OF A FLUORESCENT CHLOROPHYLL COLLOID

Abstract— A fluorescent colloid of chlorophyll a , of which some qualitative properties were noted by Krasnovsky and Brin⁽¹¹⁾, has been quantitatively characterized. The colloid is formed in neutral PO₄ buffer containing 0 1 to 8.0% Tween 20, and is stable in darkness. The extinction coefficient is 7.8 × 10⁴ 1. mole cm^-1 at the red absorption peak (668 mμ), the yield of fluorescence is ˜ 0.25, and the yield of photoautooxidative bleaching is ˜ 2 times 10^-4. The colloid sensitizes the autooxidation of paratoluenediamine with a yield of ˜0.01 to ˜0.3 depending on light intensity and substrate concentration. The yield is independent of detergent and chlorophyll concentrations. In all respects—except the dependence of yield on illumination—the colloid appears to be physically and photochemically equivalent to dissolved chlorophyll, as known in dilute solutions in organic solvents. The light dependence—the yield is inversely proportional to the cube root of absorbed intensity—could be due to a bimolecular back reaction of a chlorophyll or substrate derivative.     

LASER FLASH PHOTOKINETIC STUDIES OF ROSE BENGAL SENSITIZED PHOTODYNAMIC INTERACTIONS OF NUCLEOTIDES AND DNA

Abstract— Laser flash photolysis studies of the production of the triplet state of the xanthene dye, rose bengal (RB), have been carried out. The reactions of this state with oxygen to form singlet oxygen and the superoxide anion radical have been observed and yields measured. Quenching of RB(T₁) by oxygen leads to approximately 75% singlet oxygen and 20% superoxide. The reactivity of these species-RB(T₁), O₂(¹Δ_g) and O₂^-—with four nucleotides and DNA have been determined. Only guanine residues showed any noticeable reaction at neutral pH. At higher pH guanine rate constants increased. The consequences to biological photodynamic processes are discussed.     

1H-INDENYLFURAN AND THIOPHENE DERIVATIVES– A NEW CLASS OF SINGLET OXYGEN SENSITIZERS

Abstract— Photophysical and photochemical properties of 1H-indenylfuran and thiophene derivatives were studied. UV spectra showed that all the compounds tested absorbed UV light in the region 350–380 nm. The fluorescence spectra of 4a-e showed bands in the region 410–470 nm and quantum yields (φ_F) in the range 0.25–0.88. Attempts to calculate the triplet energy of 4a-e failed except for 4c and 4d , which showed E_T= 43–44 kcal mol^-1. These compounds are a new class of singlet oxygen sensitizers. The sensitized reaction of 2,5-dimethylfuran with singlet oxygen was followed showing that 4a and 4e are more reactive than α-terthiophene (4.0 and 2.5 times respectively), while the other compounds show the same reactivity as 1 . This behavior can be explained considering the different intersystem crossing quantum yields of compounds 4a-e . Diazabicyclo[2. 2. 2]octane is a quencher of singlet oxygen in this reaction, while we can exclude superoxide ion formation using the photooxidation of α, α-dimethylstilbene.     

PULSE RADIOLYSIS STUDIES IN MODEL LIPID SYSTEMS: FORMATION AND BEHAVIOR OF PEROXY RADICALS IN FATTY ACIDS

Abstract— Radiolytic formation and peroxidation of fatty acid radicals have been investigated by pulse radiolysis techniques in oleate, linoleate, linolenate and arachidonate systems. A strong absorption band at 280 nm associated with conjugated radicals, R_conj, formed in polyunsaturated fatty acid moieties has been used as a probe for kinetic processes occurring at doubly allylic sites in the hydrocarbon chain. Formation of R_conj by O^- has been found to be more efficient than the less selective OH radical. Peroxidation of R_conj is shown to be somewhat slower, ( k _R+ O₂˜ 3 × 10⁸ M ^-1 s^-1), than O₂ reactions with radicals in oleate ( k _R+ O₂= 1 × 10⁹ M ^-1 s^-1). Peroxy radicals generated in these reactions disappear slowly by essentially second order processes (2 k RO₁˜ 10⁷ M ^-1 s^-1). The superoxide radical, O^-₂, shows little if any reactivity towards 0.01 M linolenate or arachidonate over periods of 20 s.     

HEMATOPORPHYRIN PHOTOSENSITIZATION OF SERUM ALBUMIN and SUBTILISIN BPN'

—The photosensitized inactivation of subtilisin BPN' by free hematoporphyrin (HP) followed exponential kinetics with positive mechanistic tests for the involvement of singlet oxygen (¹O₂) as the principal intermediate. The photoinactivation quantum yield was 0.029 at 390 nm in oxygen-saturated, D₂O buffer at pH 7.0. The effects of HP binding were investigated for tryptophan oxidation in bovine serum albumin (BSA) and human serum albumin (HSA) at high protein:HP concentration ratios where the HP was > 97% complexed. The reaction kinetics were non-exponential and mimick a second-order process in the initial stages. The rate of HP photobleaching was 30-fold faster for complexed HP compared with free HP, which was shown to account for the observed kinetics. Mechanistic tests showed that ¹O₂ was the dominant photooxidizing intermediate of tryptophan residues and that it was not involved in the accompanying photobleaching of HP. The quantum yield for tryptophan oxidation in BSA was 0.11 at 390 nm in oxygen-saturated, D₂O buffer at pH 8.0. The reactivity of HSA was approximately 2-fold lower than BSA for equivalent conditions. Estimates of the reaction cross sections led to 3 Ų for the inactivation of subtilisin BPN' by ¹O₂ and 20 Ų for the oxidation of tryptophan in BSA.     

ON THE MECHANISM OF QUENCHING OF SINGLET OXYGEN IN SOLUTION

Abstract— Bimolecular rate constants for the quenching of singlet oxygen O*₂(¹Δ_g), have been obtained for several transition-metal complexes and for β-carotene. Laser photolysis experiments of aerated solutions, in which triplet anthracene is produced and quenched by oxygen, yielding singlet oxygen which then sensitizes absorption due to triplet carotene, firmly establishes diffusion-controlled energy transfer from singlet oxygen as the quenching mechanism in the case of β-carotene. The efficient quenching of singlet oxygen by two trans-planar Schiff-base Ni(II) complexes, which have low-lying triplet ligand-field states, most probably also occurs as a result of electronic energy transfer, since an analogous Pd(II) complex and ferrocene, which both have lowest-lying triplet states at higher energies than the O*₂(¹Δ_g), state, quench much less effectively.     

ELECTRIC LINEAR DICHROISM OF P700 CHLOROPHYLL U-PROTEIN COMPLEXES

Abstract— The P₇₀₀ chlorophyll a -protein complex (CPI) isolated from green plants was oriented in aqueous solutions using pulsed electric fields of up to 6700 V cm^-1. The electric linear dichroism spectrum is reported in the range of 400–720nm. Positive peaks in the linear dichroism Δ A = A _I - A ₁ (where A_I and A₁ are the absorbance components in which the polarizer orientation is parallel and perpendicular with respect to the electric field. respectively) are observed at 443 and 686 nm. The ΔA signal at 686 nm is discussed in terms of either a specialized chlorophyll form absorbing at 686 nm. or due to an exciton component absorbing at the same wavelength.     

ON THE GENERATION OF THE HYDROXYLATION AGENT FROM SUPEROXIDE RADICAL. CAN THE HABER–WEISS REACTION BE THE SOURCE OF OH RADICALS?

Abstract— In many biological systems, the role of O₂^- in hydroxylation and toxic processes was assumed to be due to the formation of OH radicals. The Haber-Weiss reaction (Haber and Weiss, 1934)—(H₂O₂+ O₂^-→ OH + OH^-+ O₂) was suggested as the origin of this activity.
In this study it is shown that this reaction pathway is too slow, and that OH is probably formed from the reaction of complexed superoxide with H₂O₂ or/and from the reduction of Fe(III), bound to biological compounds, by O₂^-; the reduced Fe(II) can then react with H₂O₂ as a Fenton reagent, to yield OH.
It is also shown that singlet oxygen cannot be formed in these biological systems neither from the dismutation of OJ nor from the reaction of O₂^- with OH. Singlet oxygen may be formed from the reduction of metal complexes by O₂^-.