THE SUPEROXIDE ANION AS ELECTRON DONOR TO THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN

Abstract— In isolated respiratory multienzyme complexes of beef heart mitochondria the b -type cytochromes can be photoreduced in presence of flavin via the superoxide anion. O^-₂ does not reduce cytochrome c ₁. In an anaerobic system, FMNH₂ formed by irradiation with blue light in presence of EDTA reduces cytochromes b and c ₁ The possible implication of O^-₂ in the electron transfer from flavin/flavoprotein to cytochrome b in blue light-controlled biological processes is discussed.     

GAMMA AND PULSE RADIOLYSIS STUDIES OF THE REACTION BETWEEN SUPEROXIDE IONS AND OXYHEMOGLOBIN-METHEMOGLOBIN SYSTEM

Abstract— The radiolytic studies of oxyhemoglobin or methemoglobin in neutral aerated aqueous solutions with formate ions, lead to three conclusions:
The oxidation of oxyhemoglobin by O^-₂ is not important. The observed low oxidation yield is probably due to the slow reaction with hydrogen peroxide produced by O^-₂ disproportionation.
The reduction of methemoglobin in γ radiolysis reaches a plateau which could be explained by structural considerations.
The reduction of methemoglobin by O^-₂ ions, if it occurs, is relatively slow: k = 1.4 × 10³ M ^-1 s^-1. But a problem remains concerning the spectral characteristics of the product.     

PHOTOINACTIVATION OF A Chlamydomonas MUTANT(NL–11) IN THE PRESENCE OF METHIONINE: ROLES OF H2O2 and O-2

Abstract— A mutant of Chlamydomonas reinhardtii (NL–11) isolated from a wild type (137c+) was inactivated in the light in the presence of methionine at concentrations where the wild type was not inactivated. The inactivation was suppressed by either catalase or superoxide dismutase (SOD). Light-induced H₂O₂ formation and nitroblue tetrazolium (NBT) reduction inNL–11 were greater than those in the wild type. Methionine stimulated both the H₂O₂ formation and the NBT reduction inNL–11 as well as the wild type. The light-induced NBT reduction inNL–11 in the presence of methionine was partially suppressed by externally added SOD suggesting the participation of O^-₂. These results suggest that the hypersensitivity ofNL–11 to methionine in the light is due to stimulated formation of H₂O₂ and O^-₂.     

MECHANISM OF PHOTOSENSITIZATION BY PHEOPHORBIDE a STUDIED BY PHOTOHEMOLYSIS OF ERYTHROCYTES AND ELECTRON spIN RESONANCE SPECTROSCOPY

Abstract Phcophorbide a (PPa), a causal substance of food intoxication, when excited by exposure to light wavelengths of over 600 nm, caused the photohemolysis of goat erythrocytes in proportion to the incubation time of the cells. The addition of N-₃, an effective scavenger of ¹O₂, to the medium markedly inhibited the hemolysis of erythrocytes in a concentration-dependent manner, whereas the addition of superoxide dismutase (SOD) and catalase, inhibitors of O^-₂ and H₂O₂ generation, respectively, to the medium had little effect on it.
Methods for converting ¹O₂ to a nitroxide radical by 2,2,6,6-tetramethyl-4-piperidone (TMPD) and for trapping O^-₂ and OH by 5,5-dimethyl-l-pyrroline-A'-oxide (DMPO) were employed to observe directly these activated oxygens by electron spin resonance (ESR). The methods provided evidence that only ¹O₂, was produced by PPa, which was excited by light wavelengths of over 600 nm. Both the addition of N₃ to the solution and the removal of oxygen from the solution inhibited the generation of ¹O₂.
These results led us to conclude that ¹O₂ was mainly responsible for the hemolysis of erythrocytes by photoexcited PPa.     

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.     

SINGLET OXYGEN: A MAJOR REACTIVE SPECIES IN THE FUROCOUMARIN PHOTOSENSITIZED INACTIVATION OF E. COLI RIBOSOMES

Abstract— The skin photosensitizing furocoumarins, 8-methoxypsoralen (MOP) and 4,5',8-trimethylpsoralen (TMP), inactivate E. coli ribosomes in vitro , on UV irradiation at 313 nm. Purging the solutions with N₂ protects the ribosomes considerably against photoinactivation (75% with MOP and 80% with TMP). In air, the ribosome photoinactivation is mainly due to singlet oxygen (¹O₂), since the presence of NaN₃ and other ¹O₂ quenchers protects the system and the inactivation is enhanced in D₂O. Although ¹O₂ dominates as the inactivating species, the possibility of additional (∼15%) minor mechanisms involving free radicals exists. However, O^-₂ does not appear to be the damaging species, since superoxide dismutase does not provide any protection.
Photosensitization of the partially purified enzyme, phe-tRNA-synthetase with MOP or TMP shows inactivation and protection curves similar to those seen with the ribosomes. On the other hand, unfrac-tionated tRNA^phc is not photosensitized under similar conditions, although it shows self-photosensitization. It is likely that in the furocoumarin-sensitized ribosomes, the primary events of photoinactivation are associated with the proteins.     

HEMOLYSIS OF HUMAN ERYTHROCYTES BY ACTIVATED OXYGEN SPECIES

Abstract— The hemolysis of human erythrocytes by irradiation at 254 nm has been studied. Neither superoxide radicals nor singlet oxygen play a significant rôle and it is likely that the major species involved are hydroxyl radicals and, indirectly, carbonate anion or formate radicals. Similarly, when erythrocytes are treated with a system commonly used as source of superoxide radicals (photoreduction of riboflavin) it has been demonstrated that O^-₂ does not participate in lysis, but that singlet oxygen (possibly with hydroxyl radicals) is a major oxygen species involved in destruction of the cell membrane.     

SUPEROXIDE RADICALS, SUPEROXIDE DISMUTASES AND THE AEROBIC LIFESTYLE

Abstract— The superoxide radical (O^-₂) is a commonplace product of the biological reduction of molecular oxygen and plays an important role in oxygen toxicity. Superoxide dismutases (SOD) catalytically scavenge this radical and are the primary defense against its cytotoxicity. The data which support these statements have been briefly reviewed. Oxygen enhances the lethality of certain antibiotics, such as streptonigrin, and of ionizing radiation as well. The role of O^-₂ in the enhancements is presented and the basis of the radioprotective effects of SOD discussed. Several more recent developments are presented in detail including: (a) The induction of the MnSOD of E. coli upon exposure to very low levels of oxygen, (b) The changes in SOD in E. coli as a function of nutritional state, during culture in a chemostat. (c) A convenient new assay and activity stain for SOD.     

PHOTOSYNTHETIC NITRITE REDUCTION BY DITHIOERYTHRITOL AND THE EFFECT OF NITRITE ON ELECTRON TRANSPORT IN ISOLATED CHLOROPLASTS

Abstract. Photosynthetic reduction of nitrite to ammonia with type C chloroplasts from the heterocont alga Bumilleriopsis filiformis was investigated using 3,6-diaminodurene/ascorbate and 3,6-diaminodurene/dithioerythritol (DAD/DTE) as electron donor couple. Rates approach 6–10 μmol NO^-₂ reduced/mg chlorophyll/h and are steady for up to 30 min. The presence of oxygen or NADP⁺ only slightly diminished the rates of nitrite reduction obtained with DAD/DTE. Illuminated chloroplasts reduce oxygen in the presence of DAD/DTE at 135 μmol/mg chlorophyll/h without acceptor supplied. Photosynthetic oxygen uptake by this system in the presence of ferredoxin and NO^-₂, however, is inhibited to 42% by nitrite reductase with concurrent nitrite reduction. NO^-₃ and NO^-₂ have no effect on photosystem I-mediated NADP⁺ reduction, NO^-₂ (10 m M ) inhibits ferricyanide-mediated oxygen evolution to 72%. Also photosystem II reactions assayed e.g. with silicomolybdate are inhibited significantly by NO^-₂ (1 m M ), but only slightly by NO^-₃. Nitrite reductase is inhibited by p -chloromercuribenzoate ( p CMB), and this inhibition is prevented by DTE. Results suggest that photosynthetic nitrite reduction can cope with low concentrations of either compound, provided relevant thiol groups are protected.     

PHOTOFRAGMENTATION OF α-OXOCARBOXYLIC ACIDS IN AQUEOUS SOLUTION. AN EPR STUDY. FORMATION OF SEMIDIONE RADICALS BY DECARBOXYLATIVE SUBSTITUTION OF α-OXOCARBOXYLIC ACIDS BY ACYL RADICALS-I: GLYOXYLIC, PYRUVIC, α-OXOBUTYRIC, α-OXOGLUTARIC AND α-OXOISOCAPROlC ACIDS

Abstract— By means of in situ photolysis EPR of aqueous solutions of α-oxocarboxylic acids (RCO-CO₂H) at pH values above 5, semidione radical anions [RC(O^-)=C(O')R] and α-hydroxy-α-carboxy alkyl radicals [RC(OH)CO₂-] were detected. C0₂ was identified as a reaction product. On photolysis of mixtures of α-oxocarboxylic acids (RCOCO₂H and R'COCC₂H), "mixed" semidione radical anions [RC(O^->=C(O)R'] were observed in addition to RC(O^-)=C(O')R, R'C(O^-)=C(O')R', RC(OH)CO₂^- and R'C(OH)CO₂^-. The experimental results are explained in terms of photodecarboxylation (α-clea-vage) of electronically excited RCOCOJ to yield RCO and CO₂. The radicals RC(OH)CO₂^- are formed by reduction of RCOCO₂^- by CO₂^-. The semidione radicals are produced by addition of RCO to RCOCO₂^- followed by decarboxylation of the intermediate adduct. This mechanism was confirmed by generating acyl radicals independently and reacting them with α-oxocarboxylic acids. Selected product studies support the mechanism suggested.     

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).     

CONCENTRATION-DEPENDENT LIFETIMES OF Cu(NN)+2 SYSTEMS: EXCIPLEX QUENCHING FROM THE ION PAIR STATE

Abstract— The concentration dependence of the lifetimes of the charge transfer excited states of Cu(dmp)⁺₂ and Cu(dpp) ⁺₂ has been investigated in CH₂C1₂ solution at 20^°C. (dmp denotes 2,9-dimethyf-1,10-phenanthroline, and dpp denotes 2,9-diphenyl-l,10-phenanthroline.) In dilute solution (< 30 μM) the lifetime of Cu(dmp)⁺₂, is 95 ± 5 ns, independent of the anion. At higher concentrations the lifetime decreases, in most cases, to a limiting value that depends upon the counterion. The measured limiting lifetimes range from 38 ± 3 ns for CIO^-₄ to 78 ± 5 ns for PF^-₆. The anion-induced quenching is attributed to exciplex quenching which is mediated by an ion pair which exists in the ground state. The results imply that the quenching ability of the anions follows the order BPh-₄ < PF -₆, < BF-₄ < CIO -₄ < NO-₃ which is consistent with previous estimates of donor strength. The lifetime of Cu(dpp)⁺₂ is also concentration dependent, but the effect is much smaller because the phenyl substituents impede attack by the anion.     

THEORY OF THE OPTICAL ABSORPTION OF LIGHT-ADAPTED BACTERIORHODOPSIN AND ITS ACIDIFIED FORMS

Abstract— We assume a model for bacteriorhodopsin chromophore such that the protonated retinal Schiff-base (PRSB) interacts with two anions in the case of light-adapted bacteriorhodopsin (bR^L), while it does with one anion in the case of the acidified form of bacteriorhodopsin (bR^acid₆₀₀). On the basis of this model, the π-electronic states of all- trans -PRSB are calculated according to our LCAO-ASMO-SCF-CI method, the anions being approximated by negative point-charges in the plane of PRSB π-system. A possible distribution of the negative point-charges around PRSB is proposed for the chromophores of bR^L, bR^acid₆₀₀, and the two irradiated forms of bR^acid₆₀₀ (the one at 3°C containing 9- cis -PRSB, and the other at — 72°C all- trans -PRSB). It is shown that the wavelength λ_max of absorption maximum observed for each form of bacteriorhodopsin can be explained reasonably well by the suggested charge distribution. Furthermore, a model for the structure of the active site of bR^L is proposed, considering that two COO⁻ groups form the anions that interact with PRSB. The calculated optical absorption of all- trans -PRSB at such a site is shown to be consistent with the observed absorption spectrum of bR^L.     

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).     

PHOTOPHYSICS AND PHOTOCHEMISTRY OF 4-DIHYDROPYRIDINONES

Dihydropyridinones (DHP) react photochemically with olefins to form cycloaddition products. The reactions proceed through the lowest excited state T ₁ of the DHPs, with rate constants which depend on the olefin and can approach the diffusional limit in the most favourable cases. Intra-and inter-molecular sensitization and quenching have been investigated, as well as spectroscopic properties such as absorption and luminescence spectra, and in particular electron energy loss spectra which have established the energy of the reactive T₁, state as 295 kj mol^-1 (3.07 eV).     

ON THE PHOTOIONIZATION ENERGY THRESHOLD OF TRYPTOPHAN IN AQUEOUS SOLUTIONS

Abstract— To investigate the existence and energy position of a photoionization threshold. tryptophan (Trp) has been photoionized in desecrated neutral aqueous or alcoholic solution under monochromatic light of variable frequency, in presence of N₂O to scavenge the photoelectron.
Present findings and some literature data converge to show the existence of a threshold for the one photon ionization process. This threshold is located at 4.5 ± 0.1 eV and 4.85 ± 0.1 cV for Trp in aqueous and ethanol solutions. respectively, which corresponds to a lowering with respect to the gas phase ionization potential of 3.4 and 3.0 eV.
The photoionization quantum yields for Trp is found about 4 times greater at 250 nm than at Λ_cx= 265 nm, where φe^-_4M=0.080±0.025. In such spectral range. at most one photoelectron out of 4–5 escaping geminate recombination would lead to Trp photodegradation in acrated solutions.
These results also point out that the neutral radical Trp. which has been previously observed for Λ_cx > 275 nm, i.e. below the ionization threshold energy—would not necessarily derive from Trp ⁺ deprotonation or cation-electron dissociative recombination. Similarly, the opening of the indole ring with formylkynurenine (FK) formation which is observed under aerobic conditions and Λ_cx >, 280 nm would not imply an electron attachment on O₂ but reactions such as Trp +³O₂ or Trp*+³O₂ or else
     

PHOTOREDUCTION OF NAD TO NADH IN SEMICONDUCTOR DISPERSIONS

Abstract— Band gap illumination of TiO₂ (anatase) dispersions in weakly alkaline solutions of nicotinamide-adenine-dinucleotide (NAD⁺) leads in the presence of rhodium trisbipyridyl complex [Rh(bipy)³⁺₃], to continuous generation of biologically active cofactor NADH. Effects of pH, NAD⁺ and Rh(bipy)³⁺₃ concentration on the efficiency of this photoconversion process are investigated. The reaction proceeds already in aqueous solution in the absence of external electron donors but it is enhanced significantly in the presence of 10% methanol.     

A PULSE RADIOLYSIS STUDY OF THE ONE-ELECTRON OXIDATION AND REDUCTION OF 5, 10-METHENYLTETRAHYDROFOLATE

Abstract— The one-electron reduction and oxidation of 5,10-methenyltetrahydrofolate has been studied in aqueous solution in the acidity range H₀= -1 to pH = 7 using the reducing species CO^-₂ and (CH₃)₂-COH and oxidising species Br^-₂, and H₂SeO+₃. The spectral and other properties of the radicals so formed were found to be indcpendent of the reductant/oxidant used. Two protolytic forms of both the oxidised and reduced radicals were observed with approximate p K , values of 0.5 ± 0.3 being determined. Both the bridged form (5.10-methenyltetrahydrofolate) and the unbridged form (5- formyltetrahydrofolate) were found to be easily oxidised, whereas only the former could be reduced.     

PHOTOINACTIVATION OF CATALASE

Abstract— Inactivation of catalase with visible light (>400nm) has been studied in purified bovine liver catalase and in peroxisomal catalase in the mitochondrial fraction of rat liver. Light corresponding to that of maximal absorbance of the heme site (405 nm) was most effective in inactivation. Although photoinactivation is O₂ dependent, scavengers of OH radical, ¹O₂ and O⁻₂ did not protect against loss of activity in either system. Superoxide dismutase partially protected purified catalase added into the mitochondrial fraction system. However, complete protection of catalase was afforded by low concentrations of substrates such as formic acid or methanol which rapidly convert Compound I to Ferricatalase.     

QUENCHING OF THE EXCITED STATES OF 2-PHENYLBENZOXAZOLE BY AZIDE ANION. FLUORESCENCE AND ESR STUDY

Electron spin resonance, spin-trapping and fluorescence techniques demonstrate that 2-phenylbenzoxazole (P) participates in photo-induced reactions with alcohols and electron donors like the azide ion. Irradiation of Pat 300 nm in deaerated ethanol produces ethoxyl and hydroxyethyl radicals which can be detected with the spin trap, 5,5-dimethyl-l-pyrroline-l-oxide (DMPO). However, irradiation of P in the presence of N^-₃ leads to the appearance of the azide radical, N^-₃, which also reacts with DMPO. Studies with the nitroxyl radical, 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), suggest that electron transfer from the azide anion to an excited state of P yields the semi-reduced sensitizer, P^-, which in turn reacts with TEMPO. The effect of sodium azide upon the fluorescence intensity and lifetime of P in aqueous ethanol has also been studied.