Dechlorination of tetrachloroguaiacol by laccase of white-rot basidiomycete Coriolus versicolor

 Degradation of tetrachloroguaiacol is catalyzed by an extracellular enzyme, the laccase of the white-rot fungus Coriolus versicolor. This enzyme catalyzes the dechlorination of tetrachloroguaiacol and release of chloride ions. The pathway for the degradation was deduced from the intermediates produced by purified laccase and ¹⁸O-labeling experiments. The first step is demethylation. The resulting tetrachlorocatechol is dechlorinated to give 2,3,5-trichloro-6-hydroxy-p-benzoquinone, 2,5-dichloro-3,6-dihydroxy-p-benzoquinone, and dichloro-6-hydroxy-p-benzoquinone. Isotopic experiments established the mechanism of dechlorination of tetrachloroguaiacol by laccase. The laccase-catalyzed dechlorination is not caused by oxidative coupling but by nucleophilic substitution in which Cl^- is released by water from cation radicals generated by laccase. Received: 25 August 1995/Received revision: 27 October 1995/Accepted: 20 November 1995     

Lipid peroxidation during the oxidation of haemoproteins by hydroperoxides. Relation to electronically excited state formation

The formation of electronically excited states during hydroperoxide metabolism is analysed in terms of recombination reactions involving secondary peroxyl radicals and scission of the O? O bond of peroxides by haemoproteins, mainly myoglobin. Both processes may be sequentially interrelated, for the cleavage of H₂O₂ by metmyoglobin leads to the formation of a strong oxidizing equivalent with the capability to promote peroxidation of polyunsaturated fatty acids. The decomposition of lipid hydroperoxides by ferryl-hydroxo complexes, as that formed during the oxidation of metmyoglobin by H₂O₂, is a source of peroxyl radicals, the recombination of which proceeds with elimination of a conjugated triplet carbonyl or singlet oxygen.     

Visible-light-promoted degradation of the antioxidants propyl gallate and t-butylhydroquinone: Mechanistic aspects

Abstract

The kinetic and mechanistic aspects of the visible-light-mediated photodegradation of the phenolic antioxidants (PA), propyl gallate (PG), and t-butylhydroquinone (TBHQ), employing riboflavin (Rf) as photosensitizer, have been studied by time-resolved and stationary techniques. The photosensitizer Rose Bengal (RB) was used for auxiliary experiments. Results show the occurrence of chemical transformations on PA with the participation of electronically excited states of Rf and different reactive oxygen species (ROS) generated from these states. With 0.02 mM Rf and 1.0 mM PA, the electronically excited triplet state of Rf is quenched by PA, in a competitive manner with the dissolved oxygen. As a consequence, a cascade of photoprocesses produces singlet oxygen (O₂(¹Δ_g)) and H₂O₂ in the case of PG and, O₂(¹Δ_g), H₂O₂ and HO^? in the case of TBHQ. The participation of these species is supported by experiments of oxygen consumption carried out in the presence of specific ROS scavengers. TBHQ has a relatively high capacity for O₂(¹Δ_g) physical deactivation and a low photodegradation efficiency by the oxidative species. Comparatively, it can be asserted that TBHQ has a higher antioxidant capacity than PG.     

Oxidative 4-dechlorination of 2,4,6-trichlorophenol catalyzed by horseradish peroxidase

 The well-known and easily available horseradish peroxidase (HRP) catalyzes the H₂O₂-dependent oxidative 4-dechlorination of the pollutant 2,4,6-trichlorophenol, which is recalcitrant to many organisms except those producing ligninases. UV-visible spectroscopy and gas chromatography-mass spectrometry identified the oxidized reaction product as 2,6-dichloro-1,4-benzoquinone. NMR and IR spectroscopic data further supported the above characterization. Experimental evidence for the elimination of HCl from the substrate was acquired by detecting the decrease in pH of the reaction mixture, and by observing the presence of the β-chlorocyclopentadienone cation fragment in the mass spectrum of 2,6-dichloro-1,4-benzoquinone. Consequently, nucleophilic attack by water on the 2,4,6-trichlorocyclohexadienone cation was proposed to give the final product. Our results indicate an oxidative dechlorination pathway catalyzed by HRP for 2,4,6-trichlorophenol, similar to that by extracellular lignin peroxidases. The relative catalytic efficiency of HRP seems higher than that of lignin peroxidases. The HRP-H₂O₂ catalytic system could be utilized in the degradation of polychlorinated phenols for industrial and biotechnological purposes. Received: 20 November 1998 / Accepted: 29 January 1999     

Characterization of photosystem II in stroma thylakoid membranes

The functional state of the PS II population localized in the stroma exposed non-appressed thylakoid region was investigated by direct analysis of the PS II content of isolated stroma thylakoid vesicles. This PS II population, possessing an antenna size typical for PS II, was found to have a fully functional oxygen evolving capacity in the presence of an added quinone electron acceptor such as phenyl-p-benzoquinone. The sensitivity to DCMU for this PS II population was the same as for PS II in control thylakoids. However, under more physiological conditions, in the absence of an added quinone acceptor, no oxygen was evolved from stroma thylakoid vesicles and their PS II centers were found to be incapable to pass electrons to PS I and to yield NADPH. By comparison of the effect of a variety of added quinone acceptors with different midpoint potentials, it is concluded that the inability of PS II in the stroma thylakoid membranes to contribute to NADPH formation probably is due to that Q_A of this population is not able to reduce PQ, although it can reduce some artificial acceptors like phenyl-p-benzoquinone. These data give further support to the notion of a discrete PS II population in the non-appressed stroma thylakoid region, PS II, having a higher midpoint potential of Q_A than the PS II population in the appressed thylakoid region, PS II. The physiological significance of a PS II population that does not produce any NADPH is discussed.Abbreviations pBQ p-benzoquinone - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCIP 2,6-dichloroindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DQ duroquinone(tetramethyl-p-benzoquinone) - FeCN ferricyanide (potassium hexacyanoferrat) - MV methylviologen - NADPH,NADP⁺ reduced or oxidized form of nicotinamide adenine dinucleotide phosphate respectively - PpBQ phenyl-p-benzoquinone - PQ plastoquinone - PS II photosystem II - PS I photosystem I - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - E microEinstein     

Photodynamically induced chemoresponses of the colorless flagellate,Astasia longa,in the presence of riboflavin

In the presence of the photosensitizer riboflavin, Astasia accumulates in illuminated fields at high fluence rates. The quenchers of riboflavin excited states, NaN₃ and KI, abolish the photodynamic effect of riboflavin. Crocetin, a ¹O₂ quencher, does not influence the photodynamic action of riboflavin while 1,4-benzoquinone very strongly depresses its effect. This indicates a type I pathway forming H₂O₂ as a photoproduct. The photodynamic effect is abolished by the addition of 10^-5 M catalase which breaks down H₂O₂. Astasia shows chemoaccumulations around the opening of a capillary filled either with riboflavin (under high intensity irradiation) or H₂O₂ which proves the hypothesis that the photobehavioral response in the presence of riboflavin is based on a chemoresponse toward H₂O₂, produced when the dye is irradiated. The accumulation around the capillary opening is not due to a direct chemotactic movement of cells but rather to a chemophobic response which prevents the cells from swimming away from the chemoattractant.     

Induction of Extracellular Hydroxyl Radical Production by White-Rot Fungi through Quinone Redox Cycling

A simple strategy for the induction of extracellular hydroxyl radical (OH) production by white-rot fungi is presented. It involves the incubation of mycelium with quinones and Fe³⁺-EDTA. Succinctly, it is based on the establishment of a quinone redox cycle catalyzed by cell-bound dehydrogenase activities and the ligninolytic enzymes (laccase and peroxidases). The semiquinone intermediate produced by the ligninolytic enzymes drives OH production by a Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺). H₂O₂ production, Fe³⁺ reduction, and OH generation were initially demonstrated with two Pleurotus eryngii mycelia (one producing laccase and versatile peroxidase and the other producing just laccase) and four quinones, 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and 2-methyl-1,4-naphthoquinone (menadione [MD]). In all cases, OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H₂O₂ levels and Fe³⁺ reduction rates observed with the four quinones. Between the two P. eryngii mycelia used, the best results were obtained with the one producing only laccase, showing higher OH production rates with added purified enzyme. The strategy was then validated in Bjerkandera adusta, Phanerochaete chrysosporium, Phlebia radiata, Pycnoporus cinnabarinus, and Trametes versicolor, also showing good correlation between OH production rates and the kinds and levels of the ligninolytic enzymes expressed by these fungi. We propose this strategy as a useful tool to study the effects of OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.White-rot fungi are unique in their ability to degrade a wide variety of organopollutants (36, 47), mainly due to the secretion of a low-specificity enzyme system whose natural function is the degradation of lignin (11). Components of this system include laccase and/or one or two types of peroxidase, such as lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP) (31). Besides acting directly, the ligninolytic enzymes can bring about lignin and pollutant degradation through the generation of low-molecular-weight extracellular oxidants, including (i) Mn³⁺, (ii) free radicals from some fungal metabolites and lignin depolymerization products (7, 22), and (iii) oxygen free radicals, mainly hydroxyl radicals (OH) and lipid peroxidation radicals (21). Although OH radicals are the strongest oxidants found in cultures of white-rot fungi (1), studies of their involvement in pollutant degradation are scarce. One of the reasons is that the mechanisms proposed for OH production still await in vivo validation.Several potential sources of extracellular OH based on the Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺) have been postulated for white-rot fungi. In one case, an extracellular fungal glycopeptide has been shown to reduce O₂ and Fe³⁺ to H₂O₂ and Fe²⁺ (45). Enzymatic sources include cellobiose dehydrogenase, LiP, and laccase. Among these, only cellobiose dehydrogenase is able to directly catalyze the formation of Fenton''s reagent (33). The ligninolytic enzymes, however, act as an indirect source of OH through the generation of Fe³⁺ and O₂ reductants, such as formate (CO₂⁻) and semiquinone (Q⁻) radicals. The first time evidence was provided that a ligninolytic enzyme was involved in OH production, oxalate was used to generate CO₂⁻ in a LiP reaction mediated by veratryl alcohol (4). The proposed mechanism consisted of the following cascade of reactions: production of veratryl alcohol cation radical (Valc⁺) by LiP, oxidation of oxalate to CO₂⁻ by Valc⁺, reduction of O₂ to O₂⁻ by CO₂⁻, and a superoxide-driven Fenton reaction (Haber-Weiss reaction) in which Fe³⁺ was reduced by O₂⁻. The OH production mechanism assisted by Q⁻ was inferred from the oxidation of 2-methoxy-1,4-benzohydroquinone (MBQH₂) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH₂) by Pleurotus eryngii laccase in the presence of Fe³⁺-EDTA. The ability of Q⁻ radicals to reduce both Fe³⁺ to Fe²⁺ and O₂ to O₂⁻, which dismutated to H₂O₂, was demonstrated (14). In this case, OH radicals were generated by a semiquinone-driven Fenton reaction, as Q⁻ radicals were the main agents accomplishing Fe³⁺ reduction. The first evidence of the likelihood of this OH production mechanism being operative in vivo had been obtained from incubations of P. eryngii with 2-methyl-1,4-naphthoquinone (menadione [MD]) and Fe³⁺-EDTA (15). Extracellular OH radicals were produced on a constant basis through quinone redox cycling, consisting of the reduction of MD by a cell-bound quinone reductase (QR) system, followed by the extracellular oxidation of the resulting hydroquinone (MDH₂) to its semiquinone radical (MD⁻). The production of extracellular O₂⁻ and H₂O₂ by P. eryngii via redox cycling involving laccase was subsequently confirmed using 1,4-benzoquinone (BQ), 2-methyl-1,4-benzoquinone, and 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone), in addition to MD (16). However, the demonstration of OH production based on the redox cycling of quinones other than MD was still required.In the present paper, we describe the induction of extracellular OH production by P. eryngii upon its incubation with BQ, 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and MD in the presence of Fe³⁺-EDTA. The three benzoquinones were selected because they are oxidation products of p-hydroxyphenyl, guaiacyl, and syringyl units of lignin (MD was included as a positive control). Along with laccase, the involvement of P. eryngii VP in the production of O₂⁻ and H₂O₂ from hydroquinone oxidation has also been reported (13). Since hydroquinones are substrates of all known ligninolytic enzymes, quinone redox cycling catalysis could involve any of them. Here, we demonstrate OH production by P. eryngii under two different culture conditions, leading to the production of laccase or laccase and VP. We also show that quinone redox cycling is widespread among white-rot fungi by using a series of well-studied species that produce different combinations of ligninolytic enzymes.     

A molecular orbital study of the metabolic pathway with hydroquinone intermediate from benzene as carcinogen

A possible metabolic activation pathway of benzenein vivo is the nonenzymatic oxidation of hydroquinone producedvia the cytochrome P-450-mediate two-step oxidation of benzene. The mechanism of the further oxidation of hydroquinone and the nature of the most reactive intermediate have not yet been clarified, although it is speculated that the intermediate isp-benzoquinone and/orp-benzosemiquinone. The theoretical result of using molecular orbital calculations (ab initio and CNDO/2 methods) indicates that although the mechanism of the nonenzymatic oxidation of hydroquinone cannot yet be determined, the intermediate is thep-benzosemiquinone anion radical. It is also suggested that active-oxygen species such as hydroxyl radical, which accelerates the nonenzymatic oxidation, play an important role in the metabolic pathway in question.     

Characterization of a glutathione conjugate of the 1,4-benzosemiquinone-free radical formed in rat hepatocytes

Rat hepatocytes treated with 1,4-benzoquinone formed 1,4-benzosemiquinone and 2-S-glutathionyl-1,4-benzosemiquinone radicals as detected by ESR spectroscopy. The 2-S-glutathionyl-1,4-benzosemiquinone radical was first obtained from the reaction of 1,4-benzoquinone with glutathione. Glutathione both reduced benzoquinone to form benzosemiquinone and conjugated benzoquinone to form 2-S-glutathionyl-1,4-benzosemiquinone radical. The ratio of these two radicals depended upon the ratio of 1,4-benzoquinone to glutathione. At near equimolar ratios, the 2-S-glutathionyl-1,4-benzosemiquinone radical was predominantly formed. This radical was characterized by computer simulation of the experimental spectra and identified by comparison of its hyperfine coupling constants with those of chemical analogues. The 2-S-glutathionyl-1,4-benzosemiquinone radicals formed inside hepatocytes, and then crossed the plasma membrane into the media.     

Electronically excited state generation during the reaction of p-benzoquinone with H2O2. Relation to product formation: 2-OH- and 2,3-epoxy-p-benzoquinone. The effect of glutathione

The reaction between H2O2 and p-benzoquinone proceeds with consumption of both reactants with second order rate constants of 1.66- and 0.77 M-1S-1, respectively. The process is mainly supported by oxygen addition reactions to the quinone resulting in the formation of both 2,3-epoxy-p-benzoquinone and 2-OH-p-benzoquinone. The former product accumulates in the assay mixture without participating in further reactions. The formation of the latter product implies free radical intermediates such as 2-OH-p-benzosemiquinone anion, which supports the generation of electronically excited states upon its oxidation by H2O2, presumably as part of an organic Fenton reaction. The relaxation of the excited state is accompanied by photoemission at 485-530 nm. Glutathione was found to counteract the oxidative aspects of the reaction between p-benzoquinone and H2O2 by a series of processes involving (a) a rapid reductive addition to the quinone with formation of a substituted p-benzohydroquinone; (b) an effective quenching of photoemission, which might be attributed to the deactivation of the excited state by the p-benzohydroquinone-glutathione adduct, and (c) the decomposition of the formed 2,3-epoxy-p-benzoquinone, also by reductive cleavage of the epoxide ring.     

Reaction centers fromRhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. II. Light-induced proton translocation

Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochromec and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ₀), a lipid-and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ₀H₂) and oxidized cytochromec. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochromec and UQ₀. At 250 and 25 µM UQ₀ and cytochromec, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.Abbreviations RC reaction center - UQ₀/UQ₀H₂ oxidized and reduced form of 2,3-dimethoxy-5-methyl-1,4-benzoquinone - D/D⁺ reduced and oxidized form of the primary electron donor of the RCs - CCCP carbonylcyanide-trichloromethoxy phenylhydrazone - UQ_A/UQ _A ^– oxidized and semiquinone form of the primary electron acceptor of the RCs - UQ_B/UQ _B ^– /UQ_BH₂ oxidized, semiquinone, and reduced form of the secondary electron acceptor of the RCs - LDAO lauryldimethylamine-N-oxide During the course of this study K.J.H. was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). This research was supported by grants from the National Institutes of Health (EY-02084) and from the Office of Naval Research (ONR-NOOO 14-79-C 0798) to M. Montal.     

Hydrogen Peroxide Inhibits Photosynthetic Electron Transport in Cells of Cyanobacteria

The effect of H₂O₂ on photosynthetic O₂ evolution and photosynthetic electron transfer in cells of cyanobacteria Anabaena variabilis and Anacystis nidulans was studied. The following experiments were performed: 1) directly testing the effect of exogenous H₂O₂; 2) testing the effect of intracellular H₂O₂ generated with the use of methyl viologen (MV); 3) testing the effect of inhibiting intracellular H₂O₂ decomposition by salicylic acid (SA) and 3-amino-1,2,4-triazole (AT). H₂O₂ inhibited photosynthetic O₂ evolution and light-induced reduction of p-benzoquinone (BQ) + ferricyanide (FeCy) in the Hill reaction. The I₅₀ value for H₂O₂ was 0.75 mM. Photosynthetic electron transfer in the cells treated with H₂O₂ was not maintained by H₂O₂, NH₂OH, 1,5-diphenylcarbazide, tetraphenylboron, or butylated hydroxytoluene added as artificial electron donors for Photosystem (PS) II. The H₂O CO₂, H₂O MV (involving PSII and PSI) and H₂O BQ + FeCy (chiefly dependent on PSII) electron transfer reactions were inhibited upon incubation of the cells with MV, SA, or AT. The N,N,N",N"-tetramethyl-p-phenylenediamine MV (chiefly dependent on PSI) electron transfer was inhibited by SA and AT but was resistant to MV. The results show that H₂O₂ inhibits photosynthetic electron transfer. It is unlikely that H₂O₂ could be a physiological electron donor in oxygenic photosynthesis.     

Mechanism of Antimicrobial Effect of Quinone Compounds

It has been observed that, in the culture medium, the toxicity of p-benzoquinone on bakers’ yeast decreases with time, and the decreasing process was examined from the view point of chemical reaction of quinone with the component of the medium.

As a result, it was shown that quinone concentration decreases by its 1,4-addition reaction with amino radicals of the component of the medium, and it was concluded that the inhibiting effect of quinone on the yeast growth is determined by the velocity of death of the yeast by the toxicity of quinone and that of inactivation of the toxicity by the addition reaction.     

Oxidation of syringic acid by extracellular peroxidase of white-rot fungus,Pleurotus ostreatus

The oxidative degradation of syringic acid by the extracellular peroxidase ofPleurotus ostreatus was studied. Three products formed in the oxidation of syringic acid by the peroxidase in the presence of O₂ and H₂O₂ were identified as 2,6-dimethoxyphenol, 2,6-dimethoxy-1,4-dihydroxybenzene, and 2,6-dimethoxy-1,4-benzoquinone. A free radical was detected as the reaction intermediate of the extracellular peroxidase-catalyzed oxidation of acetosyringone. These results can be explained by mechanisms involving the production of a phenoxy radical and subsequent decarboxylation. This is the first time that 2,6-dimethoxyphenol has been identified in extracellular peroxidase-catalyzed reactions.     

Properties of inactive Photosystem II centers

A fraction (usually in the range of 10–25%) of PS II centers is unable to transfer electrons from the primary quinone acceptor Q_A to the secondary acceptor Q_B. These centers are inactive with respect to O₂ evolution since their reopening after photochemical charge separation to the S₂O_A ^- state involves predominantly a back reaction to S₁Q_A in the few seconds time range (slower phases are also occurring). Several properties of these centers are analyzed by fluorescence and absorption change experiments. The initial rise phase F_o-F_pl of fluorescence induction under weak illumination reflects both the closure of inactive centers and the modulation of the fluorescence yield by the S-states of the oxygen-evolving system: We estimate typical relative amplitudes of these contributions as, respectively, 65 and 35% of the F_o-F_pl amplitude. The half-rise time of this phase is significantly shorter than for the fluorescence induction in the presence of DCMU (in which all centers are involved). This finding is shown to be consistent with inactive centers sharing the same light-harvesting antenna as normal centers, a view which is also supported by comparing the dependence of the fluorescence yield on the amount of closed active or inactive centers estimated through absorption changes. It is argued that the exponential kinetics of the F_o-F_pl phase does not indicate absence of excitation energy transfer between the antennas of inactive and active centers. We show that the acceptor dichlorobenzoquinone does not restore electron transfer in inactive centers, in disagreement with previous suggestions. We confirm, however, the enhancement of steady-state electron flow caused by this quinone and suggest that it acts by relieving a blocking step involved in the reoxidation of a fraction of the plastoquinone pool. Part of the discrepancies between the present results and those from previous literature may arise from the confusion of inactive centers characterized on a single turnover basis and PS II centers that become blocked under steady-state conditions because of deficient reoxidation of their secondary acceptors.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - PS photosystem     

Hydrogen production by photosystem I of Scenedesmus: Effect of heat and salicylaldoxime on electron transport and photophosphorylation

Summary Photosynthesis, photoreduction, the p-benzoquinone Hill reaction, and glucose uptake by whole cells, as well as cyclic photophosphorylation (with PMS) by chloroplast particles were strongly inhibited by 10^-2 M salicylaldoxime or by heating whole cells for 1–2 min at 55°. In contrast, H₂ photoproduction by whole cells of mutant No. 11 and wild type Scenedesmus and PS I-mediated MR reduction by chloroplast particles were either stimulated or not significantly inhibited by these agents. H₂ production by mutant No. 8 was slightly depressed by salicylaldoxime. DCMU inhibited H₂ photoproduction with 10^-2 M salicylaldoxime approximately 20%, indicating some contribution of electrons by endogenous organic compounds to photosystem II between the O₂-evolving mechanism and the DCMU-sensitive site. We conclude that photohydrogen production by PS I of Scenedesmus does not require cyclic photophosphorylation but is due to non-cyclic electron flow from organic substrate(s) through PS I to hydrogenase where molecular H₂ is released.The following abbreviations were used CI-CCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP dichlorophenol-indophenol - MR methyl red - PMS phenazine methosulfate - PS photosystem This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission to Professor H. Gaffron.     

Interactions of tertiary phosphines with p-benzoquinones; X-ray structures of [HO(CH2)3]3PC6H2(O)(OH)(MeO) and Ph3PC6H3(O)(OH)

Phosphonium zwitterions of a known type were obtained in high yield via a 1:1 reaction of p-benzoquinone or methoxy-p-benzoquinone with the tertiary phosphines R₃P [R = (CH₂)₃OH, Ph, Et, Me] and Ph₂MeP, in acetone or benzene at room temperature. In all cases, attack of the P-atom occurs at a C-atom rather than at an O-atom. The products were characterized to various degrees by elemental analysis, ³¹P{¹H}, ¹H and ¹³C NMR spectroscopies, and mass spectrometry, and two of the zwitterions, the new [HO(CH₂)₃]₃P⁺C₆H₂(O⁻)(OH)(MeO) and the known Ph₃P⁺C₆H₃(O⁻)(OH), were structurally characterized by X-ray analysis. The PEt₃ reaction also produces small amounts of the ‘dimeric’, μ-oxo co-product Et₃P⁺C₆H₂(O⁻)(OH)-O-C₆H₃(O⁻)P⁺Et₃ that is tentatively characterized by 1D- and 2D-NMR data. 2,5-Di-tert-butyl- and 2,3,5,6-tetramethyl-p-benzoquinone do not react with [HO(CH₂)₃]₃P under the conditions noted above. Heating D₂O solutions of the water-soluble zwitterions R₃P⁺C₆H₃(O⁻)(OH) [R = (CH₂)₃OH, Et] at 90 °C for 72 h leads to complete H/D exchange of the H-atom in the position ortho to the phosphonium center.     

Triplet states of bacteriochlorophyll and carotenoids in chromatophores of photosynthetic bacteria

Chromatophores from photosynthetic bacteria were excited with flashes lasting approx. 15 ns. Transient optical absorbance changes not associated with the photochemical electron-transfer reactions were interpreted as reflecting the conversion of bacteriochlorophyll or carotenoids into triplet states. Triplet states of various carotenoids were detected in five strains of bacteria; triplet states of bacteriochlorophyll, in two strains that lack carotenoids. Triplet states of antenna pigments could be distinguished from those of pigments specifically associated with the photochemical reaction centers. Antenna pigments were converted into their triplet states if the photochemical apparatus was oversaturated with light, if the primary photochemical reaction was blocked by prior chemical oxidation of P-870 or reduction of the primary electron acceptor, or if the bacteria were genetically devoid of reaction centers. Only the reduction of the electron acceptor appeared to lead to the formation of triplet states in the reaction centers.In the antenna bacteriochlorophyll, triplet states probably arise from excited singlet states by intersystem crossing. The antenna carotenoid triplets probably are formed by energy transfer from triplet antenna bacteriochlorophyll. The energy transfer process has a half time of approx. 20 ns, and is about 1 × 10³ times more rapid than the reaction of the bacteriochlorophyll triplet states with O₂. This is consistent with a role of carotenoids in preventing the formation of singlet O₂ in vivo. In the absence of carotenoids and O₂, the decay half times of the triplet states are 70 μs for the antenna bacteriochlorophyll and 6–10 μs for the reaction center bacteriochlorophyll. The carotenoid triplets decay with half times of 2–8 μs.With weak flashes, the quantum yields of the antenna triplet states are in the order of 0.02. The quantum yields decline severely after approximately one triplet state is formed per photosynthetic unit, so that even extremely strong flashes convert only a very small fraction of the antenna pigments into triplet states. The yield of fluorescence from the antenna bacteriochlorophyll declines similarly. These observations can be explained by the proposal that singlet-triplet fusion causes rapid quenching of excited singlet states in the antenna bacteriochlorophyll.     

Interaction of trout hemoglobin with H2O2: a chemiluminescence study

Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H₂O₂ using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H₂O₂ produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H₂O₂. © 1997 John Wiley & Sons, Ltd.     

Changes in [C]Atrazine Binding Associated with the Oxidation-Reduction State of the Secondary Quinone Acceptor of Photosystem II

One hypothesis of triazine-type herbicide action in photosynthetic material is that the herbicide molecule competes with a secondary quinone acceptor, B, for a binding site at the reaction center of photosystem II. The binding affinity of B has been suggested to change with its level of reduction, being most strongly bound in its semiquinone form. To test this hypothesis, [¹⁴C]atrazine binding studies have been carried out under different photochemically induced levels of B reduction in Pisum sativum. It is found that herbicide binding is reduced in continuously illuminated samples compared to dark-adapted samples. Decreased binding of atrazine corresponds to an increase in the semiquinone form of B. With flash excitation, the herbicide binding oscillates with a cycle of two, being low on odd-numbered flashes when the amount of semiquinone form of B is greatest. Treatment with NH₂OH was found to significantly decrease the strength of herbicide binding in the dark as well as stop the ability of p-benzoquinone to oxidize the semiquinone form of B. It is suggested that the mode of action of NH₂OH is disruption of quinones or their environment on both the oxidizing and reducing sides of photosystem II. Herbicide binding was found to be unaltered under conditions when p-benzosemiquinone oxidation of the reduced primary acceptor, Q⁻, is herbicide insensitive; weak herbicide binding cannot explain this herbicide insensitivity. It is concluded that the quinone-herbicide competition theory of herbicide action is correct. Also, since quinones are lipophilic the importance of the lipid composition of the thylakoid membrane in herbicide interactions is stressed.