Jin Jung

Mycosporine Glycine Protects Biological Systems Against Photodynamic Damage by Quenching Singlet Oxygen with a High Efficiency

This report concerns physiological function of mycosporinelike amino acids (MAA) as an active defense against the photooxidative effects of sunlight in marine organisms. Mycosporine glycine (MG) is a representative member of MAA family and was found to effectively suppress various detrimental effects of the Type‐II photosensitization in biological systems, such as inactivation of mitochondrial electron transport, lipid peroxidation of microsomes, hemolysis of erythrocytes and growth inhibition of Escherichia coli. The presence of MG in solutions of eosin Y or methylene blue resulted in a marked decrease in the level of singlet oxygen (1O2) produced by the sensitizers under illumination. The rate constant of 1O2 quenching by MG was determined to be 5.6 × 107M−1s−1 by the time‐resolved 1O2 luminescence decay method, which is higher than, or at least comparable to, the values for 1O2 reaction of well‐known quenchers such as 1,4‐diazabicyclo[2,2,2]octane and furfuryl alcohol. The results suggest that MG probably together with some other active MAA may play an important role in protecting marine organisms against sunlight damage by eliminating 1O2 generated from certain endogenous photosensitizers.

Photodynamic Effect of Iron Excess on Photosystem II Function in Pea Plants

An earlier mechanistic phase of iron toxicity in photosynthetic cells was interpreted in terms of enhanced photodynamic action by the cytochrome b6/f complex (Cyt b6/f) via singlet oxygen (1O2) on the photosystem II complex (PS II). Iron excess was induced in hydroponically cultured pea (Pisum sativum L.) plants, and its effect on the function of PS II in vivo as well as in vitro was studied under high‐irradiance conditions. Iron excess in plants gave rise to a significant increase in Cyt b6/f content of thylakoids. It appeared that the larger the content of Cyt b6/f, the more susceptible PS II was to photoinhibition, and the higher the rate of 1O2 photoproduction in thylakoids was. The action spectrum for degradation of the D1 protein in thylakoids revealed that photosensitization by nonporphyrin chromophore(s) was apparently associated with near UV to blue light–induced deterioration of PS II. The results are pertinent to the concept that photooxidative damage to PS II, exacerbated by iron accumulation in thylakoid membranes in the form of Cyt b6/f, is involved in the mechanism of iron toxicity in leaf cells.

Cytochrome b6/f Complex as an Indigenous Photodynamic Generator of Singlet Oxygen in Thylakoid Membranes

Abstract Possible association of photodynamic sensitization by cytochrome b6/f complex (cyt b6/f) via singlet oxygen (1O2) mechanism with photoinhibition damage to photosystem II (PS II) was studied using such subthylakoid preparations as photosystem I (PS I) particles, PS II core complex and cyt b6/f from spinach leaves. Upon exposure to bright light, PS II core complex lost photosynthetic electron transport activity to a certain extent, whose spectral dependence implied that pheophytin a is likely involved in photoinactivation of PS II core complex in itself. The presence of PS I particles exerted virtually no effect on PS II core photoinactivation. However, the inclusion of cyt b6/f in samples resulted in a marked exacerbation of the photoinactivation, particularly in UV-A and blue light. Such effect of cyt b6/f was suppressed by azide and enhanced by the medium deuteration. Photogeneration of 1O2 from cyt b6/f was confirmed by ESR and spectrophotometry, chemically trapping 1O2. Action spectra for both 1O2 photoproduction and PS II core photoinactivation by cyt b6/f bore a close resemblance to each other, seemingly carrying the absorption characteristics of the Rieske Fe-S protein. A complex deficient in the Rieske protein prepared from intact cyt b6/f showed virtually no generation of 1O2 in light, whereas an efficient photoformation of 1O2 was seen in the Rieske protein preparation. The results suggest that cyt b6/f, rather specifically the Rieske center, may play a prominent role in photoinhibition processes through type II photosensitization in thylakoids.

ACTION SPECTRA FOR THE GENERATION OF SINGLET OXYGEN FROM MITOCHONDRIAL MEMBRANES FROM SOYBEAN (Glycine max) HYPOCOTYLS

Abstract— The action spectrum for the generation of singlet oxygen (1O2) from mitochondrial membranes under aerobic conditions was measured at wavelengths between 360 and 600 nm, using sub‐mitochondrial particles (SMP) prepared from soybean hypocotyls. The spectrum, showing a peak at about 420 nm, remarkably resembles the absorption spectra of the Fe‐S centers of nonheme iron proteins. Disruption of the Fe‐S centers by treating SMP with mersalyl acid resulted in a substantial decrease in the efficiency of 1O2 generation, leaving an action spectrum whose pattern is significantly similar to the absorption spectrum of flavins, at least in the region of near UV and blue light wavelengths. Estimating the contribution of the Fe‐S centers to the generation of 1O2 from SMP, we suggest that the Fe‐S centers act as very important endogenous photosensitizers in plant cells, in so far as the type II mechanism is concerned. Possible involvement of mitochondrial flavoproteins in the generation of 1O2 is also discussed.

THE SUSCEPTIBILITY OF MUNG BEAN CHLOROPLASTS TO PHOTOINHIBITION IS INCREASED BY AN EXCESS SUPPLY OF IRON TO PLANTS: A PHOTOBIOLOGICAL ASPECT OF IRON TOXICITY IN PLANT LEAVES

In an attempt to elucidate the underlying mechanisms for iron toxicity in plants, the combined effects of iron overload and light intensities on the photosynthetic capacity of leaves were particularly focussed upon in this study, using mung bean seedlings grown under varied conditions regarding the supply of light and iron. The seedlings, when supplied with excess iron (up to 1.0 mM) and low light (40 W/m2), did not suffer any loss of photosynthesis; further, the typical symptoms of iron toxicity, as shown in the leaves grown in sunlight at ca 450 W/m2 on an average, were not seen in those. Nonetheless, excess iron supply resulted in a marked increase in photosensitivity of the low light‐adapted seedlings. A large portion of iron accumulated in chloroplasts by the supply of excess iron was found to be incorporated into thylakoids as nonheme iron (NHI), which acts as a potent sensitizer, photogenerating singlet oxygen (1O2). The generation rate of 1O2 from thylakoids linearly increased with increasing content of NHI; this was in parallel with the NHI content dependence of photoinactivation rates of photosynthetic electron transport and key enzymes of the Calvin cycle in chloroplasts. The results suggest that Fe‐dependent photosensitization reactions, occurring via the 1O2 mechanism, may be deeply involved in cellular processes leading to developing iron toxicity symptoms in plants.

Molecular and functional characterization of a unique sucrose hydrolase from Xanthomonas axonopodis pv. glycines

A novel sucrose hydrolase (SUH) from Xanthomonas axonopodis pv. glycines, a causative agent of bacterial pustule disease on soybeans, was studied at the functional and molecular levels. SUH was shown to act rather specifically on sucrose (K(m) = 2.5 mM) but not on sucrose-6-phosphate. Protein analysis of purified SUH revealed that, in this monomeric enzyme with an estimated molecular mass of 70,223 +/- 12 Da, amino acid sequences determined for several segments have corresponding nucleotide sequences in XAC3490, a protein-coding gene found in the genome of X. axonopodis pv. citri. Based on this information, the SUH gene, consisting of an open reading frame of 1,935 bp, was cloned by screening a genomic library of X. axonopodis pv. glycines 8ra. Database searches and sequence comparison revealed that SUH has significant homology to some family 13 enzymes, with all of the crucial invariant residues involved in the catalytic mechanism conserved, but it shows no similarity to known invertases belonging to family 32. suh expression in X. axonopodis pv. glycines requires sucrose induction, and insertional mutagenesis resulted in an absence of sucrose-inducible sucrose hydrolase activity in crude protein extracts and a sucrose-negative phenotype. Recombinant SUH, overproduced in Escherichia coli and purified, was shown to have the same enzymatic characteristics in terms of kinetic parameters.

INACTIVATION OF THE ACCEPTOR SIDE AND DEGRADATION OF THE D1 PROTEIN OF PHOTOSYSTEM II BY SINGLET OXYGEN PHOTOGENERATED FROM THE OUTSIDE

Abstract— The possible association of photodynamic sensitization with photoinhibition damage to the photosystem II complex (PS II) has been investigated using isolated intact thylakoids from pea leaves. For this study singlet oxygen (1O2), photoproduced by endogenous chromophores that are independent of the function of PS II, was assumed to be the major reactive intermediate involved in the photoinhibition process. When thylakoid samples preincubated with rose bengal were subjected to exposure to relatively weak green light (500–600 nm) under aerobic conditions, PS II was severely damaged. The pattern of the rose bengal‐sensitized inhibition of PS II was similar to that of high light‐induced damage to PS II: (1) the secondary quinone (QB)‐dependent electron transfer through PS II is inactivated much faster than the QB‐independent electron flow, (2) PS II activity is lost prior to degradation of the D1 protein, (3) diuron, an herbicide that binds to the QB domain on the D1 protein, prevents D1 degradation, and (4) PS II is damaged to a greater extent by the deuteration of thylakoid suspensions but to a lesser extent by the presence of histidine. Furthermore, it was observed that destroying thylakoid Fe‐S centers resulted in a marked reduction of high light‐induced PS II damage. These results may suggest that the primary processes of photoinhibition are mediated by 1O2 and that Fe‐S centers, which are located in some membrane components, but not in PS II, play an important role in photogenerating the activated oxygen immediately responsible for the initiation of photodamage to PS II.

Iron-sulfur centers as endogenous blue light sensitizers in cells: a study with an artificial non-heme iron protein.

Abstract— The possible involvement of Fe‐S clusters in photodynamic reactions as endogenous sensitizing chromophores in cells has been investigated, by using an artificial non‐heme iron protein (ANHIP) derived from bovine serum albumin and ferredoxins isolated from spinach and a red marine algae. Ferredoxins and ANHIP, when exposed to visible light, generate singlet oxygen, as measured by the imidazole plus RNO method. Irradiation with intense blue light of the ANHIP‐entrapped liposomes caused severe membrane‐damage such as liposomal lysis and lipid peroxidation. In the presence of ANHIP, isocitrate dehydrogenase and fructose‐l, 6‐diphosphatase were photoinactivated by blue light. However, all of these photosensitized reactions were significantly suppressed by a singlet oxygen (1O2) quencher, azide, but enhanced by a medium containing deuterium oxide. Further, the Fe‐S proteins with the prosthetic groups destroyed did not initiate the blue light‐induced reactions. In addition, the action spectrum for 1O2 generation from ANHIP was very similar to the visible absorption spectrum of Fe‐S centers. The results obtained in this investigation appear consistent with the suggestion that Fe‐S centers are involved in photosensitization in cells via a singlet oxygen mechanism.

In situ SUSCEPTIBILITIES OF PHOTOSYSTEMS I AND II TO PHOTOSENSITIZED DEACTIVATION via SINGLET OXYGEN MECHANISM

Abstract— A comparative study was carried out on the in situ susceptibilities to photoinactivation of the photosystem I (PS I) and II (PS II) complexes of spinach thylakoids treated with efficient type II sensitizers. While the presence of the exogenous sensitizers caused a substantial increase in the extent of photoinactivation of whole chain electron transport, it did not affect PS I activity of thylakoids in light but exerted an enhanced photoinactivating effect only on PS II. The measurements of the action spectrum for the inhibition of PS II activity of the sensitizer‐incorporated thylakoids and that for the generation of singlet oxygen (1O2) from them revealed that photosensitized inactivation of PS II is directly related to the photoproduction of 1O2 in thylakoid membranes. The results obtained in the present work clearly demonstrate an exceptional sensitivity of PS II to 1O2, providing circumstantial evidence that high light‐induced damage to PS II may result from photosensitization reactions mediated by 1O2, which is not necessarily produced within the PS II complex.

INVOLVEMENT OF THYLAKOID MEMBRANE-DEPENDENT PHOTOSENSITIZATION IN PHOTOINHIBITION OF THE CALVIN CYCLE ACTIVITY IN SPINACH CHLOROPLASTS

Photoinhibition of the light‐regulated key enzymes of the photosynthetic carbon reduction (PCR) cycle was investigated using chloroplasts isolated from spinach leaves. Light quality dependence of the light‐induced activity change (activation or inactivation) of key PCR enzymes in situ demonstrated that, while light activation is promoted mainly by red light (Λ.> 600 nm), inactivation takes place largely in the region of blue light (Λ < 500 nm). Inactivation was suppressed by a lipid soluble singlet oxygen (1O2,1Δg) quencher. When “stromal protein” was subjected to a severe photoinhibitory treatment, no significant loss of activity was observed for any PCR enzyme assayed. However, the inclusion of thylakoids in the photolysis system resulted in a substantial inactivation of the enzymes; this inactivation was significantly diminished in the presence of imidazole and enhanced to some extent by a partial deuteration of medium. In contrast, superoxide dismutase did not exert any effect. The blue light‐induced inactivation of the enzymes was remarkably decreased in the presence of thylakoids whose Fe‐S centers were destroyed. The results obtained in this study suggest that photoinactivation of the PCR enzymes in situ is mediated mainly by 1O2, which is photoproduced primarily by the Fe‐S centers of thylakoids and diffuses into the stroma.