Kozi Asada

Superoxide anion permeability of phospholipid membranes and chloroplast thylakoids

The permeability of phospholipid membranes to the superoxide anion (O-2) was determined using soybean phospholipid vesicles containing FMN in the internal space. The efflux of O-2 generated by the illumination of FMN was so slow that more than 90% of the radicals were spontaneously disproportionated within the vesicles before they could react with cytochrome c at the membrane exterior. The amount of diffused O-2 was proportional to the intravesicular concentration of O-2 over a range from 1 to 10 microM which was deduced from its disproportionation rate. The permeability coefficient of the phospholipid bilayer for O-2 was estimated to be 2.1 X 10(-6) cm s-1 at pH 7.3 and 25 degrees C. Superoxide dismutase trapped inside vesicles was not reactive with extravesicular O-2 unless Triton X-100 was added. O-2 generated outside spinach chloroplast thylakoids did not interact with superoxide dismutase or cytochrome c which had been enclosed in the thylakoids. Thus, chloroplast thylakoids also showed little permeability to O-2.

Chloroplasts: formation of active oxygen and its scavenging

Publisher Summary In chloroplasts, chlorophyll-photosensitized production of 1O2 is an unavoidable reaction. Chloroplast thylakoids univalently photoreduce molecular oxygen producing O2- through the autoxidation of an electron acceptor in photosystem I. In chloroplasts, O2- produced in thylakoids is disproportionated by superoxide dismutases (SOD) in the stroma, and the H2O2 thus produced is reduced to water by ascorbate peroxidase. Ascorbate is regenerated from its oxidation products, dehydroascorbate (DHA) and monodehydroascorbate, by photoreductants through the system shown diagrammatically in this chapter. Ascorbate peroxidase, DHA reductase, and nicotinamide adenine dinucleotide (NADH)-dependent monodehydroascorbate reductase are localized in the chloroplast stroma, as are the enzymes participating in the generation of glutathione (GSH) and NADH. The production of O2- is enhanced under conditions where the generation rate of photoreductant in thylakoids exceeds that required for CO2 reduction; low CO2 concentration and high light intensity. O2- is, however, produced even when CO2 is supplied to chloroplasts and the photoproduction of O2- appears to be indispensable for the prevention of overreduction of electron carriers in the cyclic electron transport pathway. Thus, the photorcduction of O2- is an inevitable reaction in chloroplasts. Scavenging of O2- and H2O2 is essential for chloroplasts to maintain their ability to fix CO2, because several enzymes in the CO2-reduction cycle are sensitive to active oxygen. Production of O2- and H2O2 and their scavenging system are discussed in this chapter.

Radical Production and Scavenging in the Chloroplasts

Under the conditions where leaves are exposed to high photon flux densities in excess of their photon-utilizing capacity, excess photons produce active oxygens, radicals and triplet excited pigments, which cause photoinhibition of photosynthesis as a result of oxidation of target molecules. After a brief discussion on the relationship between radicals and active oxygens, the primary target molecules of these reactive molecules are discussed in relation to photoinhibition. Subsequently, the photoproduction of active oxygens and radicals in chloroplasts is described. To avoid photoinhibition, prompt scavenging of active oxygens and radicals at the sites where they are produced is essential in the chloroplasts. The enzymes participating in this scavenging and their microcompartmentation in chloroplasts are reviewed.

CALCIFICATION IN THE ARTICULATED CORALLINE ALGA CORALLINA-PILULIFERA, WITH SPECIAL REFERENCE TO THE EFFECT OF ELEVATED CO2 CONCENTRATION

Calcification in Corallina pilulifera Postels et Ruprecht displayed diurnal variations in aerated (350 ppm CO2) culture media, with faster rates during the light than during the dark period. Addition of CO2 (air+1250 ppm) inhibited calcification. This was attributable to the decreased pH resulting from CO2 addition. Both photosynthesis and calcification were enhanced in seawater, with elevated dissolved inorganic carbon concentrations at a constant pH of 8.2.

INFLUENCE OF ENHANCED CO2 ON GROWTH AND PHOTOSYNTHESIS OF THE RED ALGAE GRACILARIA SP AND G-CHILENSIS

The influence of elevated CO2 concentrations on growth and photosynthesis ofGracilaria sp. andG. chilensis was investigated in order to procure information on the effective utilization of CO2. Growth of both was enhanced by CO2 enrichment (air + 650 ppm CO2, air + 1250 ppm CO2, the enhancement being greater inGracilaria sp. Both species increased uptake of NO3− with CO2 enrichment. Photosynthetic inorganic carbon uptake was depressed inG. chilensis by pre-culture (15 days) with CO2 enrichment, but little affected inGracilaria sp. Mass spectrometric analysis showed that O2 uptake was higher in the light than in the dark for both species and in both cases was higher inGracilaria sp. The higher growth enhancement inGracilaria sp. was attributed to greater depression of photorespiration by the enrichment of CO2 in culture.

ENHANCED GROWTH OF THE RED ALGA PORPHYRA-YEZOENSIS UEDA IN HIGH CO2 CONCENTRATIONS

Leafy thalli of the red algaPorphyra yezoensis Ueda, initiated from conchospores released from free-living conchocelis, were cultured using aeration with high CO2. It was found that the higher the CO2 concentration, the faster the growth of the thalli. Aeration with elevated CO2 lowered pH in dark, but raised pH remarkably in light with the thalli, because the photosynthetic conversion of HCO3− to OH− and CO2 proceeded much faster than the dissociation of hydrated CO2 releasing H+. Photosynthesis of the alga was found to be enhanced in the seawater of elevated dissolved inorganic carbon (DIC, CO2 + HCO3− + CO3−). It is concluded that the increased pH in the light resulted in the increase of DIC in the culture media, thus enhancing photosynthesis and growth. The relevance of the results to removal of atmospheric CO2 by marine algae is discussed.

Superoxide production in aprotic interior of chloroplast thylakoids

The site of superoxide production in spinach thylakoids was found to be the aprotic interior of the thylakoid membranes near the P700 chlorophyll a protein at the reaction center of photosystem I complexes. This conclusion was drawn from the following findings. (i) Cytochrome c reduction by illuminated thylakoids, which was confirmed to be superoxide dependent by the failure of this reaction to occur in anaerobiosis, was completely inhibited by a dibutyl catechol, but partially inhibited by a hydrophilic disulfonated derivative. (ii) P700 chlorophyll a proteins were preferentially iodinated by lactoperoxidase by the use of hydrogen peroxide that was derived from the disproportionation of superoxides in illuminated thylakoids. (iii) Hydrogen peroxide production and oxygen uptake were induced by ammonium chloride, a proton conductor that can permeate through thylakoid membranes, but whole superoxide in the bulk solution was oxidized back to molecular oxygen by cytochrome c. The effective concentration of ammonium chloride decreased to one-sixtieth of the original, when an ammonium ion ionophore, nonactin, was added. Thus, the weak acid allowed superoxide to yield hydrogen peroxide disproportionately in the thylakoid membrane interior.

Superoxide dismutases in photosynthetic organisms: Absence of the cuprozinc enzyme in eukaryotic algae

Abstract Superoxide dismutases in photosynthetic organisms at different evolutionary levels were characterized using the criterion that the Cu,Zn-enzyme is sensitive to cyanide while the Mn- and Fe-enzymes are insensitive. The effect of the antibody against spinach Cu,Zn-superoxide dismutase was also tested as a means of distinguishing the several forms of the enzyme. Superoxide dismutase activity in extracts from photosynthetic bacteria, prokaryotic algae (blue-green algae), and eukaryotic algae (red, green, and brown algae, diatoms, Euglena , and Charophyta) were insensitive to cyanide and to the antibody, suggesting the presence of the Fe- and/or Mn-enzymes and the absence of the Cu,Zn-enzyme. In contrast, ferns, mosses, and seed plants including gymnosperms and angiosperms contained the Cu,Zn-superoxide dismutase in addition to the cyanidein-sensitive enzyme in soluble or bound form. Although an aerial green alga lacks the Cu,Zn-superoxide dismutase, aquatic angiosperms and ferns, like other land plants, contain this form of superoxide dismutase. Thus the distribution of the Cu,Zn-superoxide dismutase does not reflect the habitat but, rather, the phylogeny of the organism. The relation between the oxygen concentration in the atmosphere and the appearance of various forms of superoxide dismutase during the evolution of photosynthetic organisms is discussed.

Oxidation of manganous pyrophosphate by superoxide radicals and illuminated spinach chloroplasts.

Abstract The oxidation of Mn 2+ -pyrophosphate to Mn 3+ by superoxide (O 2 − ) was quantitative as evidenced from the formation of Mn 3+ -pyrophosphate and hydrogen peroxide and from the inhibition by superoxide dismutase. Using the competitive relation between Mn 2+ -pyrophosphate and superoxide dismutase for the O 2 − , the rate constant of Mn 2+ oxidation was estimated to be about 6 × 10 6 m −1 s −1 . The oxidation of Mn 2+ -pyrophosphate by illuminated chloroplasts was also indicated to be stoichiometrically induced by O 2 − . In the presence of saturating amounts of the Mn 2+ , a double enhancement of hydrogen peroxide production and triple uptake of oxygen were found, as expected from the oxidation of Mn 2+ -pyrophosphate by O 2 − . Anaerobiosis or superoxide dismutase annuled these increments. We propose that the O 2 − generated as the sole initial step of the Mehler reaction oxidized Mn 2+ -pyrophosphate, and we discuss the role of free manganese in chloroplasts.

COOH-terminal residues of D1 and the 44 kDa CPa-2 at spinach photosystem II core complex

The COOH‐termini of the 32 kDa D1 and 44 kDa CPa‐2 were determined by protein sequencing of peptides from trypsinized photosystem II core complexes. COOH‐terminal fragments were isolated by affinity chromatography using anhydrotrypsin‐agarose. One peptide had a sequence corresponding to the segment from Asn at position 335 to Ala at position 344 of the sequence deduced from the psbA gene coding for D1. Nine amino acids may be cleaved from the COOH‐terminus of pre‐D1 during maturation. In contrast, CPa‐2 was not modified at its COOH‐terminus.