Shoots, roots, and seeds of corn (Zea mays L., cv. Michigan 500), oats (Avena sativa L., cv. Au Sable), and peas (Pisum sativum L., cv. Wando) were analyzed for their superoxide dismutase content using a photochemical assay system consisting of methionine, riboflavin, and p-nitro blue tetrazolium. The enzyme is present in the shoots, roots, and seeds of the three species. On a dry weight basis, shoots contain more enzyme than roots. In seeds, the enzyme is present in both the embryo and the storage tissue. Electrophoresis indicated a total of 10 distinct forms of the enzyme. Corn contained seven of these forms and oats three. Peas contained one of the corn and two of the oat enzymes. Nine of the enzyme activities were eliminated with cyanide treatment suggesting that they may be cupro-zinc enzymes, whereas one was cyanide-resistant and may be a manganese enzyme. Some of the leaf superoxide dismutases were found primarily in mitochondria or chloroplasts. Peroxidases at high concentrations interfere with the assay. In test tube assays of crude extracts from seedlings, the interference was negligible. On gels, however, peroxidases may account for two of the 10 superoxide dismutase forms.

Superoxide Dismutases: I. Occurrence in Higher Plants

O2- oxidizes the [4Fe-4S] clusters of dehydratases, such as aconitase, causing-inactivation and release of Fe(II), which may then reduce H2O2 to OH- +OH.. SODs inhibit such HO. production by scavengingO2-, but Cu, ZnSODs, by virtue of a nonspecific peroxidase activity, may peroxidize spin trapping agents and thus give the appearance of catalyzing OH. production from H2O2. There is a glycosylated, tetrameric Cu, ZnSOD in the extracellular space that binds to acidic glycosamino-glycans. It minimizes the reaction of O2- with NO. E. coli, and other gram negative microorganisms, contain a periplasmic Cu, ZnSOD that may serve to protect against extracellular O2-. Mn(III) complexes of multidentate macrocyclic nitrogenous ligands catalyze the dismutation of O2- and are being explored as potential pharmaceutical agents. SOD-null mutants have been prepared to reveal the biological effects of O2-. SodA, sodB E. coli exhibit dioxygen-dependent auxotrophies and enhanced mutagenesis, reflecting O2(-)-sensitive biosynthetic pathways and DNA damage. Yeast, lacking either Cu, ZnSOD or MnSOD, are oxygen intolerant, and the double mutant was hypermutable and defective in sporulation and exhibited requirements for methionine and lysine. A Cu, ZnSOD-null Drosophila exhibited a shortened lifespan.

Superoxide Radical and Superoxide Dismutases

Assaying for superoxide dismutase activity: Some large consequences of minor changes in conditions

Generation of Superoxide Radical during Autoxidation of Hydroxylamine and an Assay for Superoxide Dismutase

Inhibition of nitrite formation from hydroxylammoniumchloride: A simple assay for superoxide dismutase

Isolated cell-wall suspensions from horseradish in the presence of 5×10(-4) M MnCl2 catalyze the production of hydrogen peroxide at the expense of either NADPH or NADH. This reaction is inhibited by scavengers of the superoxide free radical ion such as ascorbate or dihydroxyphenols or by superoxide dismutase, and stimulated by monophenols such as p-coumaric acid. On comparison with isolated (commercial) horseradish peroxidase it becomes evident that (a) cell-wall-bound peroxidase(s) is (are) responsible for the production of hydrogenperoxide, involving the superoxide free radical ion as an intermediate of the complex reaction chain.

Formation of hydrogen peroxide by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.).

Photooxidation of hydroxylamine to nitrite by spinach (Spinacia oleracea L. and sugarbeet (Beta vulgaris L.) chloroplast lamellae in the presence of autoxidable electron acceptors is inhibited by either solubilized or membrane-bound superoxide dismutase (SOD). This inhibition is reversed by KCN. The rates of hydroxylamine photooxidation by chloroplast lamellae, a reaction which is apparently driven by the superoxide free-radical ion, was used for quantitating the amount of SOD bound to chloroplast lamellae, as compared to a soluble enzyme of defined concentration. After digitonin fragmentation of chloroplast lamellae, ca. 80% of the SOD activity is associated with subchloroplast particles sedimenting after 2 h centrifugation at 200 000 x g. Less than 10% of the SOD activity is associated with particles sedimenting after centrifugation for 30 min at 20 000 x g. 5-10% of the cyanide-sensitive SOD is recovered in the soluble fraction of the subchloroplast-free supernatant after centrifugation at 200 000 x g for 2 h.

Lamellar superoxide dismutase of isolated chloroplasts.

Formation of Nitrite from Hydroxylamine in the presence of illuminated chloroplast lamellae is inhibited by superoxide dismutase but not by catalase, indicating that the superoxide free radical ion and not H₂O₂ is responsible for the oxidation of hydroxylamine. Decarboxylation of α-keto acids on the other hand is strongly inhibited by catalase but only slightly by superoxide dismutase. Light-dependent hydroxylamine oxidation and decarboxylation of α-keto acids can be used, therefor, as specific and sensitive probes for the determination of either the superoxide free radical ion or hydrogen peroxide, respectively.Photosynthetic oxygen reduction in the presence of ferredoxin, (monitored by the above method) yields both H₂O₂ and O₂·⁻. The addition of an oxygen reducing factor (ORF, solubilized by heat - treatment of washed chloroplast lamellae) instead of ferredoxin, however, stimulates only the production of H₂O₂ , while O₂·⁻ - formation is not observed. The cooperation of ferredoxin and ORF during photosynthetic oxygen reduction by chloroplast lamellae apparently produces H₂O₂ not only by dismutation of O₂·⁻, but also by a separate mechanism involving ORF.

Determination of Superoxide Free Radical Ion and Hydrogen Peroxide as Products of Photosynthetic Oxygen Reduction

Ferredoxin-stimulated photosynthetic oxygen reduction and concommitant decarboxylation of glyoxylate by chloroplast lamellar systems is inhibited by ascorbate but ferredoxin dependent NADP⁺-reduction is not. In the presence of low potential electron acceptors (AQ or MV) this influence of ascorbate on glyoxylate decarboxylation by chloroplast lamellar systems is no longer observed.Using the diaphorase activity of NADP-ferredoxin reductase either bound to the chloroplast lamellar system or as an isolated enzyme fraction glyoxylate decarboxylation in the dark can be observed in the presence of NADPH + H⁺ and autooxidizable electron acceptors (AQ, MY, fer­ redoxin). The influence of ascorbate on this dark reaction, depends on whether the activity of the isolated or the lamellae-bound enzyme is measured:1. With the isolated enzyme no influence of ascorbate on AQ-, MV-or ferredoxin-stimulated glyoxylate decarboxylation is observed.2. The dark-reaction with NADPH + H⁺ as electron donor, catalyzed by the bound enzyme however is inhibited by ascorbate both in the presence of either ferredoxin or AQ. This and other observations support the view that the site of inhibition by ascorbate of oxygen reduction by chloroplast lamellar systems in the presence of ferredoxin is not identical with either the reducing side of photosystem I, ferredoxin or NADP-ferredoxin reductase. The site of inhibition by ascor­ bate is more likely connected with an additional pathway involved in photosynthetic oxygen re­ duction by chloroplast lamellar systems, which is located at the reducing side of photosystem I, i. e. in the vicinity of the NADP-ferredoxin reductase. By heat treatment of isolated chloroplast lamellar systems a factor is released showing the activity of an ascorbate-sensitive oxygen reductant upon illumination in the presence of chloroplast lamellar systems. A model for photosynthetic oxygen reduction is proposed which includes ferredoxin and a membrane-bound oxygen reductant in series. Oxygen reduction by this pathway is only operating when the available NADP is fully reduced

Adelheid Heupel

Papers

Inhibition of nitrite formation from hydroxylammoniumchloride: A simple assay for superoxide dismutase

Formation of hydrogen peroxide by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.).

Lamellar superoxide dismutase of isolated chloroplasts.

Determination of Superoxide Free Radical Ion and Hydrogen Peroxide as Products of Photosynthetic Oxygen Reduction

On the mechanism of photosynthetic oxygen reduction by isolated chloroplast lamellae.