Inactivation of Ascorbate Peroxidase in Spinach Chloroplasts on Dark Addition of Hydrogen Peroxide: Its Protection by Ascorbate

Dark addition of hydrogen peroxide to intact spinach chloroplastsresulted in the inactivation of ascorbate peroxidase accompaniedby a decrease in ascorbate contents. This was also the casein reconstituted chloroplasts containing ascorbate, NADP⁺, NAD⁺and ferredoxin. The addition of hydrogen peroxide during light,however, showed little effect on ascorbate contents and ascorbateperoxidase activity in either the intact or reconstituted chloroplasts.In contrast to ascorbate peroxidase, the enzymes participatingin the regeneration of ascorbate in chloroplasts (monodehydroascorbatereductase, dehydroascorbate reductase and glutathione reductase)were not affected by the dark addition of hydrogen peroxide.Ascorbate contents increased again by illumination of the chloroplastsafter the dark addition of hydrogen peroxide. These resultsshow that the inactivation of the hydrogen peroxide scavengingsystem on dark addition of hydrogen peroxide [Anderson et al.(1983) Biochim. Biophys. Acta 724: 69, Asada and Badger (1984)Plant & Cell Physiol. 25: 1169] is caused by the loss ofascorbate peroxidase activity. Ascorbate peroxidase activitywas rapidly lost in ascorbate-depleted medium, and protectedby its electron donors, ascorbate, isoascorbate, guaiacol andpyrogallol, but not by GSH, NAD(P)H and ferredoxin. (Received June 14, 1984; Accepted August 15, 1984)     

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.     

Phosphorylation assay in liquid scintillation counter using C̆erenkov radiation of P: Application to photophosphorylation

A simple procedure is described for the assay of phosphorylation using C?erenkov radiation to detect ³²P in a liquid scintillation counter. Unreacted ³²P_i is first removed from the reaction mixture as the phosphomolybdate complex by butanol/benzene extraction. Addition of ammonium hydroxide to the remaining aqueous fraction avoids color quenching, phase separation, and instability in the counting rate during measurement of ³²P. Application of this procedure to several photophosphorylation systems is included.     

Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxidase in Spinach Chloroplasts

Intact spinach chloroplasts scavenge hydrogen peroxide witha peroxidase that uses a photoreductant as the electron donor,but the activity of ruptured chloroplasts is very low [Nakanoand Asada (1980) Plant & Cell Physiol. 21 : 1295]. Rupturedspinach chloroplasts recovered their ability to photoreducehydrogen peroxide with the concomitant evolution of oxygen afterthe addition of glutathione and dehydroascorbate (DHA). In rupturedchloroplasts, DHA was photoreduced to ascorbate and oxygen wasevolved in the process in the presence of glutathione. DHA reductase(EC 1.8.5.1 [EC] ) and a peroxidase whose electron donor is specificto L-ascorbate are localized in chloroplast stroma. These observationsconfirm that the electron donor for the scavenging of hydrogenperoxide in chloroplasts is L-ascorbate and that the L-ascorbateis regenerated from DHA by the system: photosystem IferredoxinNADPglutathione.A preliminary characterization of the chloroplast peroxidaseis given. (Received April 16, 1981; Accepted June 3, 1981)     

Reduction of photosystem I reaction center, P-700, by plastocyanin in stroma thylakoids from spinach: Lateral diffusion of plastocyanin

The reduction kinetics of the photooxidized photosystem I reaction center (P-700⁺) by plastocyanin was studied in the stroma thylakoids prepared by the Yeda press treatment. The kinetics of the P-700⁺ reduction after flash excitation were biphasic and separated into two independent first-order reactions, the fast phase with a half-time of about 4 ms and the slow phase with a half-time of about 18 ms. Only the fast phase of the P-700⁺ reduction was sensitive to KCN and glutaraldehyde treatments of the thylakoids which block the plastocyanin site in the photosynthetic electron flow indicating that the fast phase is mediated by plastocyanin. However, the content of plastocyanin in the stroma thylakoids used was greatly decreased by the Yeda press treatment to only half that of P-700⁺ reduced in the fast phase. This indicates that one plastocyanin molecule turns over more than once in the single turnover of P-700⁺ rather than forming a fixed complex with P-700. On the other hand, the slow phase was not affected by KCN or glutaraldehyde treatment and its apparent rate constant linearly depended on the concentration of reduced dichlorophenolindophenol. These results indicate that the slow phase shows direct reduction of P-700⁺ by dichlorophenolindophenol. A second-order rate constant of 3.96 × 10⁵m^?1 s^?1 was obtained for the slow phase at pH 7.6, 25 °C. Analysis of reaction kinetics in the initial portion of the fast phase indicated initial interaction between P-700⁺ and the reduced plastocyanin and gave a half-time of 0.53 ms for the bimolecular reaction. We assumed the lateral diffusion of plastocyanin on the thylakoid membrane and calculated the two-dimensional diffusion coefficient for plastocyanin from the half-time of the initial reduction of P-700⁺ as about 2 × 10^?9 cm² s^?1.     

cDNA Cloning of Monodehydroascorbate Radical Reductase from Cucumber: a High Degree of Homology in Terms of Amino Acid Sequence between This Enzyme and Bacterial Flavoenzymes

The FAD-enzyme monodehydroascorbate (MDA) reductase catalyzesthe regeneration of ascorbate from the MDA radical using NAD(P)Has the electron donor [Hossain and Asada (1985) J. Biol. Chem.260: 12920]. We cloned a cDNA of MDA reductase from cucumberseedlings and deduced its entire sequence of amino acid residues.The cDNA library from cucumber seedlings in the expression vectorwas screened with an antiserum against cucumber MDA reductase.Inserts from three immunoscreened clones hybridized with twooligonucleotide probes designed on the basis of the sequencesof two peptide fragments from the cucumber enzyme. The nucleotidesequences of these three clones were determined and the longestone contained an open reading frame of 1,302 bp in length. Themolecular mass of the translation product predicted from theopen reading frame was 47 kDa, the same as that determined forthe purified enzyme. The amino acid sequences determined fromfragments of lysyl endopeptidase-digested MDA reductase couldbe aligned with that deduced from the open reading frame, althoughsubstitution of several residues was apparent. Thus, the openreading frame encoded an isozyme of MDA reductase of cucumberdifferent from the purified enzyme. MDA reductase has the FAD-and NAD(P)H-binding domains of flavoproteins but shares onlylimited homology in terms of amino acid sequence with flavoenzymesfrom eukaryotes. ¹Research Institute of Innovative Technology for the Earth (RITE),Kizu, Kyoto, 619-02 Japan     

Inactivation of Ascorbate Peroxidase by Thiols Requires Hydrogen Peroxide

The hydrogen peroxide-dependent oxidation of ascorbate by ascorbateperoxidase from tea leaves was inhibited by thiols, such asdithiothreitol, glutathione, mercaptoethanol and cysteine. Thesethiols themselves did not inactivate the enzyme. However, theyinactivated the enzyme when hydrogen peroxide was produced bythe metal-catalyzed oxidation of thiols or when exogenous hydrogenperoxide was added. Thiols were oxidized by ascorbate peroxidaseand hydrogen peroxide to thiyl radicals, as detected by theESR spectra of the thiyl radical-5,5'-dimethyll- pyrroline-N-oxidieadducts. Inactivation of ascorbate peroxidase by thiols andhydrogen peroxide is caused by the interaction of the enzymewith the thiyl radicals produced at its reaction center. (Received September 10, 1991; Accepted December 9, 1991)     

Methyl Viologen-Dependent Cyclic Electron Transport in Spinach Chloroplasts in the Absence of Oxygen

The methyl viologen (MV)-dependent, linear electron flow fromPS II to PS I was severely blocked in intact or broken, uncoupledchloroplasts when oxygen was removed from the suspension medium,as revealed by measurements of chlorophyll fluorescence andthe rate of photoreduction of MV. Kinetics of the reductionof pre-oxidized P700 by a saturating light pulse showed thatreduced MV in the absence of oxygen re-reduces P700⁺ via theintersystem electron transport chain. Since the re-reductionof P700⁺ was inhibited by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone,the MV-mediated cyclic electron flow, in contrast to the phenazinemethosulphate-catalyzed one, involves the plastoquinone pool.However, 2-n-heptyl-4-hydro-xyquinoline-N-oxide, 2-n-nonyl-4-hydroxyquinoline-N-oxideand antimycin A did not inhibit the MV-mediated flow. Thus,the inhibition of the linear electron flow in chloroplasts underanaerobic conditions suggested the overreduction of the plastoquinonepool as a result of the MV-mediated cyclic flow (Received February 13, 1990; Accepted March 31, 1990)     

The FAD-Enzyme Monodehydroascorbate Radical Reductase Mediates Photoproduction of Superoxide Radicals in Spinach Thylakoid Membranes

The photoreduction of dioxygen in spinach thylakoid membraneswas enhanced about 10-fold by the FAD-enzyme monodehydroascorbateradical (MDA) reductase at 1 µM. The primary photoreducedproduct of dioxygen catalyzed by MDA reductase was the superoxideradical, as evidenced by the inhibition of photoreduction ofCyt c by superoxide dismutase. The apparent K_m for dioxygenof the MDA reductase-dependent photoreduction of dioxygen was100 µM, higher by one order of magnitude than that observedwith thylakoid membranes only. Glutathione reductase, ferredoxin-NADP⁺reductase, and glycolate oxidase also mediated the photoproductionof superoxide radicals in thylakoid membranes at rates similarto those with MDA reductase. Among these flavoenzymes, MDA reductaseis the most likely mediator stimulating the photoreduction ofdioxygen in chloroplasts; its function in the protection fromphotoinhibition under excess light is discussed. (Received February 24, 1998; Accepted May 19, 1998)