Plant & Cell Physiol. 21(8): 1431-1438 (1980)

Reduction of plastocyanin with 02- and superoxide dism.utase-dependent oxidation of plastocyanin

by H202

1 The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611 and 2School of Medicine, Tottori University, Yonago, Tottori 683, japan (Received july 14, 1980)

Type I copper proteins, plastocyanin and rice blue protein, were reduced with 02-. The reduction rate constants of plastocyanins from several sources with 02- are about 106 M-1 sec-1 (1.0 X 106 M-1 sec-1 for spinach and kidney bean plastocyanins and 6.7 X 105 M-1sec-1 for japanese radish plastocyanin) at pH 7.8 at 25°C and not significantly different from that observed for rice blue protein (7.3 X 105 M-1sec-1). Reduced plastocyanin was oxidized by H202 through the peroxidase-like reaction of Cu.Znsuperoxide dismutase. Key words: Plastocyanin reduction - Rice blue protein - Superoxide radical Superoxide dismutase.

Since the discovery of SOD in 1969 (22), the superoxide radical (0 2 - ) has been shown to be involved in many biological reactions (13). In illuminated chloroplasts when intrinsic electron acceptors are exhausted, molecular oxygen is reduced univalently by the primary electron acceptor of photosystem I (3). Most of the O 2 - generated is disproportionated into O 2 and H 20 2 by the SOD contained in chloroplast thylakoids and stroma (6). Thus, chloroplast SOD plays an important role as a scavenger of O 2- and protects the chloroplasts from the deleterious action of 02- and other active species of oxygen derived from O 2 - . In addition to disproportionation by SOD, O 2 - reacts with chloroplast components although at a slow rate. We reported earlier the reduction of cytochrome f (31) and the oxidation of manganous ion (20) and glutathione (2) by O 2 - . We report here the reduction by O 2 - of plastocyanin and a rice blue protein and oxidation of reduced plastocyanin by H 20 2 catalyzed by SOD.

Materials and rnerhods

Copper proteins andenzymes Plastocyanin was purified from leaves of kidney bean (Phaseolus vulgaris), Japanese radish (Raphanus sativus L. var. acanthiformis Makino), and spinach (Spinacea oleracea) as described by Katoh et al. (18). The absorbance ratios of A595/A 278 Abbreviation: SOD, Superoxide dismutase.

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Masa-aki Takahashi 1, Yasuhisa Kono 2 and Kozi Asada 1

M. Takahashi, Y. Kono and K. Asada

1432

Measurement

of reduction of copper proteins

Reduction of copper proteins with the 02--generating xanthine oxidase system (23) was followed by the decrease in absorbance at their peak wavelengths (reference at 700 nm) using a Hitachi 356 dual wavelength spectrophotometer. The concentration of the copper proteins was assayed spectrophotometrically using the following absorbance coefficients: 4.5 mM-! cm-1 at 595 nm for kidney bean plastocyanin (24) and Japanese radish plastocyanin, 9.8 mM-l cm-1 at 597 nm for spinach plastocyanin (18), and 4.73 mM-l cm-1 at 600 nm for rice blue protein (25).

Results and discussion

Reduction

ofplastocyanin and rice blue protein by superoxide

Reduction of plastocyanins from three plant species by the 02--generating xanthine oxidase system was observed [Reaction 1] (Fig. 1). Inhibition of the reduction by SOD confirms that the reductant is O 2- (Fig. 2). plastocyanin (Cu 2+) +0 2- ---+- plastocyanin (Cu+) +0 2 -Reaction 1 Incomplete reduction (90-95%) of plastocyanin by O 2- suggested that the oxidation of the reduced plastocyanin by O 2- occurs [Reaction 2], since no spontaneous oxidation of the copper-containing proteins by H 202 was found. plastocyanin (Cut) +02-+2H+ ---+- plastocyanin (Cu 2+) +H 202 -Reaction 2 The presence of a redox shuttle composed of reactions 1 and 2 was suggested and plastocyanin showed weak 02--disproportionation activity. A blue protein from rice bran was compared with plastocyanin with respect to the reactivity with O 2-. The characteristic intense blue colour (25) showed that the rice blue protein is a Type I copper 'protein like plastocyanin (22). On the other hand, plastocyanin differs from rice blue protein in redox potential and isoelectric point, which are +350 mV and 4.0 for plastocyanin (18) and +275 mV and 9.1 for rice blue protein (25), respectively. However, reduction of the rice blue protein by O 2- was observed without any marked difference from that of plastocyanin (Compare the trace for first 10 min in Fig. 3 with that in Fig. 1). The initial reduction rate increased with the increase of the copper protein con-

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were 0.25 '(kidney bean), 0.46 (Japanese radish) and 1.0 (spinach). Rice blue protein (25) was a gift from Dr. Y. Morita. Polyacrylamide gel electrophoresis of these copper proteins followed by staining the gel for SOD (8) did not show any active band. No peroxidase activity was detectable in these copper proteins when they were assayed with guiacol as substrate (11). Reduced plastocyanin was prepared by reduction with ascorbate followed by gel filtration using Sephadex G-25. Cu,Zn-superoxide dismutase was purified from spinach leaves (6). Milk xanthine oxidase, horse heart cytochrome c and bovine liver catalase were purchased from Boehringer. Other chemicals were of reagent grade.

Plastocyanin reduction by O 2 -

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0 2 4 6 Reaction time , min Fig. 1. Reduction ofplastocyanin by 02- generated b.y xanthine-xanthine oxidase system. Reaction was started by the addition of 5 pg of xanthine oxidase (XOD) to the reaction mixture (1 ml) containing 2.7 pM kidney bean plastocyanin, 50 pM xanthine, 0.1 mM EDTA and 50 ma phosphate (pH 7.8). A few grains of sodium ascorbate (Asc) were added where indicated.

centrations; the half-maximum of the initial rate was observed at 1.6 ftM for kidney

bean and Japanese radish plastocyanins and at .--0.5 flM for rice blue protein. The second-order reaction rate constant for copper protein reduction by O 2- is determined from the competition for O 2- between the copper protein and O 2- scavenger of known reactivity with O 2- when all the O 2- generated is used for the two reactions in a steady state (4, 27). A good straight line in the competitive inhibition plot indicates the 02--plastocyanin reaction is a simple second-order reaction. The reduction rate constant with O 2- was determined for the three plastocyanins and rice blue protein from the slope of the plot by assuming the catalytic rate constant of SOD with 02- as 2 X 109 M-l sec"! at pH 7.8 and 25°C (19, 28) (Table 1). Cytochrome c underwent no redox reaction with rice blue protein (25), and therefore was used as an 02--scavenger to determine the rate constant of rice blue protein with O 2 - . Discrepancy might arise from the cytochrome c reactivity with O 2 varying somewhat from 5 X 105 M-l sec-1 (10, 29) on preparation. The reaction rate constant of plastocyanin with O 2- is higher by one to four

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M. Takahashi, Y. Kono and K. Asada

1434

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Fig. 2. Inhibition of 02--dependent reduction of plastocyanin by SOD. Spinach SOD was added to the reaction mixture containing 3.8 pM kidney bean plastocyanin. The other conditions were the same as in Fig. 1. Plastocyanin reduction rate was determined from the initial slope of the reaction trace for each SOD concentration. The ordinate scale of the insert stands for the reciprocal of the relative reduction rate (vjV) of piastocyan in, where V is the rate observed without SOD and v is that with SOD.

orders of magnitude than with a number of small molecules except with 80 2(2.7 X 107 M-I sec-I) (21) and the intrinsic electron donor, cytochrome f (3.0-3.6 X 107 M-I sec-I) (32, 33). Plastocyanin is an acidic protein with net negative charges and rice blue protein is substantially positive in the reaction mixture at pH 7.8. Considering the plastocyanin structure represented by X-ray crystallography (12) and ESR spectroscopy (9), the hydrophobic environment of the copper binding site is perhaps one reason that both basic and acidic copper proteins react similarly with negatively charged O 2-. And the nature of the copper site may explain the lower 02--reactivity with plstocyanin than with Cu,Zn-80D having solvent-accesible copper-sites (15). Molecular oxygen is reduced univalently by photosystem I (3, 5). In addition to facilitating the reduction of cytochrome f (31) and plastocyanin, the O 2 - thus Table 1 Reduction rate constants of plastocyanin and rice blue protein with 02Copper proteins

Rate constants at pH 7.8 and 25°C (M-I sec-I)

Plastocyanin (spinach)

1.0 X 106 a

Plastocyanin (kidney bean)

1.0 X 106 a

Plastocyanin (Japanese radish)

6.7 X 105 a

Blue protein (rice)

7.3 X 105 a 9.1 X 105 b

The rate constants were determined from plots of competitive inhibition by

a

SOD or

b cytochrome

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.....0 0.3

1435

Plastocyanin reduction by O 2 -

SOD-dependent oxidation

of reduced plastocyanin by H 20 2

O 2- functions uniquely both as 'a reductant and an oxidant (13). The reduction of O 2- proceeds only when the reactant carries acidic protons in aprotic media. The hydrophobic copper site of Type I-copper protein may be a cause for the suggested slow oxidation of plastocyanin with O 2- via reaction 2. The addition of Cu,Zn-SOD to the reaction mixture, in which plastocyanin or rice blue protein had been reduced by the 02--generating system, however, caused the oxidation of the copper proteins (Fig. 3). The oxidation was confirmed by the increase in the absorbance at 595 nm and its disappearance upon addition of ascorbate. More than a hundredfold concentration of SOD was required to induce the oxidation in comparison to the concentration to inhibit plastocyanin reduction. While O 2reduced the copper proteins, no H 202 was produced, but after the copper proteins

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Fig. 3. Oxidation of rice blue protein induced by SOD in the xanthine-xanthine oxidase system. Reaction conditions were the same as in Fig. 1 except that 8.1 pMrice blue protein was added in place of plastocyanin. The arrows indicate the additions of 0.2 nmole of SOD and 10 pg of catalase.

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produced encounters the following reactions in chloroplasts: disproportionation catalyzed by SOD (6); oxidation of glutathione (2), ferredoxin (1) and ascorbate (27); and glycolate formation as an oxidant (7). It was previously suggested that over 90% of the O 2- produced in chloroplasts is scavenged by SOD and only 10% of the O 2- reacts with those chloroplast components in stroma (5). However, plastocyanin and cytochrome f bind to the thylakoid and would have a greater chance to react with O 2- and to form a cyclic electron flow around photosystem I if O 2- migrates with a limited diffusion rate from the 02--generating site enclosed in the lipid matrix. In fact, 02- has been shown to supply an electron to photosystem I (26). A reaction with photosystem II has also been suggested (30). Further studies are necessary to determine whether or not such a cyclic electron flow around photosystem I occurs in intact chloroplasts.

1436

M. Takahashi, Y. Kono and K. Asada

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Fig. 4. Oxidation of plastocyanin by H202 catalyzed by SOD. The reaction mixture (1 ml) contained 50 mMphosphate (pH 7.8), 0.1 mM EDTA and 2.5 p,M kidney bean plastocyanin, half of which was in a reduced form. Where indicated, 4 p,moles ofH202 and 0.5 nmole of SOD were added.

had been reduced the 02--generating system produced H 20 2 and O 2 through

disproportionation of O 2 - , Therefore, either H 20 2 or O 2 - is an oxidant. Since catalase inhibited the oxidation of plastocyanin, H 20 2 seems to be the oxidant (Fig. 3). This is supported by the oxidation of reduced plastocyanin or rice blue protein by the coexistence of H 20 2 and SOD. An experiment with kidney bean plastocyanin is shown in Fig. 4. Thus, SOD seems to work as a peroxidase for the oxidation of copper proteins. The SOD-dependent oxidation of the copper proteins by H 20 2 is similar to the oxidation of cytochrome c, diphenylisobenzofuran and linoleic acid in the presence of SOD and H 20 2 (17). Hodgson and Fridovich have proposed an oxidation mechanism in which a reaction between H 20 2-reduced Cu,Zn-SOD and H 20 2 is involved in the generation of an oxidant (16).

References

( 1) Allen, J. F.: A two-step mechanism for the photosynthetic reduction of oxygen by ferredoxin. Biochem. Biophys. Res. Commun. 66: 36-43 (1975). ( 2) Asada, K. and S. Kanematsu: Reactivity of thiols with superoxide. Agric. Bioi. Chern. 40: 1891-1892 (1976).

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c

0

Plastocyanin reduction by 02-

1437

(22) Malkin, R.: The copper containing oxidases. In Inorganic Biochemistry 2. Edited by G. L. Eichhorn. p. 689-709. Elsevier, Amsterdam, 1973. (23) McCord, J. M. and I. Fridovich: Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprein). J. Bioi. Chem. 244: 6049-6055 (1969). (24) Milne, P. R. and]. R. E. Wells: Structural and molecular weight studies on the small copper protein, plastocyanin. ibid. 245: 1566-1574 (1970). (25) Morita, Y., A. Wadano and S. Ida: Studies on respiratory enzymes in rice kernel Part VI. Characterization of blue protein of rice bran. Agric. Biol. Chem. 35: 255-260 (1971).

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( 3) Asada, K., K. Kiso and K. Yoshikawa: Univalent reduction of molecular oxygen by spinach chloroplasts on illumination. J. BioI. Chern. 249: 2175-2181 (1974). ( 4) Asada, K., M. Takahashi and M. Nagate: Assay and inhibitors of spinach superoxide dismutase. Agric. BioI. Chem. 38: 471-473 (1974). ( 5) Asada, K., M. Takahashi, K. Tanaka and Y. Nakano: Formation of active oxygen and its fate in chloroplasts. In Biochemical and Medical Aspects of Active Oxygen. Edited by O. Hayaishi and K. Asada. p. 45-63, University of Tokyo Press, 1977. ( 6) Asada, K., M. Urano and M. Takahashi: Subcellular location of superoxide dismutase in spinach leaves and preparation of crystalline spinach superoxide dismutase. Eur. J. Biochem. 36: 257-266 (1973). ( 7) Asami, S. and T. Akazawa: Enzymatic formation of glycolate in Chlomatium. Role of superoxide radical in a transketolase-type mechanism. Biochemistry 16: 2202-2207 (1977). ( 8) Baeuchamp, C. and I. Fridovich: Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44: 276-287 (1971). ( 9) Blumberg, W. E. and]. Peisach: The optical and magnetic properties of copper in Chenopodium album plastocyanin. Biochim.Biophys. Acta 126: 269-273 (1966). (10) Butler, ]., G. G. Jayson and A. J. Swallow: The reaction between the superoxide anion radical and cytochrome C. ibid. 408: 215-222 (1975). (11) Chance, B. and A. C. Maehly: Assay of catalase and peroxidases. In Methods in Eneymol. 2. Edited by S. P. Colowick and N. O. Kaplan. p. 764-775. New York, 1955. (12) Colman, P. M., H. C. Freeman,]. M. Guss, M. Murata, V. A. Norris, ].A. M. Ramshaw and M. P. Venkattappa: X-ray crystal structure analysis of plastocyanin at 2.7 A resolution. Nature 272: 319-324 (1978). (13) Fee,]. A.: Superoxide, superoxide dismutases and oxygen toxicity. In Metal Ion Activation of Dioxygen. Edited by T. S. Spiro. p. 209-237. Wiley Interscience, 1980. (14) Fillippo, J. S., Jr., C.-I. Chern and J. S. Valentine: Oxidative cleavage of a-keto, a-hydroxy, and a-haloketones, esters and carboxylic acids by superoxide. J. Org. Chem. 41: 1077-1078 (1976). (15) Gaber, B. P., R. D. Brown, S. H. Konig and]. A. Fee: Nuclear magnetic relaxation dispersion in protein solutions V. Bovine erythrocyte superoxide dismutase. Biochim. Biophys. Acta 271: 1-5 (1972). (16) Hodgson, E. K. and I. Fridovich: The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: Inactivation of the enzyme. Biochemistry 14: 5294-5299 (1975). (17) Hodgson, E. K. and I. Fridovich: The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: chemiluminescence and peroxidation. ibid. 14: 5299-5303 (1975). (18) Katoh, S., I. Shiratori and A. Takamiya: Purification and some properties of spinach plastocyanin. J. Biochem. 51: 32-40 (1962). (19) Klug, D.,]. Rabani and I. Fridovich: A direct demonstration of the catalytic action of superoxide dismutase through the use of pulse radiolysis. J. Biol. Chem. 247: 4839-4842 (1972). (20) Kono, Y., M. Takahashi and K. Asada: Oxidation of manganous pyrophosphate by superoxide radicals and illuminated chloroplasts. Arch. Biochem. Biophys. 174: 454-462 (1976). (21) Lambeth, D. o. and G. Palmer: The kinetics and mechanism of reduction of electron transfer proteins and other compounds of biological interest by dithionite. J. Biol. Chem. 248: 60956103 (1973).

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(26) Muallem, A. and S. Malkin: Anomalous oxygen uptake from isolated chloroplasts inhibited in photosystem II and without external electron donors. Biochim. Biophys. Acta 546: 175-182 (1979). (27) Nishikimi, M.: Oxidation of ascorbic acid with superoxide anion generated by the xanthinexanthine oxidase system. Biochem. Biophys. Res. Commun. 63: 463-468 (1975). (28) Rotilio, G., R. C. Bray and E. M. Fielden: A pulse radiolysis study of superoxide dismutase. Biochim. Biophys. Acta 268: 605-609 (1972). (29) Simic, M. G., 1. A. Taub,]. Tocci and P. A. Hurwitz: Free radical reduction offerricytochrome c. Biochem. Biophys. Res. Commun. 62: 161-167 (1975). (30) Takahama, U. and M. Nishimura: Light-induced chemiluminescence of luminol in spinach chloroplast fragments: Reaction of 02- with electron transfer components. Plant & Cell Physiol. 18: 1139-1148 (1977). (31) Tanaka, K., M. Takahashi and K. Asada: Isolation of monomeric cytochromejfrom]apanese radish and a mechanism of autoreduction. J. Biol. Chem. 253: 7397-7403 (1979). (32) Tanaka, K., M. Takahashi and K. Asada: Oxidation rate of monomeric radish cytochromej with plastocyanin. Plant & CellPhysiol. 22: (1981) (in press). (33) Wood, P. M.: Rate of electron transfer between plastocyanin, cytochrome}; related proteins and artificial redox reagents in solution. Biochim. Biophys. Acta 357: 370-379 (1974).

Reduction of plastocyanin with O2- and superoxide dismutase-dependent oxidation of plastocyanin by H2O2.

Type I copper proteins, plastocyanin and rice blue protein, were reduced with O2 (-). The reduction rate constants of plastocyanins from several sourc...
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