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ISSN 1607-6729, Doklady Biochemistry and Biophysics, 2006, Vol. 408, pp. 113–116. © Pleiades Publishing, Inc., 2006 Original Russian Text © M.M. Mubarakshina, S.A. Khorobrykh, M.A. Kozuleva, B.N. Ivanov, 2006, published in Doklady Akademii Nauk, 2006, Vol. 408, No. 1, pp. 118–121.

BIOCHEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY

Intramembrane Formation of Hydrogen Peroxide during Oxygen Reduction in Thylakoids of Higher Plants M. M. Mubarakshina, S. A. Khorobrykh, M. A. Kozuleva, and B. N. Ivanov Presented by Academician V.A. Shuvalov November 7, 2005 Received December 22, 2005

DOI: 10.1134/S160767290603001X

Reactive oxygen species (ROS)—superoxide anion •– radical (superoxide) O 2 and hydrogen peroxide H2O2—are currently regarded not only as inducers of destructive processes in cells but also primarily as factors involved in redox signaling. These factors ensure both coordination of intracellular processes and adequate response of the entire organism to diseases and environmental changes: it is believed that the stable and mobile H2O2 molecule is the key signaling agent [1–3]. Photosynthetic electron transport chain (PETC) of chloroplasts is one of the key ROS producers in plants [4, 5]. The primary product of é2 reduction in PETC is superoxide, which is generated both in the acceptor side of photosystem I (PSI) [4] and in the plastoquinone •– pool [6, 7]. It was established that O 2 can be generated within the thylakoid membrane [8], whereas H2O2 is •–

thought to be the result of dismutation of O 2 outside the membrane. In this study, we showed that part of H2O2 is generated in the membrane, which may be essential for the implementation of the signal function by these molecules. Thylakoids were isolated from pea leaves as described in [6] and suspended in a medium containing 0.4 M sucrose, 20 mM NaCl, 5 mM MgCl2, 25 mM HEPES–KOH (pH 7.6). The suspension was added to a reaction medium containing 0.1 M sucrose, 20 mM NaCl, 5 mM MgCl2, 50 mM HEPES–KOH (pH 7.8), and 1 µM gramicidin D to a chlorophyll concentration of 10 µg/ml. To render granum thylakoids unstacked, the media used for their isolation and suspension, as well as the reaction medium, contained no NaCl and MgCl2, and the concentration of ä+ in the reaction medium, which in these experiments contained 25 mM HEPES, was 10–12 mM. The absence of grana was Institute of Fundamental Problems in Biology, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia

monitored by measuring light scattering and chlorophyll fluorescence quantum yield in the presence of diuron in suspensions of the control and unstacked thylakoids [9]. The content of chlorophyll in thylakoids was determined by 95% ethanol extract [10]. The reaction medium (21°ë) was illuminated through a KC-10 red light filter (λ > 600 nm) with light with an intensity of 500 µmol photons s–1 m–2. The content of é2 in vessel was measured with a Clark-type oxygen electrode. Cytochrome c reduction was determined by changes in the optical density ∆A550–540 using the differential extinction coefficient 19 mM–1 cm–1 [6]. It was found that the rate of cytochrome c reduction reaches a plateau at concentrations of ~30 µM. Cytochrome c, an oxidant (scavenger) of superoxide radicals that does not penetrate the membrane, was added to thylakoids to prevent H2O2 formation in the light as a result of dismutation of these radicals generated on the outer surface of the membrane or released from the membrane to the medium. An addition of catalase to thylakoid suspension after illumination even in the presence of cytochrome c led to oxygen evolution (Figs. 1a, 1b), whereas an addition of catalase before illumination had no effect. Thus, the production of H2O2 in spite of scavenging of “external” superoxide by cytochrome Ò was not suppressed completely, suggesting that H2O2 is generated inside the thylakoids. It was found that the rate of H2O2 generation, estimated by the amount of O2 evolved after catalase addition, was 7.4 µmol/h per mg Chl, which corresponds to the rate of electron transport through this pathway equal to 14.8 µeq/h per mg Chl (Fig. 1b). To estimate which part of the total electron transport along PETC is accounted for by this transport, the rates of changes in O2 concentration ( V O2 ) and cytochrome c reduction (Vcyt) were measured in the same suspension (Table 1). The last value, taking into account that one molecule of evolved O2 corresponds to four electrons entered in PETC from water (this stoichiometry of cytochrome Ò reduction under our conditions was verified experimentally), gave

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Figure 1c shows that considerable H2O2 production in the presence of cytochrome Ò takes place in unstacked thylakoids. The proportion of electrons involved in such H2O2 formation decreases, on average, by 10% (data not shown) compared to normal thylakoids. It cannot be ruled out that this change results •– from the fact that the reaction of O 2 with cytochrome Ò in grana is hampered; however, it is more likely to be the consequence of changes in interaction between PETC carriers under the conditions of thylakoid unstacking [12].

D

4 µM O2

(‡)

The addition of potassium cyanide to the suspension (Table 1, experiment I) or sodium azide (not shown)— inhibitors of plant superoxide dismutases (SODs), had no effect on the number of electrons involved in H2O2 formation in the presence of cytochrome Ò. This finding indicates that the formation of H2O2 is not the result of

+ catalase

1 min

L

•–

D

O 2 dismutation catalyzed by SOD that might contaminate thylakoids during isolation.

(b) + catalase

L

D

(c) + catalase Fig. 1. Light-induced changes in the oxygen concentration in the suspension of (a, b) intact and (c) unstacked thylakoids in the (a) absence and (b, c) presence of 60 µM cytochrome c. The final concentration of catalase was 500 U/ml. Designations: L, light; D, darkness.

the rate of O2 evolution during electron transfer to cytochrome Ò. By subtracting this rate from V O2 , the rate of uptake of O2 molecules incorporated into H2O2 was determined. Taking into account the known stoichiometry between this uptake and electron transfer from water (1O2 : 4–) [11], the electron flow (Ve, H2O2) involved in H2O2 formation in the presence of cytochrome Ò was calculated, and its proportion in the total electron flow (êth, Pth = V e, H2 O2 /Ve) was found (Table 1). This value varied from 35 to 55% and constituted, on average, 40% (n = 12).

Hydrogen peroxide can be produced in the presence of cytochrome c in the internal space of thylakoids •– (lumen) if part of O 2 enters the lumen and dismutates in it. To assess the amount of superoxide involved in H2O2 formation in the lumen, the rate of oxygen uptake in the presence and absence of ascorbate in the suspension were measured in the presence of SOD, which pre•– vents the reaction between O 2 and ascorbate outside thylakoids. When superoxide is reduced by ascorbate, the stoichiometry between O2 uptake and electron transfer from water for such superoxides becomes 3O2 : 4– [11], which leads to an increase in the oxygen uptake rate, with the electron transport rate remaining unchanged (which is limited in this case by the rate of O2 reduction [7]). The increment in the rate of oxygen uptake after ascorbate addition allowed us to calculate the proportion in the total flow of those electrons that •– produce O 2 , which react with ascorbate in the lumen where SOD does not penetrate (Table 2). It was, on average, 9% (n = 5). This is the maximum estimate, because the donation of electrons for PETC by ascorbate cannot be ruled out [13], which may additionally (not as a result of change in the O2 : – stoichiometry) increase the rate of oxygen uptake due to its reduction in PSI. A possible contribution of H2O2 production in the lumen into its overall production in thylakoids was also assessed indirectly, without addition of a reductant to the reaction medium. It is known that changes in pH lead to significant changes in the rate constant of the superoxide dismutation reaction. In view of this, it could be assumed that, if superoxide enters the lumen, the proportion of electrons involved in H2O2 formation should depend on pH value in the lumen. The pH value in the lumen was varied by exclusion of gramicidin D from the reaction medium (it is known that, at pH 7.8 in

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Table 1. Electron flow involved in intrathylakoid H2O2 formation (Pth , percent of the total electron transfer from water) Experiment I

II

V O2 , µmol Vcyt. c , µmol cyt. V e, H2 O2 , µeq Ve , µeq/h per mg Additive to reaction medium Chl O2/h per mg Chl c/h per mg Chl O2/h per mg Chl – + cyt. c + cyt. c; + KCN – + cyt. c + cyt. c; – gram. D

–5.5 ± 0.3 1.9 ± 0.2 2.4 ± 0.3 –6.0 ± 0.7 2.3 ± 0.3 2.5 ± 0.4

22 16.4 15.2 24 13.3 13.8

24 ± 1.1 24.9 ± 0.1 22.5 ± 1.1 23.8 ± 0.3

22.0 40.4 40.1 24.0 35.8 37.6

Pth , % 75* 40 38 55* 37 37

Note: The final concentrations of cytochrome c and KCN in the main reaction medium were 40 µM and 1 mM, respectively. Sign “–” shows a decrease in the oxygen content in medium; The asterisk shows the values that were calculated using the V e, H O values, 2

2

determined in this experiment in the presence of cytochrome c, and the Ve value determined in the absence of cytochrome c.

Table 2. Electron flow involved in H2O2 formation in the lumen (Plu , percent of the total electron transfer from water) Additive to reaction medium + SOD + SOD; + ascorbate + cyt. c

V O2 , µmol Vcyt. c , µmol cyt. c/h Ve , µeq/h per mg per mg Chl Chl O2/h per mg Chl –12.5 ± 0.3 –13.8 ± 0.4 4.1 ± 0.4

50 50* 85.1

50.7 ± 1.7

Plu , %

Pth , %

5 40

Note: Plu = [1/2(B – A)/A] × 100, %, where A and B are the rates of oxygen uptake in the absence and presence of ascorbate, respectively. The experiment with the thylakoid preparation in the presence of cytochrome c is shown for a comparison. The final concentrations of cytochrome c, sodium ascorbate, and SOD were 40 µM, 1 mM, and 100 U/ml, respectively. The asterisk show the value that was taken equal to that measured in the absence of ascorbate (see text).

medium, the pH value in the lumen in the light is lower in the absence of gramicidin D than in its presence [14]). However, varying pH value in the lumen had no marked effect on the above value (Table 1, experiment II). As seen from Table 1, the rate of electron transfer along PETC is greater in the presence of cytochrome c than in its absence, apparently, owing to the transfer of additional electrons to cytochrome c from PETC. However, in the presence of cytochrome c, the overall electron transfer rate even was considerably lower than the ability of PETC to transfer electrons, because under similar conditions in the presence of methylviologen, an effective electron acceptor, the rate of electron transfer reached 600 µeq/h per mg Chl [7]. This assumes saturation of all potential pathways of electron transfer both in the presence and absence of cytochrome c. Therefore, the electron flow involved in H2O2 formation inside thylakoids in the absence of cytochrome c is, at least, no lower than that measured in its presence. Using the last value, it is possible to estimate the proportion of this flow in the total electron transfer in the absence of cytochrome c (numbers with asterisk in Table 1). It was found to be, on average, 63% (n = 10). Thus, a considerable part of H2O2 formed in the course of oxygen reduction in thylakoids is generated inside the thylakoid membranes. However, the rate of DOKLADY BIOCHEMISTRY AND BIOPHYSICS

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O 2 dismutation in them is low due to electrostatic repulsion in media with low permittivity. Recently, we found that the contribution of the plastoquinone pool to oxygen reduction may reach 70% in actinic light and assumed that H2O2 is generated in the thermodynami•–

cally favorable reaction of O 2 reduction by plastoquinone [7]. Numerous data indicate that the development of adaptive responses of plants depend on the redox state of the plastoquinone pool [5, 15]. The H2O2 molecules formed with the involvement of this pool may serve as signals for the expression of chloroplast and nuclear genes. Hydrogen peroxide generation within membrane is of principal importance, because the molecules formed there may be released into the stroma in any site of the membrane, bypassing the systems of H2O2 detoxication concentrated in the vicinity of PSI [4]. Furthermore, H2O2 may possibly quit the chloroplast without being released into the stroma. ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research, project no. 05-04-48629. 2006

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