Participation of ferredoxin in oxygen reduction by the ... - Springer Link

1 downloads 0 Views 132KB Size Report
INTRODUCTION. The Mehler reaction—reduction of O2 by the components of the photosynthetic electron transport chain (PETC)—amounts to 30% of the ...
ISSN 0006-3509, Biophysics, 2007, Vol. 52, No. 4, pp. 393–397. © Pleiades Publishing, Inc., 2007. Original Russian Text © M.A. Kozuleva, I.A. Naidov, M.M. Mubarakshina, B.N. Ivanov, 2007, published in Biofizika, 2007, Vol. 52, No. 4, pp. 650–655.

CELL BIOPHYSICS

Participation of Ferredoxin in Oxygen Reduction by the Photosynthetic Electron Transport Chain M. A. Kozuleva, I. A. Naidov, M. M. Mubarakshina, and B. N. Ivanov Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia Received July 27, 2006; in final form, December 22, 2006

Abstract⎯Oxygen reduction by isolated pea thylakoids was studied in the presence of ferredoxin (Fd), Fd + NADP, and cytochrome c. At Fd concentrations optimal for NADP reduction, it contributed 30–50% of the reducing equivalents (as deduced by comparing the rates of oxygen reduction and light oxidation of reduced Fd). The oxygen reduction rate in the presence of Fd + NADP was 3–4 times lower than with Fd alone, and comparable to that with cyt c. It is supposed that the process involves a photosystem I component whose reaction with oxygen depends on the rate of electron efflux from the PS I terminal acceptors, and that this component is phylloquinone. DOI: 10.1134/S0006350907040069 Key words: photosynthesis, thylakoid membrane, photosystem I, O2 reduction, ferredoxin, phylloquinone

INTRODUCTION

which is interpreted as acceleration of electron transfer to oxygen owing to Fd mediation between the PS I and O2. Oxygen uptake with or without Fd is suppressed to zero by catalase, indicating that it results from reduction of O2 by the electrons fed into PETC:

The Mehler reaction—reduction of O2 by the components of the photosynthetic electron transport chain (PETC)—amounts to 30% of the overall PETC throughput in the chloroplasts of C3 plants under conditions optimal for CO2 fixation, as indicated by mass spectrometric measurements [1, 2]. The interest in this process generating reactive oxygen species has recently increased in view of the problem of intracellular redox signaling.

water oxidation in PS II, H 2 O − 2e − → 1 / 2O 2 + 2H + ;

(1)

superoxide radical generation

The primary product of O2 reduction in PETC is superoxide anion radical O −2⋅ [3], and it is supposed that the main reductants are the components of the acceptor part of photosystem I with the lowest redox potentials among the PETC carriers [4]. However, the mechanism of such reduction (the redox potential of the O2/O −2⋅ pair in water is –160 mV [4]) remains obscure. One opinion is that the major participant is ferredoxin (Fd) [5], a water-soluble protein with a redox potential of –420 mV accepting electrons from the membrane-bound PS I components. Addition of Fd to isolated thylakoids increases O2 uptake [6–8],

2O 2 + 2e − → 2O −2⋅

(2)

2O −2⋅ + 2H + → H 2 O 2 + O 2 ;

(3)

and dismutation

peroxide destruction by catalase 2H 2 O 2 → 2H 2 O + O 2 .

(4)

Yet, Fd oxidation by oxygen is known to be slow [9, 10], the rate constant being 10–3 M–1 s–1 [10]. The other opinion [3, 4, 11] is that Fd is unrelated to O2 reduction even if present, and the process involves PS I terminal acceptors such as FX or FA/B. Thus, despite long research, the role of Fd remains ambiguous. Here we measured the electron flux to oxygen through Fd when either O2 was the sole final

Abbreviations: Fd, ferredoxin; PETC, photosynthetic electron transport chain; PS, photosystem. 393

394

KOZULEVA et al.

8.97 mM–1 cm–1. The redox state of Fd was determined by measuring ΔA463 and using the difference extinction coefficient of 5.4 mM–1 cm–1 [15]. The measuring light path was 5 mm. The cuvettes were illuminated in the spectrophotometer (Hitachi-557) compartment in the perpendicular direction (suspension thickness, 4 mm) through a KS-14 filter (λ > 620 nm) at an irradiance of 350 μmol quanta s–1 m–2 (measured at the cell front with LiCor-260); these conditions provided an equal rate of Fd reduction in both cells. The photomultiplier was protected from stray light with an SZS-21 filter. Redox state of ferredoxin in a thylakoid suspension upon switching on (↑) and off (↓) the light (λ > 620 nm, 350 μmol quanta s–1m–2) without (a) and with 0.5 mM NADP (b); see comments in text.

acceptor or the system included NADP+ as the main physiological electron acceptor in chloroplasts. EXPERIMENTAL Thylakoids and Fd were isolated from pea leaves [12, 13]. Membranes were suspended (0.1 M sucrose, 20 mM NaCl, 5 mM MgCl2, 50 mM HepesKOH pH 7.8 with 1 μM gramicidin D) to 10 μg/ml chlorophyll (determined using 95% ethanol extraction [14]). Molecular oxygen in the thylakoid suspension was monitored with a Clark electrode in a thermostated (21°C) cell. The overall Fd concentration was determined using the extinction coefficient ε463 = Table 1. Rates of oxygen reduction in an illuminated thylakoid suspension VOe and ferredoxin oxidation in the dark 2 (VFdox ) at different Fd concentrations, and the effect of 1 μM antimycin A (AmA) [Fd], V e , μeq (mg VFdox , μeq (mg V ox /V , % AmA O 2 –1 –1 O2 Fd μM Chl) h Chl)–1 h–1 5

15

30



114.8 ± 0.5

23.58 ± 0.7

20.5 ± 0.5

+

125.3 ± 2.2

24.1 ± 0.9

19.3 ± 1.0



173.9 ± 1.3

59.7 ± 4.2

34.4 ± 2.7

+

192.3 ± 1.9

65.3 ± 2.0

34 ± 1.2



230.9 ± 1.6

109.3 ± 2.4

47.3 ± 1.4

+

263.7 ± 1.2

122.2 ± 3.3

46.3 ± 1.0

In accordance with the stoichiometry in Eqs. (1)–(3), the oxygen reduction rate VOe2 was calculated as 4(VO2 ) upt (oxygen uptake rate). In the presence of NADP the latter was found by measuring the rate of [O2] change in the thylakoid suspension without and with catalase [16]: (VO2 ) upt = VO−2cat − VO+2cat .

(5)

With catalase, the observed oxygen evolution is proportional to NADP reduction, and the overall electron transport along the chain is 4 × (VO+2cat + + VO−2cat − VO+2cat ). The rate of light-induced O2 uptake in the presence of cyt c was found as elsewhere [17] as the rate of [O2] change minus the rate of oxygen evolution (calculated from cyt c reduction measured in the same cell as ΔA550 and a difference extinction coefficient of 19 mM–1 cm–1). RESULTS Upon illumination of the thylakoid suspension, added Fd is reduced to a steady state (figure) amounting to 10–15% of its total concentration (15 μM). The steady-state rate of Fd oxidation in the light, VFdox , was determined by plotting a tangent to the initial part of the redox curve upon switching off the light. Table 1 shows the influence of antimycin A, a potent inhibitor of the Fd-dependent cyclic electron transport around PS I (CET), on VFdox and (VO2 ) upt at different Fd concentrations. Upon inhibition of CET, Fd is oxidized only by oxygen, and VFdox exactly corresponds to the rate of Fd-dependent O2 reduction. If CET was operative in the absence of antimycin, the share of this process in the overall O2 reduction in the presence of antimycin should have increased, but this is not BIOPHYSICS

Vol. 52

No. 4

2007

FERREDOXIN IN PHOTOSYNTHETIC OXYGEN REDUCTION

395

Table 2. Rates of oxygen reduction (VO 2 ) and ferredoxin oxidation (VFdox ) and the effect of NADP (+) Oxygen reduction not involving Fd

NADP, 0.5 mM

VOe , μeq (mg 2 Chl)–1 h–1

Steady-state [Fdred], μmol (mg Chl)–1

VFdox , μeq (mg Chl)–1 h–1

μeq (mg Chl)–1 h–1

%





85.9 ± 3.2





85.9

100

15



241.1 ± 2.2

0.185 ± 0.0167

85.7 ± 3.6

155.4

64.4

+

62.7

0.053 ± 0.0036

24.4

38.3

61.1



363.1 ± 3.3

0.375 ± 0.0263

173.4 ± 5.4

189.7

52.2

+

112.0

0.093 ± 0.0042

43.2

68.8

61.4

[Fd], μM

30

observed. Besides, VFdox rises much less than (VO2 ) upt . Hence, VFdox may be regarded as equal to the rate of O2 reduction involving Fd without antimycin as well. The rise in (VO2 ) upt , i.e., electron transport rate, may be due to a significant smaller energy dissipation in the PS II antenna in the presence of antimycin, resulting in a greater energy influx to the reaction center. Fd oxidation by O2 is a second-order reaction, but considering that [O2] (250–300 μM) in the suspension is much higher than [Fd] (5–30 μM], it can be presented as a pseudofirst-order reaction, VFdox = k1[Fdred]. From the experiments exemplified in the figure, k1 was found as the ratio of VFdox to the steady-state [Fdred] in the light. It varied from 0.098 to 0.128 s–1 for different Fd preparations and was independent of [Fd]. These values are commensurate with those obtained for oxidation of dithionite-reduced Fd without thylakoids, 0.09 [9] and 0.28 s–1 [10]. Table 2 lists the results of an experiment with one suspension and one Fd sample. Averaged over seven such experiments, the contribution of Fd-dependent pathway to the overall oxygen reduction was 41% at 15 μM Fd and increased somewhat at higher concentration. The steady-state [Fdred] in the light markedly decreases in the presence of NADP (figure) as electrons are drawn off by this efficient acceptor. In this case (“+” in Table 2) the rate of Fd oxidation by oxygen was obtained using the [Fdred] and the k1 determined for this Fd sample. Over five experiments, the share of the Fd-dependent process in overall oxygen reduction with 15 μM Fd and 0.5 mM NADP was 32%. The balance, i.e., the share of the electron flux to oxygen from the PETC membrane components (right column in Table 2) was much the same with and BIOPHYSICS

Vol. 52

No. 4

2007

without NADP, but the actual O2-reducing flux was 3–4 times lower in the presence of NADP. At the same time, the overall PETC throughput with NADP and [Fd] optimal for NADP reduction was enhanced four- to sixfold ([16] and Table 3). Obviously, it is this increased “draw-off” onto an efficient acceptor that attenuates the number of electrons transferred to oxygen from the membrane components. Table 3 reports an experiment with one thylakoid suspension and different acceptor systems. Cyt c but slightly increased the PETC rate, Fd was more efficient, and the Fd + NADP pair produced a great effect. The O2 reduction rate measured with cyt c (asterisked in Table 3) reflects only the superoxide radicals that are generated and give rise to H2O2 within the membrane [17]. The superoxides born on the outer membrane surface or having left the membrane are oxidized by cyt c, and their contribution (measured as SOD-dependent cyt c reduction) is about 20% of the overall electron transport (not shown). Thus, the oxygen reduction in the presence of cyt c (due solely to the Table 3. Influence of electron acceptor on oxygen reduction in PETC Acceptor

Overall electron Reduction by memtransport, μeq brane carriers, μeq (mg Chl)–1 h–1 (mg Chl)–1 h–1

O2

74.8 ± 2.3

74.8 ± 2.3

O2 + cyt c (40 μM)

83.5 ± 1.6

45.2* ± 1.9

O2 + Fd (15 μM)

211.2 ± 2.1

126.8 ± 3.4

O2 + Fd (15 μM) + NADP (0.5 mM)

913.8 ± 1.3

55 ± 1.7

* Intramembrane production of superoxide (see text).

396

KOZULEVA et al.

membrane PETC components, as in the absence of acceptors) was about 62 μeq (mg Chl)–1 h–1; this is close to the value for Fd + NADP, and both are markedly lower than with Fd alone. DISCUSSION In vivo in the presence of CO2 and in vitro at maximal NADP reduction the concentration of reduced Fd is small, but oxygen is reduced at an appreciable rate [5, 19]. It remained unknown heretofore to what extent Fd participates in this process. Our data show that at 15 μM Fd, when the NADP reduction rate is maximal, just a little less than a half of the reducing electrons go through Fd (Tables 1, 2). Therewith both the NADP and the O2 reduction rates in our experiments are close to those observed in plant leaves [5]. Which membrane carriers reduce O2 concurrently with Fd? No data are available in the literature to assess the possible competitiveness of Fd and O2 as acceptors. Though Fd is not firmly associated with the thylakoid membrane, it is a very potent specific acceptor, because it is known that in vivo electrons are readily supplied through Fd to all metabolic demands. Considering that the PS I acceptors FA and FB, the immediate Fd reductants, reside on the PsaC subunit protruding from the membrane [20], we suppose that all the electrons that without Fd would go to O2 (and cause O −2⋅ formation outside the membrane) would be passed to Fd when it is available, thus providing for the Fd-dependent O2 and NADP reduction. Superoxide generated within the membrane may also get into the medium, but it has been found [12] that the release of O −2⋅ arising in the plastoquinone pool (the second possible place of O2 reduction in the PETC) is negligible. It has been hypothesized [12, 21] that in both locations such O −2⋅ is reduced by plastohydroquinone to generate intramembrane H2O2. The dependence of the “membrane-dependent” O2 reduction on the rate of electron efflux through the chain suggests the involvement of a PETC component whose reduced lifetime depends on this rate. Such a component may be phylloquinone, the A1 intermediate between P700 and FA/B with a redox potential of –820 mV, embedded in the membrane depth [20]. If the PS I iron–sulfur centers are oxidized rapidly (as with Fd + NADP), electrons from phylloquinone quickly go to FX and FA/B. If FA/B oxidation is hindered

and electrons are “stalled” thereon, the resulting electrostatic field may prevent phylloquinone reduction. At an intermediate FA/B oxidation rate, the steadystate level of phylloquinone reduction can be most high. Such a situation, favoring phylloquinone reaction with O2, may develop in the presence of Fd alone (Table 3). In this case the rate of membrane-dependent O2 reduction (mean 133 μeq (mg Chl)–1 h–1 in five experiments) was even higher than in the absence of other acceptors (72 μeq (mg Chl)–1 h–1). In vivo, conditions for predominant Fd oxidation by oxygen arise under shortage of NADP+, e.g., when stomata close under stress. Thus, we have shown that the rates and scales of oxygen reduction by ferredoxin and by the membrane carriers are quite comparable. The intramembrane process yielding H2O2 may be important for transmitting a signal about the PETC state to the regulatory systems of the cell [22]. ACKNOWLEDGMENTS The work was supported by the Russian Foundation for Basic Research (05-04-48629. REFERENCES 1. C. B. Osmond and C. E. Grace, J. Exper. Bot. 46, 1351 (1995). 2. M. R. Badger, S. von Caemmerer, S. Ruuska, and H. Nakano, Phil. Trans. R. Soc. Lond. 355, 1433 (2000). 3. K. Asada, K. Kiso, and K. Yoshikawa, J. Biol Chem. 249, 2175 (1974). 4. K. Asada, Ann. Rev. Plant Physiol. Plant Mol. Biol. 50, 601 (1999). 5. J. M. Robinson, Physiol. Plantarum 72, 666 (1988). 6. J. Allen, Nature 256, 599 (1975). 7. B. N. Ivanov, T. P. Red’ko, V. L. Shmeleva, and E. N. Mukhin, Biokhimiya 45, 1425 (1980). 8. R. T. Furbank and M. R. Badger, Biochim. Biophys. Acta 723, 400 (1983). 9. B. Hosein and G. Palmer, Biochim. Biophys. Acta 723, 383 (1983). 10. J. H. Golbeck and R. Radmer, in Adv. Photosynth. Res., Ed. by C. Sybesma (The Hague, The Netherlands: MarBIOPHYSICS

Vol. 52

No. 4

2007

FERREDOXIN IN PHOTOSYNTHETIC OXYGEN REDUCTION

397

tinus Nijhoff/Dr W. Junk publishers, 1984), Vol. 1, pp. 561–564.

16. T. P. Red’ko, V. L. Shmeleva, B. N. Ivanov, and E. N. Mukhin, Biokhimiya 47, 1695 (1982).

11. C. Miyake, U. Schreiber, H. Hormann, et al., Plant Cell Physiol. 39, 821 (1998).

17. M. M. Mubarakshina, S. A. Khorobrykh, M. A. Kozuleva, and B. N. Ivanov, Dokl. RAN 408, 1 (2006).

12. S. A. Khorobrykh and B. N. Ivanov, Photosyhth. Res. 71, 209 (2002).

18. B. N. Ivanov, Y. Kobayashi, N. K. Bukhov, and Heber, Photosynth. Res. 57, 61 (1998).

13. E. N. Mukhin, E. A. Akulova, and V. K. Gins, in Methods of Isolating and Studying Protein Components of the Photosynthetic Apparatus, Ed. by V. B. Evstigneev (AN SSSR, Pushchino, 1973), pp. 20–32 [in Russian].

19. J. P. Hosler and C. F. Yocum, Biochim. Biophys. Acta 808, 21 (1985). 20. P. Fromme, P. Jordan, and N. Kraub, Biochim. Biophys. Acta 1507, 5 (2001).

14. H. K. Lichtenthaler, Meth. Enzymol. 148, 350 (1987).

21. S. Khorobrykh, M. Mubarakshina, and B. Ivanov, Biochim. Biophys. Acta 1657, 164 (2004).

15. R. E. Cleland and D. S. Bendall, Photosynth. Res. 34, 409 (1992).

22. B. Ivanov and S. Khorobrykh, Antioxidants & Redox Signalling 5, 43 (2003).

BIOPHYSICS

Vol. 52

No. 4

2007