Proc. Natl. Acad. Sci. USA Vol. 94, pp. 1597–1602, February 1997 Plant Biology

Redox chains in envelope membranes: Spectroscopic evidence for the presence of electron carriers, including iron–sulfur centers (quinones͞flavins͞desaturation͞EPR spectroscopy)

PASCALE JA¨GER-VOTTERO*, ALBERT-JEAN DORNE*†,JEANNE JORDANOV‡,ROLAND DOUCE*, AND JACQUES JOYARD*

*Laboratoire de Physiologie Cellulaire Ve´ge´tale, De´partement de Biologie Mole´culaireet Structurale and ‡Laboratoire de Spectroscopie des Complexes Polyme´talliques et Me´talloprote´ines, De´partementde Recherche Fondamentale sur la Matie`reCondense´e, Unite´ de Recherche Associe´eCentre National de la Recherche Scientifique nЊ576, Universite´Joseph Fourier et Commissariat a`l’Energie Atomique–, 17 rue des Martyrs, F-38054, Grenoble ce´dex9, France

Communicated by Pierre Joliot, Institute of Physico-Chemical Biology, Paris, France, October 15, 1996 (received for review May 28, 1996)

ABSTRACT We have shown that envelope membranes NADP oxidoreductase, stearoyl-ACP desaturase (for review see from spinach contain (i) semiquinone and fla- ref. 4). An n-6 -linked desaturase, probably involving ferre- vosemiquinone radicals, (ii) a series of iron-containing elec- doxin:NADPH oxidoreductase, has been characterized in chlo- tron-transfer centers, and (iii) flavins (mostly FAD) loosely roplast envelope membranes (5). Experiments on chloroplasts (6) associated with . In contrast, we were unable to detect suggest that O2 could be the final electron acceptor, whereas any cytochrome in spinach chloroplast envelope membranes. reduced ferredoxin (EЈ0 ϭϪ0.4 V) could be the source of 3؉ In addition to a high spin [1Fe] type associated with electrons for the reduction of O2 to H2O(EЈ0 ϭϩ0.81 V). we observed two iron–sulfur centers, Because ferredoxin delivers only one electron at a time, the ,4.3 ؍ an EPR signal at g a [4Fe-4S]1؉ and a [2Fe-2S]1؉, associated with features, envelope desaturase has to oxidize two reduced ferredoxins, and which were detected store the first electron before the double bond is formed (4). This ,1.935 ؍ and g 1.921 ؍ respectively, at g after reduction by NADPH and NADH, respectively. The is possible only in the presence of a complex electron-transfer [4Fe-4S] center, but not the [2Fe-2S] center, was also reduced chain, which has not been detected yet in envelopes. by dithionite or 5-deazaflavin͞oxalate. An unusual Fe-S cen- Desaturation is not the only envelope enzymatic process that -was also would require an electron-transfer chain. For instance, opti ,2.057 ؍ ter, named X, associated with a signal at g detected, which was reduced by dithionite but not by NADH or mization of photosynthesis in chloroplasts is dependent on the NADPH. Extremely fast spin–relaxation rates of flavin- and maintenance of pH gradients between the stroma and both the quinone-free radicals suggest their close proximity to the thylakoid lumen and the cytosol (7). In the latter case, the -4Fe-4S] cluster or the high-spin [1Fe]3؉ center. Envelope inner envelope probably contains an energy-transducing pro] membranes probably contain enzymatic activities involved in ton pump as a primary mechanism facilitating the formation the formation and reduction of semiquinone radicals (quinol of stroma-cytosol ⌬pH (8, 9). A possible role for an envelope oxidase, NADPH-quinone, and NADPH-semiquinone reduc- electron-transfer chain could be to maintain this ⌬pH. tases). The physiological significance of our results is dis- To date, the only known envelope constituents that could play cussed with respect to (i) the presence of desaturase activities a role in electron transfer are prenylquinones: ␣-tocopherol and in envelope membranes and (ii) the mechanisms involved in plastoquinone-9 (10, 11). Because quinone radicals are often the export of protons to the cytosol, which partially regulate involved in redox chains and since semiquinones are paramag- the stromal pH during photosynthesis. The characterization netic and therefore detectable by EPR spectroscopy, we investi- of such a wide variety of electron carriers in envelope mem- gated envelope membranes from spinach chloroplasts by EPR branes opens new fields of research on the functions of this spectroscopy. In this article, we report for the first time the membrane system within the plant cell. presence in envelope membranes of: (i) several iron–sulfur pro- teins, (ii) semiquinones, and (iii) flavins that could be compo- The two envelope membranes that surround chloroplasts contain nents of one or several electron-transfer chains. numerous enzymes involved in the biosynthesis of specific membrane constituents: glycerolipids, prenylquinones, chloro- MATERIALS AND METHODS phyll precursors, carotenoids (for review see ref. 1). The forma- Purification of Envelope Membranes from Intact Spinach tion of polyunsaturated fatty acids and of colored carotenoids Chloroplast. Chloroplasts were isolated from spinach (Spinacia involve desaturation steps that are only poorly understood. For oleracea L.) leaves and further purified by centrifugation in instance, sequential desaturation of the colorless carotenoid Percoll gradients (12). Envelope membranes, thylakoids, and precursor phytoene leads to the formation of lycopene, a colored stroma were purified from chloroplasts, lysed in hypotonic me- carotenoid with 11 double bonds. Quinones and factors regulat- dium, by centrifugation through a step-sucrose gradient (12). ing the redox state of quinones may play a major role in phytoene They were stored (in liquid nitrogen) at 10 mg of protein per ml desaturation, whereas in vitro molecular oxygen is the terminal in 10 mM 4-morpholinepropanesulfonic acid (MOPS)–NaOH electron acceptor (2, 3). Most of our knowledge on fatty acid (pH 7.8), or lyophilized for pentane treatment. Extensive analyses desaturation in chloroplast concerns the soluble components of of purified envelope fractions have shown (for review see ref. 12) the 18:0 to 18:1 desaturation system: ferredoxin, ferredoxin: that they were totally devoid of membranes derived either from thylakoids, mitochondria, endoplasmic reticulum, or any other The publication costs of this article were defrayed in part by page charge extraplastidial membranes that could exhibit EPR signals. payment. This article must therefore be hereby marked ‘‘advertisement’’ in Pentane Treatment of Chloroplast Envelope Membranes. One accordance with 18 U.S.C. §1734 solely to indicate this fact. milliliter of chilled (0–5ЊC) distilled pentane (13) was added to

Copyright ᭧ 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424͞97͞941597-6$2.00͞0 †To whom reprint requests should be addressed. e-mail: PNAS is available online at http:͞͞www.pnas.org. [email protected].

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lyophilized envelope membranes (10 mg protein). The mixture Protein Analyses and Determination. Thylakoid and enve- was vortexed in a glass tube for 5 min under argon at 0–5ЊC and lope polypeptides (corresponding to 120 ␮g protein) were finally centrifuged at 1000 ϫ g (Kubota, Tokyo). The supernatant analyzed by SDS͞PAGE, as described by Chua (21). Cyto- was discarded. Pentane extraction of the pellet was repeated four chromes could be directly visualized on the gels by following times (14). The remaining pentane was removed from the enve- their peroxidase activity with H2O2 and 3,3Ј,5,5Ј-tetramethyl- lope pellet by evaporation under a stream of argon during 1 hr at benzidine (TMBZ), as described by Thomas et al. (22). Protein 0–5ЊC followed by 1 hr at 20ЊC. Envelope proteins were finally concentration was determined according to Lowry et al. (23), rehydrated in 1 mM MOPS–NaOH (pH 7.8). using BSA as a standard. EPR Measurements. EPR spectra were recorded on a Varian E 109 spectrometer coupled to a Hewlett–Packard 9826 calcu- RESULTS ؍ lator and equipped with a Varian Gaussmeter and an EIP 548A Chloroplast Envelope Membranes Show EPR Signals at g Fig. 1A shows that native envelope .2 ؍ microwave-frequency counter, for calibration of the magnetic 4.3 and Around g field and of the frequency. The samples were cooled with a liquid membranes present a signal at g ϭ 4.3 and another complex signal helium transfer system (ESR 900, Oxford Instrument) to variable around g ϭ 2, dominated by a major isotropic feature at g ϭ 2.003 temperatures starting from 4.2 K. The temperature was measured (Fig. 1B). Pentane-treated membranes were then used to increase with a gold–iron͞chromel thermocouple located about 2 cm the signal-to-noise ratio. Pentane is a nonpolar molecule, which below the bottom of the EPR sample in the flowing helium gas does not affect the integrity of the membranes. Indeed, after stream. Samples of envelope membranes (150 ␮l, 1.5–6 mg pentane treatment of the membranes their EPR features remain protein) were placed in EPR quartz tubes, rapidly frozen in liquid unchanged. The g ϭ 2 region was resolved in a series of signals nitrogen, and stored at 77 K. with maxima at g values of 2.167, 2.077, 2.017, 1.961, 1.929, and Reduction of Chloroplast Envelope Membranes with Chem- 1.875, as well as an isotropic signal at 2.003 (Fig. 1C). Some of ical Agents. The samples were reduced by addition of either these could be associated with Mn2ϩ (I ϭ 5͞2), as suggested by dithionite or 5-deazaflavin͞oxalate. Aliquots of a stock solu- the six-line pattern of the signal and by the hyperfine splitting tion of dithionite were progressively added to the same enve- lope sample to obtain a range of concentration (up to 5 mM). Envelope membranes and dithionite at given concentrations were incubated together for 3 min at 20ЊC and then rapidly frozen in liquid nitrogen for EPR analysis. After recording its spectrum, the sample was thawed for the next addition of dithionite. Reduction with the photoactivatable catalyst 5-dea- zaflavin (20 ␮M) in the presence of sodium oxalate (25 mM) as electron donor was performed as described by Jouanneau et al. (15). The mixture containing envelope membranes together with 5-deazaflavin͞oxalate was irradiated for 30 min at 30 cm from a white light source (250 W) to start the photoreduction. Control experiments were also run in the presence of 5-dea- zaflavin͞oxalate, but the samples were placed in the dark instead of being irradiated. After incubation, the samples were rapidly frozen in liquid nitrogen for EPR analysis. Oxidation or Reduction of Chloroplast Envelope Membranes with Physiological Mediators. NADH or NADPH (EЈ0 ϭϪ0.32 V) and oxygen (EЈ0 ϭϩ0.81 V) were used, respectively, to reduce or oxidize envelope electron carriers. NADPH or NADH were progressively added to the same envelope sample to obtain a range of concentration (up to 500 ␮M). After each addition, envelope membranes were incubated for 10 min at 25ЊC and then rapidly frozen in liquid nitrogen for EPR analysis, as described above. After recording the spectrum, the sample was thawed for the next addition of NADPH or NADH. Oxidation of envelope electron carriers was achieved under a stream of oxygen during an increasing length of time (up to 20 min). Aliquots of the suspension were taken at 0, 5, 10, and 20 min and rapidly frozen in liquid nitrogen for EPR analysis. Carotenoid and Prenylquinone Determination. Carotenoids and prenylquinones were analyzed as described by Block et al. (16) and Lichtenthaler et al. (10), respectively. Flavin Determination. Flavins were analyzed in envelope membranes as described by Faeder and Siegel (17). Protein- bound flavins were first extracted from membranes as described by Yagi (18), prior to flavin determination. Fluorescence inten- sities (␭ex ϭ 450 nm, ␭em ϭ 530 nm) of the mixtures containing flavins were first determined at pH 7.7 with a Spex Fluoromax FIG. 1. EPR signals of native (A and B) and pentane-treated (C) spectrofluorimeter, then the pH of the mixtures was adjusted to envelope membranes from spinach chloroplasts. Protein concentra- 2.6 by addition of 1 N HCl for another fluorescence analysis. The tion: 10 mg͞ml (A and B)or45mg͞ml (C). EPR conditions: field, amounts of FMN and FAD present in the samples were then 2500 Ϯ 2500 Gauss (A) or 3250 Ϯ 400 Gauss (B and C); time constant, 0.128 sec (A) or 0.25 sec (B and C); microwave power, 1 mW; calculated by using the equations of Faeder and Siegel (17). modulation amplitude, 12.5 G; frequency, 9.22 GHz; temperature, 4.2 Nonheme Iron and Sulfur Determination. Nonheme iron and K; gain, 104. The six-line pattern under spectrum C indicates the sulfur were determined in envelope membranes as described by hyperfine lines due to Mn2ϩ; the distances between these lines are Doeg and Ziegler (19) and Beinert (20), respectively. indicated by the different values, expressed in Gauss. Downloaded by guest on September 29, 2021 Plant Biology: Ja¨ger-Vottero et al. Proc. Natl. Acad. Sci. USA 94 (1997) 1599

value of Ϸ96 Gauss. Indeed, manganese was found (Ϸ0.5 nmol per mg protein) in purified envelope membranes (24). The signals observed in the g ϭ 2 region could also be associated with iron–sulfur proteins. In support of this suggestion, analyses of five different preparations have shown that envelope membranes contain 3.2 Ϯ 0.3 nmol Fe and 5.3 Ϯ 0.4 nmol S per mg protein. Pentane-treated envelope membranes are highly concen- trated compared with native membranes (on average 40–50 mg protein per ml instead of 10–15). This is essential for EPR analyses of envelope preparations, since the signals detected have a low signal-to-noise ratio. One should keep in mind (i) that envelope membranes have a much higher lipid-to-protein ratio (1.2–1.5 mg lipid per mg protein) compared with other cell membranes (i.e., thylakoids, 0.5 mg lipid per mg protein) and (ii) that chloroplast envelope membranes are likely to have a low content of redox centers, thus making very difficult a complete characterization of the centers involved. Is Characteristic of 4.3 ؍ The Envelope EPR Signal at g High-Spin Fe3؉. This cation could be present in envelope vesicles either as ‘‘free’’ Fe3ϩ or associated with a protein in a [1Fe]3ϩ center. For instance, a g ϭ 4.31 signal was observed in the oxidized form of rubredoxin, a [1Fe]3ϩ protein, from Pseudomo- nas oleovorans (25). In envelope membranes, the g ϭ 4.3 signal is FIG. 2. Evolution of the amplitude of the chloroplast envelope probably not due to contaminating free Fe3ϩ, since (i) its ampli- EPR signal at g ϭ 2.003 as a function of temperature (A) and tude did not decrease when envelope membranes were washed microwave power (B). Protein concentration: 50 mg͞ml. EPR condi- extensively by EDTA (not shown) and (ii) it was still very visible tions: see Fig. 1B, except that the temperature varied from 4.2 to 46 when the spectra were recorded at 15 K instead of 4.2 K (not K(A) and the microwave power varied from 0.01 mW to 50 mW; temperature, 4.2 K (B). shown). This last observation is indicative of a Fe3ϩ species with 3ϩ different relaxation properties from free Fe . Therefore, the second signal was tentatively assigned to a flavosemiquinone 3ϩ Fe center giving rise to the g ϭ 4.3 signal in envelope mem- species (for example see ref. 29). branes is likely to be covalently linked to proteins. Interestingly, Chloroplast Envelope Membranes Are Devoid of Cyto- a lipoxygenase, which is an iron-containing protein and therefore chromes. The low- and high-spin hemes of cytochromes are could contribute to such a signal, has recently been characterized characterized by EPR signals in the g ϭ 3 and g ϭ 6 regions, in chloroplast envelope membranes (26). respectively. The lack of any signal in these regions strongly -This feature suggests that envelope membranes may be devoid of cyto .2.003 ؍ Characterization of the EPR Signal at g is isotropic and has a band width (peak-to-peak) of 12 Ϯ 1 Gauss chromes. The presence of cytochromes in chloroplast membranes (Fig. 1B). This value is indicative of a semiquinone radical, rather was also investigated after SDS͞PAGE analysis of envelope and than of a flavin radical, also because no wings on either side of thylakoid polypeptides. Cytochromes b6 and f from thylakoids the resonance transition were visible (27). In addition, we ob- were easily visualized on the gels by following their peroxidase served that the signal at g ϭ 2.003 was present in membranes activity with H2O2 and TMBZ. In contrast, no reaction was prepared at pH ranging from 6 to 8 (not shown). The presence of observed with purified envelope membranes (not shown). These such a stabilized semiquinone radical in envelope membrane is two sets of results are in good agreement with our previous rather surprising, because semiquinone radicals are in general spectrophotometric observations (30); the low temperature (77 transient species that are formed during oxidoreduction pro- K) difference spectrum resulting from dithionite-reduced minus cesses. The stabilization of a semiquinone intermediate is prob- oxidized envelope preparations does not show any absorption ably dependent on its binding to a specific protein (for example peak due to cytochromes. Therefore, we have been unable to see ref. 28). Quinones that are loosely bound to proteins can be detect cytochromes in purified envelope membranes by using extracted by pentane (14). Fig. 1C shows that after the pentane three different methods. This raises some doubt on the putative treatment the g ϭ 2.003 signal was still visible. Its relative intensity identification of a cytochrome as the product of the chloroplastic was much smaller than in the native envelope, because the gene cemA, which was found to be localized in the inner envelope pentane-treated envelope contained 4.5 times more protein per membrane from pea chloroplasts (31). milliliter than native envelope preparations, whereas the g ϭ Reduction of Envelope Membranes by Sodium Dithionite. 2.003 signal was increased only by 1.2-fold. This result suggests, Addition of dithionite to pentane-treated envelope mem- that either the envelope semiquinone was partially extracted by branes leads to major changes of the EPR spectrum. With the pentane treatment or the redox state of the envelope semiqui- increasing concentrations of dithionite (from 10 ␮M to 5 mM), none was affected by a change in its environment brought about we first observed the progressive disappearance of the g ϭ by the pentane treatment. The first hypothesis is rather unlikely 2.003 signal (not shown), probably because the semiquinone since we observed that pentane treatment was unable to remove radical was reduced to the quinol state, which is EPR-silent, envelope prenylquinones, in contrast to carotenoids which were thus revealing more clearly a new signal, named Y, with a either totally or partially extracted by pentane (not shown). maximum at g ϭ 2.017. This new signal is shown in Fig. 3 for The g ϭ 2.003 signal arises from a rapidly relaxing radical, as the pentane-treated envelope recorded after addition of excess shown by the important decrease of its amplitude when raising the amounts (5 mM) of dithionite. Unfortunately, further identi- temperature from 4.2 to 46 K (Fig. 2A). Its power-saturation fication of the center associated with the g ϭ 2.017 signal is (from 0.01 mW to 50 mW) behavior produces a biphasic curve almost impossible, since its observation requires the complete (Fig. 2B). One component, with the peak-to-peak width of 12 Ϯ disappearance of the semiquinone signal, which can be ob- 1 Gauss, saturates at 2 mW and was assigned to the semiquinone tained only at high dithionite concentration. radical. Beyond 2 mW, a second component is revealed, with a With increasing concentrations of dithionite (from 10 ␮Mto5 larger peak-to-peak width of 21 Ϯ 1 Gauss, which does not mM), we also noticed an increase of a broad (68 Ϯ 2 Gauss) saturate before 40–50 mW. The radical associated with this signal, named X, centered at g ϭ 2.057 (Fig. 3A). Its maximum Downloaded by guest on September 29, 2021 1600 Plant Biology: Ja¨ger-Vottero et al. Proc. Natl. Acad. Sci. USA 94 (1997)

intensity occurred in the presence of 100 ␮M of dithionite (not shown). When the spectrum was recorded at 40 K, this signal was not observed any longer (not shown), suggesting that it could be associated with an iron–sulfur center. However, no iron–sulfur protein exhibiting such a high gy value in the reduced state has yet been described in any other biological system. Indeed, the protein involved has EPR characteristics that are different from those associated with traditional [4Fe-4S]1ϩ, [3Fe-4S]1ϩ, or [2Fe-2S]1ϩ centers (for review see ref. 32). When recording the EPR spectrum of dithionite-reduced pentane-treated envelope under different conditions (tempera- ture, 15 K; microwave power, 5 mW), signal Y, with a maximum at g ϭ 2.017, became more intense and a new signal appeared, centered at g ϭ 1.921 (Fig. 3B). This last signal was already visible, but very weak, in the native envelope membrane (see Fig. 1C) and was further analyzed after photoreduction with 5-deazaflavin͞ oxalate, which allows for reduction of iron–sulfur proteins with a very low redox potential. Similar signals have been extensively reviewed in the literature (for example see ref. 33). Photoreduction of Envelope Membranes by 5-Deazaflavin͞ Oxalate. Fig. 4 shows that after photoreduction with 5-deazafla- vin͞oxalate the envelope semiquinone is reduced to the quinol state since the signal at g ϭ 2.003 is abolished, thus unmasking the g ϭ 2.017 signal of the Y species. Also, when recording the FIG. 4. Effect of photoreduction by 5-deazaflavin oxalate on spectrum both at a higher temperature (15 K) and a higher ͞ chloroplast envelope EPR signals. Protein concentration: 10 mg͞ml. microwave power (5 mW), both amplitudes of the Y signal and EPR conditions: see Fig. 1B, except for spectrum IIb and IIc, in which of the signal at g ϭ 1.921 were considerably increased. The latter the microwave power was 5 mW and the temperature 15 K and 35 K, could be associated with either a [4Fe-4S] or a [2Fe-2S] cluster respectively. (34). These two centers differ markedly in their relaxation properties: a [4Fe-4S] cluster has a faster relaxation and is a diagnostic tool for distinguishing the centers, provided that therefore no longer visible at 30 K, whereas a [2Fe-2S] cluster magnetic interactions with other paramagnetic species are absent. relaxes more slowly and can still be detected at 70 K. This provides Fig. 4 indicates that the signal at g ϭ 1.921 is completely abolished at 35 K, and therefore that envelope membranes contain a [4Fe-4S] center, paramagnetic when reduced, with a global charge of ϩ1, i.e., a [4Fe-4S]1ϩ center. To provide a more physiological view of the importance of envelope components that may be involved in electron trans- fer, we investigated whether NADPH, NADH, and oxygen could modulate the amplitude of the chloroplast envelope EPR signals. Oxidation and Reduction of Envelope Membranes with Physiological Mediators. A new signal, centered at g ϭ 1.935, was revealed upon the addition of 10 ␮M of NADH to pentane-treated envelope membranes (Fig. 5). This feature was still visible when the spectrum was recorded at 45 K, suggesting that it could be associated with a [2Fe-2S]1ϩ center. Surprisingly, it was not observed after reduction with 5-dea- zaflavin͞oxalate, which is able to reduce centers with a much lower redox potential than those reduced by NADH. This difference could be due to either to a very high affinity of the envelope [2Fe-2S]1ϩ center for NADH (possibly via a dehy-

FIG. 3. Effect of dithionite on chloroplast envelope EPR signals. Protein concentration: 35 mg͞ml (A)or45mg͞ml (B). EPR conditions: see Fig. 1B, except for B, in which the microwave power was 5 mW and the temperature 15 K. The signal centered at g ϭ 2.003 was no longer FIG. 5. Effect of reduction with NADH on chloroplast envelope visible in dithionite-treated samples, and a signal centered at g ϭ 1.921 was EPR signals. Pentane-treated envelope corresponding to 30 mg of visible in the dithionite-treated sample only when recorded at high protein͞ml was used. EPR conditions: see Fig. 1B, except that the microwave power and temperature (as indicated in B). temperature was set at 15 K. Downloaded by guest on September 29, 2021 Plant Biology: Ja¨ger-Vottero et al. Proc. Natl. Acad. Sci. USA 94 (1997) 1601

drogenase) or to a limitation in the accessibility of the center envelope preparations was analyzed: mean values of 300 and 45 by deazaflavin. Since NADH is not, in general, a true physi- pmol͞mg protein, respectively, for FAD and FMN, were deter- ological donor for most of the chloroplast enzymes, it is mined, making possible the presence of a flavosemiquinone possible that the [2Fe-2S]1ϩ center reduced by NADH could radical in envelope membranes (see above). be localized in a compartment only accessible from the cytosol. Therefore, the protein associated with the [2Fe-2S]1ϩ center DISCUSSION can be located either on the outer envelope membrane or on EPR-detectable centers have been identified in chloroplast the outer face of the inner envelope membrane. envelope membranes. First, envelope membranes contain Table 1 summarizes some effects of NADPH and͞or oxygen on semiquinone radicals. In vitro, the quinone seems to function the amplitude of the semiquinone signal and of the signal mostly between the quinol and the semiquinone state. Enve- associated with the [4Fe-4S]1ϩ center. First, addition of 10 ␮Mof lope membranes probably contain several enzymatic activities NADPH (in the presence of oxygen) to envelope membranes led involved in the formation and reduction of semiquinone to the appearance of the g ϭ 1.921 signal. This signal was also radicals, namely a quinol oxidase (which catalyzes the oxygen- observed after reduction with 5-deazaflavin͞oxalate (Fig. 4), but dependent conversion of quinol into semiquinone), an NAD- not after reduction by NADH (Fig. 5). Another effect of NADPH PH–quinone reductase (which catalyzes the reduction of qui- addition (in the absence of oxygen) was the decrease of the none into semiquinone), and an NADPH–semiquinone reduc- semiquinone signal at g ϭ 2.003 (Table 1). These results suggest tase (which catalyzes the reduction of semiquinone into that the semiquinone is reduced to its quinol form by NADPH, quinol). The semiquinone is probably stabilized because of the probably owing to NADPH–semiquinone reductase activity. In possible existence of an active quinol oxidase, which could contrast, when NADPH was added in the presence of saturating transfer electrons from quinol to oxygen. levels of oxygen, the amplitude of the semiquinone signal in- Second, envelope membranes contain a series of iron- creased significantly (Table 1). Finally, when envelope mem- containing electron-transfer proteins, but are certainly lacking branes were placed for 20 min under a stream of oxygen in the any detectable cytochrome, which makes this membrane sys- absence of NADPH, the amplitude of the semiquinone signal at tem quite unique among plant membranes. In addition to one g ϭ 2.003 increased at least by a factor of 4 (Table 1). Although (or several) [1Fe]3ϩ protein(s) which is͞are associated with the pure ␣-tocopherol is indeed able to generate an EPR-active EPR signal centered at g ϭ 4.3, we observed an unusual Fe-S ␣-tocopheroxyl radical (not shown), the possibility that the oxy- center, named X, associated with a signal centered at g ϭ 2.057, gen-induced increase of the g ϭ 2.003 signal could be due to the which was reduced by dithionite but not by NADH or NADPH. formation of an ␣-tocopheroxyl radical is rather unlikely. We Two additional Fe-S centers, a [4Fe-4S]1ϩ center, associated observed that the g ϭ 2.003 signal formed during the oxygenation with a feature at g ϭ 1.921, and a [2Fe-2S]1ϩ center, associated of envelope membranes was almost completely abolished after with a feature at g ϭ 1.935, were detected after reduction, further addition of NADPH under argon (Table 1), in good respectively, by NADPH and NADH. The [4Fe-4S] center, but agreement with our previous observations of the reduction of the not the [2Fe-2S] center, was also reduced by dithionite or semiquinone by this cofactor. In fact, ␣-tocopheroxyl radicals are 5-deazaflavin͞oxalate. Signal Y, with a maximum at g ϭ 2.017, recycled back to ␣-tocopherol with either ascorbate or reduced remained unidentified. Third, envelope membranes contain glutathione to generate either monodehydroascorbate or gluta- protein-associated FAD and FMN, which could be responsible thionyl (GS⅐) radicals. Furthermore, these radicals are themselves for the flavosemiquinone radical observed. thought to be recycled by NADPH (35). Because neither ascor- These electron carriers are likely to be associated into one or bate nor glutathione were present in our experiments, we can several electron-transfer chains. At this stage of the work, com- conclude that the oxygen-induced increase of the g ϭ 2.003 signal plete description of putative electron-transfer chains in envelope is probably not due to the formation of an ␣-tocopheroxyl radical. membranes is obviously speculative. However, a possible working More likely, this increase is due to an oxidation of quinol to hypothesis is shown in Fig. 6, which provides some reasonable semiquinone, probably owing to a quinol oxidase activity. clues for the interpretation of our results. Until thermodynamic Finally, NADH and NADPH are able to transfer two profiles of both FAD and Q are analyzed, one cannot decide electrons, whereas iron–sulfur centers can accept only one. whether parallel electron-transfer chains branched at the flavin Therefore, a link between electron donors and acceptors is site are necessary, or if a single chain with both flavin and quinone necessary: flavins that can transfer either one or two electrons sites functioning as n ϭ 2 7 n ϭ 1 converters (37) is present. (36) are good candidates for such a function. We therefore analyzed the flavin content of envelope membranes. Analyses of the Flavin Content of Envelope Membranes. Only between one-third and one-half of the envelope flavins could be extracted by a classical aqueous treatment, whereas they were all solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS) (33 mM). No -linked flavins were further released by proteolytic digestion of envelope proteins. This demonstrates that in envelope membranes a major proportion of the flavins is probably stabilized by proteins in a noncovalent manner. The FAD and FMN content of 17 different FIG. 6. Scheme for a possible electron transfer chain in chloroplast ⅐ Table 1. Effect of oxidation by oxygen and reduction by NADPH envelope membranes. FAD͞FADH ͞FADH2 is the link between on g ϭ 1.921 and g ϭ 2.003 chloroplast envelope EPR signals NADPH and the iron–sulfur centers X and [4Fe-4S]1ϩ (steps 1–3). NADPH–quinone reductase (step 4) and NADPH–semiquinone re- Oxygen ductase (step 5) allow the oxidation of FADH2 and FADH⅐ by the Signal No NADPH then iron–sulfur centers X and [4Fe-4S]1ϩ. In this scheme the [4Fe-4S] g addi- NADPH ϩ NADPH center is shown to be involved in the reoxidation of FADH2 into value tion NADPH ϩ argon oxygen Argon Oxygen ϩ argon FADH⅐ (step 2) and the X center in the reoxidation of FADH⅐ into 1.921 Ϸ0 ϩϩ Ϸ0 ϩϩ Ϸ0 Ϸ0 Ϸ0 FAD (step 3). The symmetric situation (i.e., steps 2 and 3 involving the centers X and [4Fe-4S], respectively) is also possible. Regeneration of 2.003 ϩϩ ϩ(ϩ) ϩ ϩϩϩ Ϸ0 ϩϩϩϩϩ ϩ the semiquinone radical by oxygen is due to a quinol oxidase (step 6). The relative amplitude of the chloroplast envelope EPR signals is Oxidation of the semiquinone radical to its quinone form (step 7) has indicated by a more or less large number of plusses. not yet been demonstrated. Downloaded by guest on September 29, 2021 1602 Plant Biology: Ja¨ger-Vottero et al. Proc. Natl. Acad. Sci. USA 94 (1997)

However, some preliminary observations, such as the oxygen- are pumped by isolated intact chloroplasts not only from the dependent formation of semiquinone radicals and an oxygen- stroma into the intrathylakoid compartment, but also across the independent regeneration of the quinone from the semiquinone envelope into the external medium (1, 7). The processes involved radical, could favor a branched electron chain. in the regulation of the stromal pH during photosynthesis are still Flavins, and especially FAD, are able to receive two elec- unknown (8, 9) and our results could provide some clues toward trons from NADPH. FAD is reduced by NADPH in a single an understanding of this regulation; for example, NADPH– step (step 1) into FADH2 (Fig. 6). FADH2 is then reoxidized quinone reductase of the chloroplast envelope could be involved into FAD in a two-step process via FADH⅐ (steps 2 and 3). Two in the transfer of protons from the stroma to the cytosol. of the iron–sulfur centers, i.e., X and [4Fe-4S], are probably involved in this reoxidation process since quinones or semiqui- We would like to thank Dr. Tomoko Onishi for the very wise nones are, in general, not directly involved in such reactions. comments provided during the course of this work. For instance, the [4Fe-4S] center could be involved in the ⅐ 1. Douce, R. & Joyard, J. (1990) Annu. Rev. Cell Biol. 6, 173–216. reoxidation of FADH2 into FADH (step 2) and the X center ⅐ 2. Beyer, P., Mayer, M. & Kleinig, H. (1989) Eur. J. Biochem. 184, 141–150. in the reoxidation of FADH into FAD (step 3) or vice versa. 3. Mayer, M. P., Beyer, P. & Kleinig, H. (1990) Eur. J. Biochem. 191, 359–363. NADPH–quinone reductase (step 4) and NADPH– 4. Heinz, E. (1993) in Lipid Metabolism in Plants, ed. Moore, T. S. (CRC, Boca semiquinone reductase (step 5) are, respectively, involved in Raton, FL), pp. 33–89. the formation of the semiquinone radical and of the quinol 5. Schmidt, H., Dresselhaus, T., Buck, F. & Heinz, E. (1995) Plant Mol. Biol. 26, 631–642. form. In the presence of NADPH, the (probably) high turnover 6. Norman, H. A., Pillai, P. & St. John, J. B. (1991) Phytochemistry 30, of center X does not permit to follow its reduction, whereas the 2217–2222. [4Fe-4S] center and the quinone are both observed to be at 7. Heber, U. & Heldt, H. W. (1981) Annu. Rev. Plant Physiol. 32, 139–168. least partially reduced, suggesting that the quinone is involved 8. Robinson, S. P. (1985) Biochim. Biophys. Acta 806, 187–194. in the oxidation of center X (step 4) and thus converted into 9. Berkowitz, G. A. & Peters, J. S. (1993) Plant Physiol. 102, 261–267. 10. Lichtenthaler, H. K., Prenzel, U., Douce, R. & Joyard, J. (1981) Biochem. semiquinone. On the other hand, the semiquinone is likely to Biophys. Acta 64, 99–105. be reduced to the quinol state by oxidation of the reduced 11. Soll, J., Schultz, G., Joyard, J., Douce, R. & Block, M. A. (1985) Arch. [4Fe-4S]1ϩ center (step 5). Biochem. Biophys. 238, 290–299. The last step is the oxygen-dependent formation of semiqui- 12. Douce, R. & Joyard, J. (1982) in Methods in Chloroplast Molecular Biology, eds. Edelman, M., Hallick, R. & Chua, N. H. (Elsevier, Amsterdam), pp. none radicals (step 6). In general, cytochromes are the redox 239–256. intermediates between quinol and oxygen (24, 38), since the 13. Shriver, D. F. (1969) The Manipulation of Air-Sensitive Compounds difference between the redox potential of the quinone͞ (McGraw–Hill, New York). semiquinone (0 V) and of the O ͞H O(ϩ0.81 V) couples is too 14. Szarkowska, L. (1966) Arch. Biochem. Biophys. 113, 519–525. 2 2 15. Jouanneau, Y., Meyer, C., Gaillard, J. & Vignais, P. M. (1990) Biochem. large for a direct link to exist between these molecules. Since Biophys. Res. Commun. 171, 273–279. envelope membranes are devoid of cytochrome, enzymes such as 16. Block, M. A., Joyard, J. & Douce, R. (1980) Biochim. Biophys. Acta 631, a quinol oxidase (see above) could play a role in this process. For 210–219. instance, in plant mitochondria, the quinol oxidase that could be 17. Faeder, E. J. & Siegel, L. M. (1973) Anal. Biochem. 53, 332–336. 18. Yagi, K. (1971) Methods Enzymol. 18, 290–296. linked to the cyanide-insensitive pathway is an EPR-silent fla- 19. Doeg, K. & Ziegler, D. (1962) Arch. Biochem. Biophys. 97, 37–40. voprotein (39, 40). Finally, we have no information about the 20. Beinert, H. (1983) Anal. Biochem. 131, 373–378. mechanisms that could be involved in the regeneration of the 21. Chua, N. H. (1980) Methods Enzymol. 69, 434–446. quinone from the semiquinone radical (step 7); very likely, the 22. Thomas, P. E., Ryan, D. & Lewin, W. (1976) Anal. Biochem. 75, 167–170. 23. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. natural electron acceptor is missing. Since we never observed any Chem. 193, 265–275. decrease of the g ϭ 2.003 signal in the presence of oxygen, the 24. Joyard, J. & Douce, R. (1976) Physiol. Ve´g. 14, 31–48. participation of oxygen in this process is rather unlikely. 25. Peisach, J., Blumberg, W. E., Lode, E. T. & Coon, M. J. (1971) J. Biol. The physiological significance of our results could be first Chem. 246, 5877–5881. 26. Ble´e,E. & Joyard, J. (1996) Plant Physiol. 110, 445–454. related to the presence of desaturase activities in envelope 27. Bowyer, J. R. & Ohnishi, T. (1985) in Coenzyme Q, ed. Lenaz, G. (Wiley, membranes. For instance, phytoene desaturase is a FAD- New York), pp. 409–432. containing flavoprotein (41, 42), its activity requires quinones, 28. Lin, C., Robertson, D. E., Ahmad, M., Raibekas, A. A., Jorns, M. S., oxygen, and factors regulating the redox state of quinones (2, 3, Dutton, P. L. & Cashmore, A. R. (1995) Science 269, 968–969. 43). Concerning fatty acid desaturation, the mechanism involved 29. Vanoni, M. A., Edmonson, D. E., Zanetti, G. & Curti, B. (1992) Biochem- istry 31, 4613–4623. in chloroplasts is probably very different from the microsomal 30. Douce, R., Holtz, R. B. & Benson, A. A. (1973) J. Biol. Chem. 248, desaturase system. In microsomes, the immediate electron donor 7215–7222. to the desaturase, an iron-containing protein, is a b5 cytochrome, 31. Sasaki, Y., Sekiguchi, K., Nagano, Y. & Matsuno, R. (1993) FEBS Lett. 316, which in turn can receive electrons from two different flavopro- 93–98. 32. Cammack, R. (1992) Adv. Inorg. Chem. 38, 281–322. teins, one using NADH as a cofactor the other NADPH (4). In 33. Naud, I., Vincon, M., Garin, J., Gaillard, J., Forest, E. & Jouanneau Y. contrast, it seems that lipid-linked desaturation in chloroplasts (1994) Eur. J. Biochem. 222, 933–939. requires NADPH and O2 as the final electron acceptor and 34. Orme-Johnson, W. H. & Sands, R. H. (1973) in Iron–Sulfur Proteins, ed. reduced ferredoxin as the source of the additional two electrons Lowenberg, W. (Academic, New York), Vol. 2, pp. 195–235. 35. Asada, K. & Takashi, M. (1987) in Photoinhibition, eds. Kyle, D. J., necessary to reduce O2 to H2O (4). No cytochrome seems to be Osmond, B. & Arntzen, C. J. (Elsevier, Amsterdam), pp. 227–280. involved in this process, which is in agreement with our results. 36. Rubinstein, B. & Luster, D. G. (1993) Annu. Rev. Plant Physiol. Mol. Biol. Membrane-bound desaturases from plants, cyanobacteria, yeast, 44, 131–155. and mammals all contain homologous regions with the general 37. Hederstedt, L. & Ohnishi, T. (1992) in Molecular Mechanisms in Bioener- getics, ed. Ernster, L. (Elsevier, Amsterdam), pp. 163–198. sequence His-Xaa-Xaa-Xaa-His, which may provide metal- 38. Rich, P. R. (1984) Biochim. Biophys. Acta 768, 53–79. chelating ligands contributing to the binding of oxygen in the 39. Douce, R. (1985) Mitochondria in Higher Plants: Structure, Function and reaction center (5, 44). For phytoene desaturase and for the n-6 Biogenesis (Academic, Orlando, FL). 40. Siedow. J. N. & Umbach, A. L. (1995) Plant Cell 7, 821–831. lipid-linked desaturase, the participation of O2 as the final elec- 41. Bartley, G. E., Kumle, A., Beyer, P. & Scolnik, P. A. (1993) Methods tron acceptor suggests that they have lower redox potentials than Enzymol. 214, 374–385. oxygen. They could therefore be involved in the oxygen- 42. Hugueney, P., Ro¨mer, S., Kuntz, M. & Camara, B. (1992) Eur. J. Biochem. dependent oxidation of quinol to semiquinone (Fig. 6, step 6). 209, 399–407. Physiological and biochemical evidence for such a mechanism to 43. Nievelstein, V., Vandekerchove, J., Tadros, M. H., Lintig, J. V., Nitschke, operate in chloroplast envelope membranes remain to be ob- W. & Beyer, P. (1995) Eur. J. Biochem. 233, 864–872. 44. Shanklin, J., Whittle, E. J. & Fox, B. G. (1995) in Plant Lipid Metabolism, tained. In addition, desaturation is not the only envelope reaction eds. Kader, J. C. & Mazliak, P. (Kluwer, Dordrecht, The Netherlands), pp. that requires an electron flow. For instance, in the light, protons 18–20. Downloaded by guest on September 29, 2021