Proc. Natl. Acad. Sci. USA Vol. 94, pp. 1597–1602, February 1997 Plant Biology Redox chains in chloroplast envelope membranes: Spectroscopic evidence for the presence of electron carriers, including iron–sulfur centers (quinonesyflavinsydesaturationyEPR 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 n8576, Universite´Joseph Fourier et Commissariat a`l’Energie Atomique–Grenoble, 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 chloroplasts contain (i) semiquinone and fla- ref. 4). An n-6 lipid-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 proteins. 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 (E90 520.4 V) could be the source of 31 In addition to a high spin [1Fe] type protein associated with electrons for the reduction of O2 to H2O(E90 510.81 V). an EPR signal at g 5 4.3, we observed two iron–sulfur centers, Because ferredoxin delivers only one electron at a time, the a [4Fe-4S]11 and a [2Fe-2S]11, associated with features, envelope desaturase has to oxidize two reduced ferredoxins, and respectively, at g 5 1.921 and g 5 1.935, which were detected store the first electron before the double bond is formed (4). This 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-deazaflavinyoxalate. An unusual Fe-S cen- Desaturation is not the only envelope enzymatic process that ter, named X, associated with a signal at g 5 2.057, was also would require an electron-transfer chain. For instance, opti- 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]31 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 DpH (8, 9). A possible role for an envelope oxidase, NADPH-quinone, and NADPH-semiquinone reduc- electron-transfer chain could be to maintain this DpH. 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: a-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 plastid 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 (0–58C) distilled pentane (13) was added to Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y941597-6$2.00y0 †To whom reprint requests should be addressed. e-mail: PNAS is available online at http:yywww.pnas.org. [email protected]. 1597 Downloaded by guest on September 29, 2021 1598 Plant Biology: Ja¨ger-Vottero et al. Proc. Natl. Acad. Sci. USA 94 (1997) 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 0–58C and lope polypeptides (corresponding to 120 mg protein) were finally centrifuged at 1000 3 g (Kubota, Tokyo). The supernatant analyzed by SDSyPAGE, 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 3,39,5,59-tetramethyl- lope pellet by evaporation under a stream of argon during 1 hr at benzidine (TMBZ), as described by Thomas et al. (22). Protein 0–58C followed by 1 hr at 208C. 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 5 microwave-frequency counter, for calibration of the magnetic 4.3 and Around g 5 2. Fig. 1A shows that native envelope field and of the frequency. The samples were cooled with a liquid membranes present a signal at g 5 4.3 and another complex signal helium transfer system (ESR 900, Oxford Instrument) to variable around g 5 2, dominated by a major isotropic feature at g 5 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–ironychromel 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 ml, 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 5 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 Mn21 (I 5 5y2), as suggested by dithionite or 5-deazaflavinyoxalate. 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 208C 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 mM) in the presence of sodium oxalate (25 mM) as electron donor was performed as described by Jouanneau et al.