EPR Studies on 6-Benzoylaminopurine and Thenoyltrifluoroacetone Inhibition Sites of Succinate Dehydrogenase in Plant Mitochondria

Plant Cell Physiol. 37(7): 914-921 (1996) JSPP © 1996 EPR Studies on 6-Benzoylaminopurine and Thenoyltrifluoroacetone Inhibition Sites of Succinate Dehydrogenase in Plant Mitochondria Michele Chauveau and Jean Roussaux Laboratoire de Biologie Cellulaire Vdgetale, Universite Pierre et Marie Curie, 12, rue Cuvier, 75005 Paris, France Key words: Cytokinins — EPR — Plant mitochondria — Solarium tuberosum — Succinate dehydrogenase — Thenoyltrifluoroacetone. Cytokinins and other adenine derivatives can specifically interact with the electron transport chain of plant and animal mitochondria (Miller 1980, Roussaux et al. 1986). In plants, the cyanide-insensitive electron transport pathway is inhibited by the synthetic cytokinin 6-benzyladenine (Dizengremel et al. 1982), while other adenine derivatives exert a specific inhibitory effect on the cyanide-sensitive Abbreviations: AA, antimycin A; BOAP, 6-benzoylaminopurine; DCPIP, 2,6-dichlorophenol-indophenol; EPR, electron paramagnetic resonance; FeCN, potassium ferricyanide; HiPIP, high potential iron-protein; PMS, phenazine methosulfate; SDH, succinate dehydrogenase; TTFA, thenoyltrifluoroacetone. electron transport pathway (Roussaux et al. 1986). Thus, alkylaminopurines (-CH2-AP) such as benzyladenine or kinetin selectively inhibit the NADH-ubiquinone reductase (complex I) activity of the respiratory chain. On the contrary, acylaminopurines (-CO-AP) such as 6-benzoylaminopurine (BOAP) act on the succinate-ubiquinone reductase segment (complex II). The mammalian and bacterial succinate dehydrogenases (SDH) are composed of two subunits: a 70-kDa flavoprotein which contains FAD, and a 30-kDa ironsulfur protein (Hederstedt and Ohnishi 1992). In addition, two smaller polypeptides anchor the SDH to the mitochondrial inner membrane, making up the whole succinate-ubiquinone oxidoreductase system (complex II). Three Fe-S clusters have been detected by EPR in complex II of mammalian mitochondria. Both centers S-l (2Fe-2S) and S-2 (4Fe-4S) exhibit an EPR spectrum of rhombic symmetry in their reduced form around g=1.94 (Beinert et al. 1975, Ohnishi et al. 1976a). The third one, center S-3 (3Fe-4S), of the high potential type (HiPIP), is nearly isotropic and EPR-detectable at g = 2.01 in its oxidized form only (Ohnishi et al. 1976b). Centers S-l and S-3 are succinate-reducible whereas center S-2 can be reduced by dithionite only. These Fe-S clusters are probably all located in the 30 kDa subunit (Hederstedt and Ohnishi 1992). The SDH inhibitor thenoyltrifluoroacetone (TTFA) is a chelator for transition metals, and it has been suggested that its effect is due to the chelation of Fe (Nelson et al. 1971). However, other chelators have generally no effect on iron-sulfur proteins (Malkin 1973, Ulvik and Romslo 1975). Moreover TTFA (and the competitive inhibitors carboxamides) could also interfere with center S-3 by modifying its environment (Mowery et al. 1976, 1977). EPR studies have shown that inhibition by TTFA is due to a perturbation of the interaction between center S-3 of SDH and ubisemiquinones, the inhibitor quenching the ubiquinone signal completely (Ingledew and Ohnishi 1977, Salerno and Ohnishi 1980). Finally, experiments with labelled carboxin derivatives have identified the TTFA and carboxin site of action to the O2 side of SDH (Coles et al. 1978). This site includes SDH together with the small polypeptides of complex II and even lipids (Coles et al. 1978, Ramsay et al. 1981). In plant mitochondria complex II has not been isolated (Maeshima et al. 1987), and SDH has been much less studied. However, the composition of plant SDH is similar 914 6-Benzoylaminopurine (BOAP) inhibits succinate oxidation at the level of complex II (succinate-ubiquinone reductase) in the respiratory chain of plant mitochondria. In order to identify its site of action, the effects of BOAP on mitochondrial membranes of potato tubers were studied using electron paramagnetic resonance (EPR), and compared to those of thenoyltrifluoroacetone (TTFA), an inhibitor of complex II. Under aerobic conditions, BOAP induced no change in the EPR signal of the Fe-S center S-3 of succinate dehydrogenase (SDH), unlike TTFA which decreased the height and modified the spectrum of the signal. In the presence of ferricyanide (FeCN), BOAP weakly enhanced the S-3 signal whereas TTFA changed only its shape but not its height. Under anaerobic conditions, center S-l was completely reduced in the presence of BOAP, while in the presence of TTFA center S-l was less reduced than in the control, and the center S-3 signal was not different from that under aerobic conditions. Reoxidation of anaerobic preparations was obtained by O2 or FeCN addition. Oxygen was ineffective as an oxidant in the presence of either inhibitor, but a partial reoxidation of center S-3 was obtained with FeCN in the presence of BOAP. These observations point to two different sites for TTFA and BOAP. Both inhibitors act on the O2 side of center S-3, BOAP interfering with the environment of the Fe-S cluster of S-3, while TTFA would act on the cluster itself. Inhibition site of 6-benzoylaminopurine on SDH of the applied magnetic field in the range 0-0.1 T. The temperature of the samples was controlled with a variable temperature cryostat (Oxford Instruments) and measured with a gold iron/ chromel thermocouple. The samples (about 3 mg protein in 200 n\) maintained at 4°C were placed in a quartz tube (3 mm in diameter) and rapidly frozen at 13 K, directly in the cryostat. The different experimental procedures are specified in the text or figure legends (see Fig. 1). O2 consumption was measured at 4°C with an oxygen electrode (Hansatech) in 15 mM phosphate (pH 7.3) in the presence of 100 mM succinate. Succinate-Cyt c, succinate-ferricyanide and succinate-ubiquinone/DCPIP reductase activities were measured spectrophotometrically at 25°C in 5 mM phosphate (pH 7.3) as previously described (Roussaux and Chauveau 1993). The ubiquinone used was decylubiquinone (Sigma) with DCPIP as the ultimate electron acceptor. The ubiquinol-Cyt c reductase activity was measured under the same conditions as succinate-Cyt c reductase activity with decylubiquinone reduced by dithionite (100/iM) as substrate instead of succinate. TTFA and BOAP were dissolved in dimethylsulfoxide and antimycin A (AA) in ethanol. The solvents at the volumes added (less than 20 ft\) had no effect on the EPR signals and enzyme activities. Protein was determined by the Bradford method according to Fanger (1987). Materials and Methods Potato tuber (Solanum tuberosum L., cv. Bintje) mitochondria were isolated according to Dizengremel and Lance (1976) and purified on a 26% Percoll density gradient (Moreau and Romani 1982). The membrane fraction was obtained by freezing at -30°C overnight, thawing, and centrifugation at 150,000xg for 1 h. The pellet was resuspended in 15 mM mono-potassium and di-sodium phosphate (pH 7.3) containing 1 mM EDTA to a final conce (...truncated)