MINIREVIEW 2-Oxo acid dehydrogenase complexes in redox regulation Role of the lipoate residues and thioredoxin Victoria I. Bunik A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia A number of cellular systems cooperate in redox regulation, providing metabolic responses according to changes in the oxidation (or reduction) of the redox active components of a cell. Key systems of central metabolism, such as the 2-oxo acid dehydrogenase complexes, are important participants in redox regulation, because their function is controlled by the NADH/NAD + ratio and the complex-bound dihydro- lipoate/lipoate ratio. Redox state of the complex-bound lipoate is an indicator of the availability of the reaction substrates (2-oxo acid, CoA and NAD + ) and thiol-disulfide status of the medium. Accumulation of the dihydrolipoate intermediate causes inactivation of the first enzyme of the complexes. With the mammalian pyruvate dehydrogenase, the phosphorylation system is involved in the lipoate- dependent regulation, whereas mammalian 2-oxoglutarate dehydrogenase exhibits a higher sensitivity to direct regula- tion by the complex-bound dihydrolipoate/lipoate and external SH/S-S, including mitochondrial thioredoxin. Thioredoxin efficiently protects the complexes from self- inactivation during catalysis at low NAD + . As a result, 2-oxoglutarate dehydrogenase complex may provide succi- nyl-CoA for phosphorylation of GDP and ADP under conditions of restricted NAD + availability. This may be essential upon accumulation of NADH and exhaustion of the pyridine nucleotide pool. Concomitantly, thioredoxin stimulates the complex-bound dihydrolipoate-dependent production of reactive oxygen species. It is suggested that this side-effect of the 2-oxo acid oxidation at low NAD + in vivo would be overcome by cooperation of mitochondrial thioredoxin and the thioredoxin-dependent peroxidase, SP-22. Keywords: dihydrolipoate; 2-oxo acid dehydrogenase complex; radical species; redox state; thioredoxin. Introduction Regulation of metabolism dependent on the cellular redox state has attracted increasing attention [1–5]. The redox state is characterized by the degree of oxidation or reduction of various redox-active species of a cell. Among these species, pyridine nucleotides and thiol/disulfide compounds are of special significance, as they interconnect many enzymes of the multifaceted metabolic network. On the one hand, the steady-state ratios of NAD(P)H/NAD(P) + and SH/S-S mediate the redox regulation through direct effects on proteins. Activities of many enzymes depend on the redox state of the pyridine nucleotide pools, while proteins with essential SH/S-S groups can be regulated by post-translational modification involving cellular thiols and disulfides [1,5–7]. On the other hand, the NAD(P)H/ NAD(P) + and SH/S-S ratios are intimately related to the cellular level of ROS. Reduced pyridine nucleotides and thiols participate both in ROS formation and degradation. The former process is effected by different NAD(P)H oxidases [8] and upon thiol oxidation [9,10], while the glutathione- and thioredoxin-dependent peroxidase reac- tions use NADPH and thiols to scavenge hydrogen peroxide and limit formation of radical species [11]. At low concentrations, ROS are essential participants of the cellular redox regulation [3,11,12]. Their extremely high reactivity allows for the fast local modification of proteins. This can provide transient oxidative modifications against the reducing potential of the medium, e.g. formation of a protein disulfide bond in the reducing cytoplasm [13]. However, the high reactivity also leads to cell destruction if cellular capacity to scavenge ROS is compromised. This happens under pathological conditions where NAD(P)H/ NAD(P) + and SH/S-S ratios are decreased [14–17]. While experiments with intact cells enable us to assess the net effects of redox perturbations that reflect integrated metabolic responses, dissecting the mechanisms of these overall responses requires investigation of the separate components of the metabolic network. Among cellular systems, the 2-oxo acid dehydrogenase multienzyme com- plexes occupy key positions for redox regulation. In the overall process (see reactions 1–5 in scheme below) involving sequential action of 2-oxo acid dehydrogenase (E1), dihydrolipoamide acyltransferase (E2) and dihydrolipo- amide dehydrogenase (E3), they split a carbon-carbon bond of the 2-oxo acid preserving its energy in acyl-CoA Correspondence to V. Bunik, A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia. Fax: + 7 095 939 31 81, Tel.: + 7 095 939 14 56, E-mail: bunik@genebee.msu.su Abbreviations: E1, 2-oxo acid dehydrogenase; E2, dihydrolipoamide acyltransferase; E3, dihydrolipoamide dehydrogenase; EPR, electron paramagnetic resonance; ROS, reactive oxygen species. (Received 23 July 2002, revised 10 December 2002, accepted 11 December 2002) Eur. J. Biochem. 270, 1036–1042 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03470.x and NADH, where R defines pyruvate, 2-oxoglutarate or the 2-oxo acids with branched carbon chain: Thus, the catalytic action of the 2-oxo acid dehydro- genase complexes directly influences the NADH/NAD + ratio and involves the important biological SH/S-S com- pounds, lipoic acid and CoA, with one of them (lipoate) being covalently bound to the complexes. Sensitivity of the 2-oxo acid dehydrogenase complexes to NADH/NAD + has long been recognized as a mechanism of feedback control [18,19]. Being operative in vivo [20], it increases fluxes through pyruvate dehydrogenase complex under more oxidizing conditions [2]. However, the SH/S-S- dependent regulation of the complexes, in particular, by the complex-bound lipoate/dihydrolipoate ratio, has received little attention, although some experiments suggested the interaction of the complex-bound dihydrolipoate with externaldisulfides[21,22].Atthesametime,thereare numerous studies of the antioxidant properties of free lipoate [23–25]. Free dihydrolipoate efficiently reduces transcription factors [26] and thioredoxin [27], which are well-known components of the redox regulation [5,11,28]. Because cellular lipoate/dihydrolipoate is mostly localized to the 2-oxo acid dehydrogenase complexes, the interplay between the complexes and other participants of redox signaling is of great interest. An essential feature of the complex-bound lipoate/ dihydrolipoate couple is that its redox state is linked to the irreversible reaction of the 2-oxo acid oxidation. Because of this, the lipoate redox state in vivo is defined by the steady-state concentrations of the overall reaction sub- strates (2-oxo acid, CoA and NAD + ) rather than thermodynamic equilibrium with cellular thiols. Another essential feature of the lipoate residues within the 2-oxo acid dehydrogenase complexes arises from the ÔcrowdingÕ effect. Confinement of the complex-bound lipoate within the volume of the enzyme complexes may result in unusual properties of the compound, when compared to the same quantity of the lipoate molecules distributed in bulk solution. In particular, neighbouring sulfur greatly increa- ses the stability of the thiyl radical in dithiols compared to monothiols [29]. Additional stabilization of the lipoate thiyl radicals may be expected within the network of the interacting complex-bound lipoate residues. Thus, the complexes provide an excellent opportunity to study in vitro the consequences of cellular compartmentalization of biologically active SH/S-S. Taking into account multiple pharmacological effects of free lipoate [23–25], we suggested that the lipoate clustering within the 2-oxo acid dehydrogenase complexes may be significant not only for the typical enzymatic catalysis, but also for cellular redox regulation, including cellular protec- tion against oxidative damage. Our idea that the complex- bound lipoate possesses a function beyond its role as a catalytic intermediate in the oxidative decarboxylation is in good agreement with certain data about the complexes, which have not previously been given an adequate explan- ation. Even a limited number of organisms from which the 2-oxo acid dehydrogenase complexes have been isolated have revealed structural variations of these systems that go beyond those essential for catalytic performance. The variations are linked to different lipoate content in the complexes, including different degrees of oligomerization, different stoichiometries of catalytic components (in parti- cular, lipoyl moieties) and different localization of the lipoate residues. They may be incorporated not only in the established lipoate holder, E2, but also in other components [30,31]. Depending on the source and presence of substrates, the mostly investigated ÔclassicÕ complexes (i.e. those containing lipoate residues in E2 only) are built around oligomeric cores of 3, 24 or 60 E2 subunits. Different types of E2 may bear up to three lipoate residues, which is genetically determined [30,32,33]. Surprisingly, more than half of the lipoyl moieties of the E2 oligomer [34–36], or two of the three lipoyl-bearing domains of E2 [37,38], may be removed without significant change in the overall activity in vitro. Yet microorganisms possessing pyruvate dehydro- genase complex with a decreased number of lipoyl domains are at a physiological disadvantage. They exhibit decreased growth rates and are eventually Ôwashed-outÕ from the mixed population containing the mutant and wild type cells [38,39]. The physiological behaviour, however, is main- tained even with a reduced number of lipoyl groups as long as the ÔreachÕ of the lipoyl moieties is not decreased. That is, the mutant strain possessing E2 bearing the three lipoyl domains with only the outermost one lipoylated (the two inner lipoyl domains in this case do not contain the lipoyl group) behaved identically to the wild type under the conditions employed [38]. Because such a mutant complex catalyzes pyruvate oxidation even 25% less efficiently than the complexes which are unable to provide the normal growth rates (with E2 containing one or two fully lipoylated domains) [38], the physiological advantage of the E2 with the three lipoyl domains cannot be ascribed entirely to the catalytic role of these domains. Rather, the advantage appears to depend on the ability of the lipoyl group to protrude from the inner core of the complex, indicative of the biological significance of the complex-bound lipoate interaction with the surrounding medium. This paper reviews experimental evidence for the involve- ment of the complex-bound lipoate in such ÔparacatalyticÕ reactions, i.e. those where the complex-bound lipoate escapes the catalytic route (reactions 1–5). The reactions underlie the SH/S-S-dependent regulation of the 2-oxo acid dehydrogenase complexes on different levels. The basic level corresponds to self-regulation of the complexes by the complex-bound lipoate/dihydrolipoate ratio. Involvement Ó FEBS 2003 Redox regulation of 2-oxo acid dehydrogenation (Eur. J. Biochem. 270) 1037 of external components in the lipoate-dependent reactions extends this regulation to a higher level. For instance, production of ROS by the complexes and interchange of the complex-bound dihydrolipoate/lipoate with external thiols and disulfides, including thioredoxin, may be important for ROS-dependent signaling. Thus, the redox state of the complex-bound lipoate creates a sensitive link between the 2-oxo acid dehydrogenase reaction and surrounding medium. Self-regulation of the complexes by the redox state of the lipoate residues As follows from the scheme of the overall 2-oxo acid dehydrogenase reaction (reactions 1–5), the steady-state ratio of the complex-bound lipoate/dihydrolipoate is a func- tion of (a) concentrations of the reaction substrates and products, (b) kinetic properties of the component enzymes, E1, E2 and E3, and (c) their stoichiometry and interactions within the complex. During catalysis in the physiological direction, the complex-bound lipoate is reduced to dihydro- lipoate by the 2-oxo acid and CoA (reactions 1–3) and the dihydrolipoate is reoxidized by NAD + in a FAD-dependent process (reactions 4,5). The lipoate may be also reduced in the backward reactions (5 and 4), upon preincubation with NADH. In the mitochondrial 2-oxoglutarate dehydrogenase complex, this induces strong cooperativity among the active sites of its first component, 2-oxoglutarate dehydrogenase, upon 2-oxoglutarate binding, and complicates the kinetic dependence of the reaction rate on 2-oxoglutarate [40]. Isolated from the complex, the 2-oxoglutarate dehydro- genase component did not show such changes in response to NADH. However, the changes were observed after the enzyme reduction with dihydrolipoate. Similar concentra- tion of other cellular thiols, such as glutathione, cysteine or CoA, were ineffective [40]. Thus, the lipoate residues of the complex mediate the regulation of its first component by the last product of the overall reaction, NADH. Reduction of the lipoate residues of the complexes in the forward direction, i.e. by 2-oxo acid and CoA (reactions 1–3), when the following reoxidation by NAD + (reaction 5) is restricted, is accompanied by an irreversible inactivation of E1 [41]. The inactivation is observed both in the presence and absence of O 2 .When the complex-bound lipoate was reduced under anaerobic conditions, the complex-bound thyil radical and a radical fragment of 2-oxo acid were detected in spin trapping experiments with a-phenyl-N-tert-butylnitrone and 5,5¢- dimethyl-1-pyrroline-N-oxide, respectively [42]. Thus, the E1 inactivation occurs upon 1e – reduction of the thiyl radical of the complex-bound dihydrolipoate by the E1 catalytic intermediate (E1*S): The resulting substrate-derived radical fragment (S Æ ) likely causes the observed E1 inactivation due to a site- directed modification. Efficiency of reaction 6 is provided by the protein–protein interactions evolved to enable the catalytic 2e – reduction of the lipoate by E1*S (reaction 2). In the absence of O 2 , the dihydrolipoate thiyl radical is transiently formed upon equilibration of the complex redox centers. In the presence of O 2 , the E3-bound FAD catalyzes 1e – oxidation of the complex-bound dihydrolipo- ate by oxygen, resulting in superoxide anion radical production [42]. The thiyl radical of the complex-bound dihydrolipoate is an intermediate of this side reaction (Fig. 1). The superoxide production by the complexes is competitive with the NAD + reduction. Under conditions where less NAD + is available, more superoxide is produced, and this leads to a higher steady-state concen- tration of complex-bound thiyl radicals and a concomi- tantly greater extent of enzyme inactivation by the 2-oxo acid plus CoA. Saturation by NAD + protects from the 1e – oxidation of the dihydrolipoate intermediate by oxygen and prevents inactivation [41]. Resistance of the overall activity to the superoxide anion radical produced is documented by the fact that superoxide dismutase does not prevent the inactivation. This is in good accord with the independence of the inactivation on the presence of oxygen. The dihydrolipoate-mediated inactivation of E1 at low NAD + concentrations is more pronounced in mammalian than bacterial complexes [43]. In contrast, inhibition of E3 by over-reduction with NADH is less efficient in mamma- lian complexes [44]. Thus, the E3 inhibition seems to be the main response of bacterial complexes to NADH accumu- lation. The mammalian complexes develop the E1-directed mechanisms of NADH- and dihydrolipoate-dependent regulation. It is the initial stage of the substrate transfor- mation which is then affected. The 2-oxoglutarate dehydrogenase complex is more sensitive to the 2-oxo acid, CoA-induced inactivation than the pyruvate dehydrogenase complex [43]. This agrees with the difference in the regulation of the two complexes by Fig. 1. Production of superoxide anion radical by the 2-oxo acid dehy- drogenase complexes. Oxidation of the complex-bound dihydrolipoate by oxygen is catalyzed by the E3-bound FAD. Superoxide anion radical is detected in the reaction medium by appearance of the EPR signal corresponding to its reaction with the spin trap a-phenyl-N-tert- butylnitrone. Appearance of the EPR signal is blocked either by the modification of the complex-bound FAD or by addition of superoxide dismutase. 1038 V. I. Bunik (Eur. J. Biochem. 270) Ó FEBS 2003 phosphorylation/dephosphorylation. The latter mechanism controls the function of eukaryotic pyruvate dehydro- genase, whereas 2-oxoglutarate dehydrogenase is not phos- phorylated. Remarkably, the efficiency of the pyruvate dehydrogenase phosphorylation depends on the state of the complex-bound dihydrolipoate. Thus, the redox regulation of the eukaryotic pyruvate dehydrogenase is mediated by the phosphorylation/dephosphorylation system, which thus becomes the main transducer of multiple metabolic signals. Regarding the mammalian 2-oxoglutarate dehydrogenase complex, the 2-oxo acid, CoA-dependent inactivation through the complex-bound dihydrolipoate intermediate appears to be the biologically relevant mechanism of redox regulation. The concentrations of 2-oxoglutarate and CoA determined in mitochondria [45] are saturating for the complex, while NADH/NAD + ratio varies depending on the metabolic state. Moreover, NAD + is a substrate of many mitochondrial enzymes, with the competition between them further reducing the effective NAD + concentration available for 2-oxoglutarate oxidation. Hence, estimation of the substrate ratio existing in vivo shows that it is in the range where the self-inactivation of the 2-oxoglutarate dehydrogenase complex upon accumulation of the dihydro- lipoate intermediate may occur. As a result, the complex-bound lipoate allows the starting component of the complexes, E1, to respond to the state of the mitochondrial NAD + and NADH pool. The E1 activity is regulated both upon accumulation of NADH and decrease of NAD + . The E1 inactivation at low NAD + concentration prevents the side production of dihydrolipo- ate-dependent reactive oxygen species (Fig. 1) at the expense of the 2-oxo acid oxidation. Because a decrease in concentration of mitochondrial pyridine nucleotides induces antioxidant defense mechanisms [15,16], inactivation of the 2-oxo acid dehydrogenases under these conditions may be a part of the integrated response. It also may explain the reduction of the 2-oxoglutarate dehydrogenase complex activity observed under different pathological states [46]. Thiol-disulfide exchange between the complex-bound lipoate and external thiols/disulfides In studies of mitochondria, disulfides inhibited mitochond- rial respiration at the level of the 2-oxoglutarate dehydro- genase reaction [21,22]. Investigation of the purified 2-oxoglutarate dehydrogenase complex confirmed the inhi- bition of the overall reaction by the low molecular mass disulfides [41]. The data pointed to the exchange of redox equivalents between the complexes and the medium, involving the dihydrolipoate intermediate. Such an exchange also enabled thiols or disulfides to protect the complexes from the inactivation at low levels of NAD + [43,47]. With free disulfides which are substrates for E3 (R-lipoate), the flow of reducing equivalents from 2-oxo acids to the disulfides was catalyzed by E3 [48,49]. Because dihydrolipoate is an efficient reductant of thioredoxin [27] which may further direct reducing equivalents to different processes [28], the 2-oxo acid dehydrogenase reaction coupled to free lipoate reduction in the presence of thio- redoxin may be a source of reducing equivalents for not only NADH-dependent, but also thioredoxin-dependent pathways. For instance, the reaction provides reduction of disulfides in proteins such as insulin and thioredoxin reductase [48,49]. Recently, an antioxidant defense system in mycobacteria was discovered where the 2-oxoglutarate dehydrogenase complex provides reducing equivalents to the peroxiredoxin alkyl hydroperoxide reductase through a thioredoxin-like protein [50]. Discovery of a specific mitochondrial thioredoxin with unknown protein targets [51] stimulated our interest in the potential interplay between the 2-oxo acid dehydrogenase complexes and thioredoxin via the complex-bound lipoate. Unraveling an in vivo function of a thioredoxin species is complicated by the high chemical reactivity of its dithiol/ disulfide group, as it allows thioredoxin to participate in a number of redox processes in vitro. Study of cross- reactivity of thioredoxins and potential target proteins from different species helps to solve this problem through revealing specific protein–protein interactions promoting chemical reactions. In particular, mitochondrial thio- redoxin rather inefficiently regulates the enzymes which are known to depend on the thioredoxin action [52]. In contrast, it efficiently protects the 2-oxo acid dehydro- genase complexes from the 2-oxo acid, CoA-dependent inactivation [47,52]. Studies using four types of the 2-oxo acid dehydrogenase complexes and 11 thioredoxin species support the biological relevance of this protection [43,47]. Mitochondrial complexes are much more sensitive to the thioredoxin regulation than their bacterial counterparts. This is due to a greater sensitivity of the mitochondrial complexes to the 2-oxo acid, CoA-induced inactivation, as the thioredoxin effect is related to alleviation of this inactivation. On the other hand, among 11 thioredoxin species with comparable activity in the nonspecific insulin reduction test, mitochondrial thioredoxin is by far the most effective in protecting the complexes. While some of thioredoxins are inactive or even decrease the complex activity, mitochondrial thioredoxin is protective down to 10 )7 M concentrations. Correlation of the thioredoxin effects and protein structures revealed the following structural determinants of the specific action of mito- chondrial thioredoxin on the complexes [47]: (a) active site disulfide/dithiol group and the residues modulating its properties, (b) the interaction between the a3/3 10 and a1 helices and the length of the a1 helix and (c) the three charged residues on the thioredoxin surface opposite to the active site, which significantly influence polarization of the molecule. Experimentally observed effects of different thioredoxins on the complexes (increase or decrease in the complex activity, or none) correlate with the dipole direction, while the effective thioredoxin concentrations correlate with the dipole magnitude. It is known that steering effects of the long-range interactions between the electrostatic dipoles increase the number of effective collisions, i.e. collisions which may be stabilized by short-range interactions [53]. The observed correlation between polarization of the thioredoxin molecule and efficiency of its protection of the 2-oxo acid dehydroge- nase complexes points to long-range interactions as the basis for the effect of thioredoxin on the activity of the complex. This relationship suggests coevolution of the interacting proteins, which would not be possible if the interactions were not relevant in vivo. Ó FEBS 2003 Redox regulation of 2-oxo acid dehydrogenation (Eur. J. Biochem. 270) 1039 Thioredoxin protection from the 2-oxo acid, CoA- induced inactivation of the dehydrogenase complexes, and the recently published data on the high stability of the thioredoxin thyil radical, which allows thioredoxin to prevent the pro-oxidant action of the radical [54], support the proposed mechanism of the inactivation (reaction 6). Catalysing the dismutation of the dihydrolipoate thyil radicals (Fig. 2), thioredoxin prevents their adverse action upon E1. The main component of cellular thiol buffer, glutathione, also protects the complexes in vitro [41]. However, unlike thioredoxin, low molecular mass thiols do not specifically bind to the complexes and their thiyl radicals are known to possess pro-oxidant action [10,55,56]. Hence, regarding the overall mitochondrial metabolism, glutathione cannot be an efficient scavenger of the complex- bound thiyl radicals of dihydrolipoate. Protected by thioredoxin, the 2-oxoglutarate dehydro- genase complex can produce energy at low NAD + not only in the form of NADH, but also in the form of a macroergic compound, succinyl-CoA. The latter supports the only reaction of substrate phosphorylation in the Krebs cycle, catalyzed by succinyl thiokinase. This may be especially important in cases where leakage of pyridine nucleotides or accumulation of NADH occurs due to disturbances in the respiratory chain. However, switching off the self-regulation of the complexes by the dihydrolipoate thiyl radical, thioredoxin also stimulates the side reaction of the super- oxide anion radical production by the complexes (Fig. 1). In this regard, it is worth noting that mitochondrial thiore- doxin is a substrate of mitochondrial thioredoxin peroxi- dase, SP-22 [57]. Reduced by the complex-bound dihydrolipoate and coupled to SP-22, thioredoxin would be able to scavenge hydrogen peroxide, which is formed after dismutation of the superoxide anion radical produced by the complexes as shown below: Thus, thioredoxin interaction with the 2-oxo acid dehy- drogenase complexes under conditions of an increased steady-state concentration of dihydrolipoate may provide a dual positive effect: relief of the pro-oxidant action of dihydrolipoate on E1 and scavenging of ROS produced by E3. As a result, cooperation of the 2-oxo acid dehydro- genase complexes, thioredoxin and SP-22 (reaction 7) enables oxidation of 2-oxo acids under increased concen- tration of the dihydrolipoate intermediate without accumu- lation of ROS. The antioxidant action of mitochondrial thioredoxin upon the 2-oxoglutarate dehydrogenase may be involved in the thioredoxin antiapoptotic action when cells are treated with tert-butyl hydroperoxide [58]. Selective targeting of the 2-oxoglutarate dehydrogenase complex under oxidative stress [59] and inactivation of the 2-oxoglutarate dehydro- genase by tert-butyl hydroperoxide [60] favour this inter- pretation. Concluding remarks Participationofthe2-oxoaciddehydrogenasecomplexes in redox regulation is summarized in Fig. 3. The interac- tion of the complexes with the surrounding medium may be realized through the lipoate-dependent ÔparacatalyticÕ reactions. Such reactions allow the 2-oxo acid dehydro- genase complexes to transform a signal in the form of metabolite concentrations into chemical reactions such as ROS production, thioredoxin reduction and E1 modifi- cation. This network of reactions provides not only self- regulation of the complexes (Fig. 3, boxed region), but also their interaction with the surrounding medium, which may be used in different signaling pathways. Our data on the interplay between the mitochondrial complexes and thioredoxin favors participation of the complexes in the redox-dependent signaling through the thioredoxin sys- tem. Other forms of such participation are known. For example, the E1 subunit of the pyruvate dehydrogenase Fig. 3. Participation of the 2-oxo acid dehydrogenase complexes in the redox regulation of metabolism. The redox state of the surrounding medium is sensed by the complexes through the concentrations of 2-oxo acid, CoA, NAD(H) and oxygen. This external signal is trans- formed into the ratio of the complex-bound lipoate/dihydrolipoate. Dependent on the input, this redox couple may regulate the activity of the starting component E1 (a), produce output to surrounding medium in the form of ROS (b) and reduce thioredoxin and disulfides (c). Thioredoxin interferes with the self-regulation of the complexes (a), concomitantly stimulating ROS production (b). The latter may be overcome in the presence of SP-22. Fig. 2. Thioredoxin catalysis of the dismutation of the thiyl radicals of the complex-bound dihydrolipoate intermediate. 1040 V. I. Bunik (Eur. J. Biochem. 270) Ó FEBS 2003 complex from Azotobacter vinelandii wasshowntobindto the fpr promoter region DNA, which is activated upon cellular response to oxidative stress [61]. In Escheri- chia coli, this promoter is activated by the redox-depend- ent transcription factor SoxS. In view of the redox-sensing function of the lipoate/dihydrolipoate couple of the complex (Fig. 3) and the intimate link between this couple and E1, the E1 dissociation from the complex to bind DNA may represent another form of the lipoate- dependent response under conditions of oxidative stress. Acknowledgements This work was supported by grants from DFG (438 17-159-92), Volkswagen (I/69766) and Alexander von Humboldt (IV RUS 1003594 STP) Foundations. Critical reading of the manuscript by Prof J. J. 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