Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.     

Genomic organization and identification of a novel alternative splicing variant of mouse mitochondrial thioredoxin reductase (TrxR2) gene

Eukaryotic mitochondria are equipped with a complete thioredoxin system, composed of thioredoxin and thioredoxin reductase, which has been implicated in the protection against the reactive oxygen intermdiates generated during the respiratory process in this organelle. Like its cytosolic counterpart, mammalian mitochondrial thioredoxin reductase is a homodimeric selenoprotein. We report here the genomic organization of the mouse mitochondrial thioredoxin gene (TrxR2) that spans 53 kb and consists of 18 exons ranging from 20 to 210 bp. All splicing sites conformed to the GT/AG rule with the exon-intron boundaries located exactly at the same position as the human TrxR2 gene, the only mammalian mitochondrial thioredoxin reductase gene whose genomic structure has been elucidated to date. In addition, we have identified a novel mRNA splicing variant lacking intron 14 resulting in a protein subunit with a shorter interface domain. This new splicing variant provides a framework for further analysis of this important enzyme as its predicted homodimeric conformation can now be expanded to a putative heterodimeric structure as well as a small subunit homodimer with the obvious implications at the regulatory level.     

Crystal structure of human peroxiredoxin 5, a novel type of mammalian peroxiredoxin at 1.5 A resolution

The peroxiredoxins define an emerging family of peroxidases able to reduce hydrogen peroxide and alkyl hydroperoxides with the use of reducing equivalents derived from thiol-containing donor molecules such as thioredoxin, glutathione, trypanothione and AhpF. Peroxiredoxins have been identified in prokaryotes as well as in eukaryotes. Peroxiredoxin 5 (PRDX5) is a novel type of mammalian thioredoxin peroxidase widely expressed in tissues and located cellularly to mitochondria, peroxisomes and cytosol. Functionally, PRDX5 has been implicated in antioxidant protective mechanisms as well as in signal transduction in cells. We report here the 1.5 A resolution crystal structure of human PRDX5 in its reduced form. The crystal structure reveals that PRDX5 presents a thioredoxin-like domain. Interestingly, the crystal structure shows also that PRDX5 does not form a dimer like other mammalian members of the peroxiredoxin family. In the reduced form of PRDX5, Cys47 and Cys151 are distant of 13.8 A although these two cysteine residues are thought to be involved in peroxide reductase activity by forming an intramolecular disulfide intermediate in the oxidized enzyme. These data suggest that the enzyme would necessitate a conformational change to form a disulfide bond between catalytic Cys47 and Cys151 upon oxidation according to proposed peroxide reduction mechanisms. Moreover, the presence of a benzoate ion, a hydroxyl radical scavenger, was noted close to the active-site pocket. The possible role of benzoate in the antioxidant activity of PRDX5 is discussed.     

Reduction of thioredoxin significantly decreases its partial specific volume and adiabatic compressibility.

The partial specific volume and adiabatic compressibility were determined at several temperatures for oxidized and reduced Escherichia coli thioredoxin. Oxidized thioredoxin had a partial specific volume of 0.785-0.809 mL/g at the observed upper limit for all proteins whereas the partial specific volume of reduced thioredoxin was 0.745-0.755 mL/g, a value in the range found for a majority of proteins. The adiabatic compressibility of oxidized thioredoxin was also much larger (9.8-18 x 10(-12) cm2 dyne-1) than that of the reduced protein (3.8-7.3 x 10(-12)). Apart from the region immediately around the small disulfide loop, the structures of the oxidized (X-ray, crystal) and reduced protein (nuclear magnetic resonance, solution) are reported to be very similar. It would appear that alterations in the solvent layer in contact with the protein surface must play a major role in producing these large changes in the apparent specific volumes and compressibilities in this system. Some activities of thioredoxin require the reduced structure but are not electron transfer reactions. The large changes in physical parameters reported here suggest the possibility of a reversible metabolic control function for the SS bond.     

Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes

Alterations in mitochondrial structure and function are a hallmark of cancer cells compared to normal cells and thus targeting mitochondria has emerged as an novel approach to cancer therapy. The mitochondrial thioredoxin 2 (Trx2) system is critical for cell viability, but its role in cancer biology is not well understood. Recently some cationic triphenylmethanes such as brilliant green (BG) and gentian violet were shown to have antitumor and antiangiogenic activity with unknown mechanisms. Here we demonstrate that BG killed cells at nanomolar concentrations and targeted mitochondrial Trx2, which was oxidized and degraded. HeLa cells were more sensitive to BG than fibroblasts. In HeLa cells, Trx2 down-regulation by siRNA resulted in increased sensitivity to BG, whereas for fibroblasts, the same treatments had no effect. BG was observed to accumulate in mitochondria and cause a rapid and dramatic decrease in mitochondrial Trx2 protein. With a redox Western blot method, we found that treatment with BG caused oxidation of both Trx1 and Trx2, followed by release of cytochrome c and apoptosis-inducing factor from the mitochondria into the cytosol. Moreover, this treatment resulted in an elevation of the mRNA level of Lon protease, a protein quality control enzyme in the mitochondrial matrix, suggesting that the oxidized Trx2 may be degraded by Lon protease.     

Solution structure of thioredoxin h1 from Arabidopsis thaliana

Present in virtually every species, thioredoxins catalyze disulfide/dithiol exchange with various substrate proteins. While the human genome contains a single thioredoxin gene, plant thioredoxins are a complex protein family. A total of 19 different thioredoxin genes in six subfamilies has emerged from analysis of the Arabidopsis thaliana genome. Some function specifically in mitochondrial and chloroplast redox signaling processes, but target substrates for a group of eight thioredoxin proteins comprising the h subfamily are largely uncharacterized. In the course of a structural genomics effort directed at the recently completed A. thaliana genome, we determined the structure of thioredoxin h1 (At3g51030.1) in the oxidized state. The structure, defined by 1637 NMR-derived distance and torsion angle constraints, displays the conserved thioredoxin fold, consisting of a five-stranded beta-sheet flanked by four helices. Redox-dependent chemical shift perturbations mapped primarily to the conserved WCGPC active-site sequence and other nearby residues, but distant regions of the C-terminal helix were also affected by reduction of the active-site disulfide. Comparisons of the oxidized A. thaliana thioredoxin h1 structure with an h-type thioredoxin from poplar in the reduced state revealed structural differences in the C-terminal helix but no major changes in the active site conformation.     

Determination of the positions of bound water molecules in the solution structure of reduced human thioredoxin by heteronuclear three-dimensional nuclear magnetic resonance spectroscopy.

The presence of bound water molecules in the solution structure of reduced human thioredoxin has been investigated using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy. It is demonstrated that the backbone amide protons of Lys21, Lys39, Lys82, Gly83 and Asn102, as well as the side-chain amide group of Asn102, are in close proximity to bound water molecules. Examination of the high-resolution solution structure of reduced human thioredoxin reveals that these results are best accounted for by four bound water molecules. Subsequent simulated annealing calculations carried out on the basis of interproton distance and hydrogen bonding restraints to the bound water molecules, supplemented by the original set of experimental restraints used in the calculation of the three-dimensional structure of human thioredoxin, permit a more precise localization of the bound water positions. Potential hydrogen bonds to these water molecules are described and a comparison is made to corresponding bound water molecules in the crystal structure of oxidized Escherichia coli thioredoxin.     

Reduction of mitochondrial protein mitoNEET [2Fe–2S] clusters by human glutathione reductase

The human mitochondrial outer membrane protein mitoNEET is a newly discovered target of the type 2 diabetes drug pioglitazone. Structurally, mitoNEET is a homodimer with each monomer containing an N-terminal transmembrane α helix tethered to the mitochondrial outer membrane and a C-terminal cytosolic domain hosting a redox-active [2Fe–2S] cluster. Genetic studies have shown that mitoNEET has a central role in regulating energy metabolism in mitochondria. However, the specific function of mitoNEET remains largely elusive. Here we find that the mitoNEET [2Fe–2S] clusters can be efficiently reduced by Escherichia coli thioredoxin reductase and glutathione reductase in an NADPH-dependent reaction. Purified human glutathione reductase has the same activity as E. coli thioredoxin reductase and glutathione reductase to reduce the mitoNEET [2Fe–2S] clusters. However, rat thioredoxin reductase, a human thioredoxin reductase homolog that contains selenocysteine in the catalytic center, has very little or no activity to reduce the mitoNEET [2Fe–2S] clusters. N-ethylmaleimide, a potent thiol modifier, completely inhibits human glutathione reductase from reducing the mitoNEET [2Fe–2S] clusters, indicating that the redox-active disulfide in the catalytic center of human glutathione reductase may be directly involved in reducing the mitoNEET [2Fe–2S] clusters. Additional studies reveal that the reduced mitoNEET [2Fe–2S] clusters in mouse heart cell extracts can be reversibly oxidized by hydrogen peroxide without disruption of the clusters, suggesting that the mitoNEET [2Fe–2S] clusters may undergo redox transition to regulate energy metabolism in mitochondria in response to oxidative signals.     

Assignment of the proton NMR spectrum of reduced and oxidized thioredoxin: sequence-specific assignments, secondary structure, and global fold

Complete proton assignments are reported for the 1H nuclear magnetic resonance (NMR) spectrum of Escherichia coli thioredoxin in the oxidized (with active-site disulfide bridge) and reduced (with two sulfhydryl groups) states. The assignments were obtained by using an integrated assignment strategy in which spin systems were identified from a combination of relayed and multiple quantum NMR techniques prior to sequential assignment. Elements of secondary structure were identified in each protein from characteristic nuclear Overhauser effects (NOE), coupling constants, and slowly exchanging amide protons. In both oxidized and reduced thioredoxin, approximately 33% of the 108 amino acid residues participate in a beta-sheet containing four major strands (three antiparallel and one parallel). A further short beta-strand is connected in a parallel fashion at the N-terminal end of the sheet. Two of the antiparallel beta-strands are connected by a 7-residue beta-bulge loop. Three helical segments, also containing approximately 33% of the amino acid residues, are well-defined in both oxidized and reduced thioredoxin. The remaining third of the molecule apparently consists of reverse turns and loops with little defined secondary structure. The global folds of oxidized and reduced thioredoxin are shown to be essentially identical. Both NOE connectivities and chemical shift values for the two proteins are very similar, except in the immediate vicinity of the active site where significant variations in the chemical shift indicate subtle conformational changes. While the overall fold of oxidized thioredoxin is the same in solution and in the crystalline state, some small differences in local conformation are apparent.     

The modulation of thiol redox state affects the production and metabolism of hydrogen peroxide by heart mitochondria

In rat heart mitochondria, auranofin, arsenite, diamide, and BCNU increase H2O2 formation, further stimulated by antimycin. However, in submitochondrial particles, H2O2 formation and oxygen uptake are not affected, indicating that these substances do not alter respiration. Mitochondria are also able to rapidly metabolize added H2O2 in a process partially prevented by BCNU or auranofin. Calcium does not modify the production of H2O2 and the mitochondrial thioredoxin system is not affected by calcium ions. Auranofin, arsenite, and diamide determine a large mitochondrial permeability transition, while BCNU and acetoacetate are ineffective. Thiols and glutathione are modified only by BCNU and diamide. However, all the compounds tested cause the release of cytochrome c that occurs also in the absence of mitochondrial swelling. In conclusion, the compounds utilized share the common feature of shifting the mitochondrial thiol-linked redox balance towards a more oxidized condition that is responsible of the observed effects.     

Crystal structure of the catalytic domain of a human thioredoxin-like protein.

Thioredoxin is a ubiquitous dithiol oxidoreductase found in many organisms and involved in numerous biochemical processes. Human thioredoxin-like protein (hTRXL) is differentially expressed at different development stages of human fetal cerebrum and belongs to an expanding family of thioredoxins. We have solved the crystal structure of the recombinant N-terminal catalytic domain (hTRXL-N) of hTRXL in its oxidized form at 2.2-A resolution. Although this domain shares a similar three-dimensional structure with human thioredoxin (hTRX), a unique feature of hTRXL-N is the large number of positively charged residues distributed around the active site, which has been implicated in substrate specificity. Furthermore, the hTRXL-N crystal structure is monomeric while hTRX is dimeric in its four crystal structures (reduced, oxidized, C73S and C32S/C35S mutants) reported to date. As dimerization is the key regulatory factor in hTRX, the positive charge and lack of dimer formation of hTRXL-N suggest that it could interact with the acidic amino-acid rich C-terminal region, thereby suggesting a novel regulation mechanism.     

Escherichia coli thioredoxin folds into two compact forms of different stability to urea denaturation

The urea-induced denaturation of Escherichia coli thioredoxin and thioredoxin variants has been examined by electrophoresis on urea gradient slab gels by the method of Creighton [Creighton, T. (1986) Methods Enzymol. 131, 156-172]. Thioredoxin has only two cysteine residues, and these form a redox-active disulfide at the active site. Oxidized thioredoxin-S2 and reduced thioredoxin-(SH)2 each show two folded isomers with a large difference in stability to urea denaturation. The difference in stability is greater for the isomers of oxidized than for the isomers of reduced thioredoxin. At 2 degrees C, the urea concentrations at the denaturation midpoint are approximately 8 and 4.3 M for the oxidized isomers and 4.8 and 3.7 M for the reduced isomers. The difference between the gel patterns of samples applied in native versus denaturing buffer, and at 2 and 25 degrees C, is characteristic for the involvement of a cis-proline-trans-proline isomerization. The data very strongly suggest that the two folded forms of different stabilities correspond to the cis and trans isomers of the highly conserved Pro 76 peptide bond, which is cis in the crystal structure of oxidized thioredoxin. Urea gel experiments with the mutant thioredoxin P76A, with alanine substituted for proline at position 76, corroborate this interpretation. The electrophoretic banding pattern diagnostic for an involvement of proline isomerization in urea denaturation is not observed for oxidized P76A. In broad estimates of delta G degree for the native-denatured transition, the difference in delta G degree (no urea) between the putative cis and trans isomers of the Ile 75-Pro 76 peptide bond is approximately 3 kcal/mol for oxidized thioredoxin and approximately 1.5 kcal/mol for reduced thioredoxin. Since cis oxidized thioredoxin is much more stable than trans, folded oxidized thioredoxin is essentially all cis. In folded reduced thioredoxin, cis and trans interconvert slowly, on the minute time scale at 2 and 25 degrees C. In the absence of urea, the folded reduced thioredoxin is less than a few percent trans. Three additional mutants with additions or substitutions at the active site also show electrophoresis banding patterns consistent with a difference in stability between cis and trans isomers.     

Overlapping roles of the cytoplasmic and mitochondrial redox regulatory systems in the yeast Saccharomyces cerevisiae

Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.     

Lysosomal thiol reductase negatively regulates autophagy by altering glutathione synthesis and oxidation

Redox regulation is critical for a number of cellular functions and has been implicated in the etiology and progression of several diseases, such as cardiovascular diseases, neurodegenerative diseases, and cancer. It has been shown that, in the absence of gamma-interferon inducible lysosomal thiol reductase (GILT), cells are under increased oxidative stress with higher superoxide levels and decreased stability, expression, and function of mitochondrial manganese superoxide dismutase (SOD2). Here, we further elucidate the role of GILT in the homeostatic regulation of oxidative stress. We show that GILT-deficient fibroblasts exhibit reduced glutathione levels, shift in GSSG/GSH ratio toward the oxidized form, and accumulate dysfunctional mitochondria. Redox-sensitive pathways involving Erk1/2 activation and nuclear high mobility group box 1 (HMGB1) protein cytosolic translocation are both activated and associated with increased autophagy in GILT−/− fibroblasts. We hypothesize that these events are responsible for degrading the damaged mitochondria and mitochondrial SOD2 in the absence of GILT. This is the first time to our knowledge that a lysosomal enzyme has been implicated in global effects within the cell.     

Mitochondrial thioredoxin reductase in bovine adrenal cortex its purification, properties, nucleotide/amino acid sequences, and identification of selenocysteine.

Mitochondrial thioredoxin reductase was purified from bovine adrenal cortex. The enzyme is a first protein component in the mitochondrial thioredoxin-dependent peroxide reductase system. The purified reductase exhibited an apparent molecular mass of 56 kDa on SDS/PAGE, whereas the native protein was about 100 kDa, suggesting a homodimeric structure. It catalysed NADPH-dependent reduction of 5, 5'dithiobis(2-nitrobenzoic acid) and thioredoxins from various origins but not glutathione, oxidized dithiothreitol, DL-alpha-lipoic acid, or insulin. Amino acid and nucleotide sequence analyses revealed that it had a presequence composed of 21 amino acids which had features characteristic of a mitochondrial targeting signal. The amino acid sequence of the mature protein was similar to that of bovine cytosolic thioredoxin reductase (57%) and of human glutathione reductase (34%) and less similar to that of Escherichia coli (19%) or yeast (17%) enzymes. Human and bovine cytosolic thioredoxin reductase were recently identified to contain selenocysteine (Sec) as one of their amino acid constituents. We also identified Sec in the C-terminal region of mitochondrial (mt)-thioredoxin reductase by means of MS and amino acid sequence analyses of the C-terminal fragment. The four-amino acid motif, Gly-Cys-Sec-Gly, which is conserved among all Sec-containing thioredoxin reductases, probably functions as the third redox centre of the enzyme, as the mitochondrial reductase was inhibited by 1-chloro-2,4-dinitrobenzene, which was reported to modify Sec and Cys covalently. It is known that mammalian thioredoxin reductase is different from bacterial or yeast enzyme in, for example, their subunit molecular masses and domain structures. These two different types of enzymes with similar activity are suggested to have evolved convergently. Our data clearly show that mitochondria, which might have originated from symbiotic prokaryotes, contain thioredoxin reductase similar to the cytosolic enzyme and different from the bacterial one.     

Thioredoxin 2 haploinsufficiency in mice results in impaired mitochondrial function and increased oxidative stress

The mitochondrial form of thioredoxin, thioredoxin 2 (Txn2), plays an important role in redox control and protection against ROS-induced mitochondrial damage. To evaluate the effect of reduced levels of Txn2 in vivo, we measured oxidative damage and mitochondrial function using mice heterozygous for the Txn2 gene (Txn2(+/-)). The Txn2(+/-) mice showed approximately 50% decrease in Trx-2 protein expression in all tissues without upregulating the other major components of the antioxidant defense system. Reduced levels of Txn2 resulted in decreased mitochondrial function as shown by reduced ATP production by isolated mitochondria and reduced activity of electron transport chain complexes (ETCs). Mitochondria isolated from Txn2(+/-) mice also showed increased ROS production compared to wild type mice. The Txn2(+/-) mice showed increased oxidative damage to nuclear DNA, lipids, and proteins in liver. In addition, we observed an increase in apoptosis in liver from Txn2(+/-) mice compared with wild type mice after diquat treatment. Our results suggest that Txn2 plays an important role in protecting the mitochondria against oxidative stress and in sensitizing the cells to ROS-induced apoptosis.     

Possible existence of lysosome-like organella within mitochondria and its role in mitochondrial quality control

The accumulation of unhealthy mitochondria results in mitochondrial dysfunction, which has been implicated in aging, cancer, and a variety of degenerative diseases. However, the mechanism by which mitochondrial quality is regulated remains unclear. Here, we show that Mieap, a novel p53-inducible protein, induces intramitochondrial lysosome-like organella that plays a critical role in mitochondrial quality control. Mieap expression is directly regulated by p53 and is frequently lost in human cancer as result of DNA methylation. Mieap dramatically induces the accumulation of lysosomal proteins within mitochondria and mitochondrial acidic condition without destroying the mitochondrial structure (designated MALM, for Mieap-induced accumulation of lysosome-like organelles within mitochondria) in response to mitochondrial damage. MALM was not related to canonical autophagy. MALM is involved in the degradation of oxidized mitochondrial proteins, leading to increased ATP synthesis and decreased reactive oxygen species generation. These results suggest that Mieap induces intramitochondrial lysosome-like organella that plays a critical role in mitochondrial quality control by eliminating oxidized mitochondrial proteins. Cancer cells might accumulate unhealthy mitochondria due to p53 mutations and/or Mieap methylation, representing a potential cause of the Warburg effect.     

High-resolution three-dimensional structure of reduced recombinant human thioredoxin in solution

The solution structure of recombinant human thioredoxin (105 residues) has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Approximate interproton distance restraints were derived from nuclear Overhauser effect (NOE) measurements. In addition, a large number of stereospecific assignments for beta-methylene protons and torsion angle restraints for phi, psi, and chi 1 were obtained by using a conformational grid search on the basis of the intraresidue and sequential NOE data in conjunction with 3JHN alpha and 3J alpha beta coupling constants. The structure calculations were based on 1983 approximate interproton distance restraints, 52 hydrogen-bonding restraints for 26 hydrogen bonds, and 98 phi, 71 psi, and 72 chi 1 torsion angle restraints. The 33 final simulated annealing structures obtained had an average atomic rms distribution of the individual structures about the mean coordinate positions of 0.40 +/- 0.06 A for the backbone atoms and 0.78 +/- 0.05 A for all atoms. The solution structure of human thioredoxin consists of a five-stranded beta-sheet surrounded by four alpha-helices, with an active site protrusion containing the two redox-active cysteines. The overall structure is similar to the crystal and NMR structures of oxidized [Katti, S. K., LeMaster, D. M., & Eklund, H. (1990) J. Mol. Biol. 212, 167-184] and reduced [Dyson, J. H., Gippert, G. P., Case, D. A., Holmgren, A., & Wright, P. (1990) Biochemistry 29, 4129-4136] Escherichia coli thioredoxin, respectively, despite the moderate 25% amino acid sequence homology. Several differences, however, can be noted. The human alpha 1 helix is a full turn longer than the corresponding helix in E. coli thioredoxin and is characterized by a more regular helical geometry. The helix labeled alpha 3 in human thioredoxin has its counterpart in the 3(10) helix of the E. coli protein and is also longer in the human protein. In contrast to these structural differences, the conformation of the active site loop in both proteins is very similar, reflecting the perfect sequence identity for a stretch of eight amino acid residues around the redox-active cysteines.     

Human mitochondrial thioredoxin reductase reduces cytochrome c and confers resistance to complex III inhibition

The ubiquitously expressed mammalian thioredoxin reductases are selenoproteins that together with NADPH regenerate active reduced thioredoxins and are involved in diverse actions mediated by redox control. Two main forms of mammalian thioredoxin reductases have been isolated, one cytosolic (TrxR1) and one present in mitochondria (TrxR2). Although the principal target for TrxRs is thioredoxin, the cytosolic form can regenerate several important antioxidants such as ascorbic acid, lipoic acid, and ubiquinone. In this study we demonstrate that cytochrome c is a substrate for both TrxR1 and TrxR2. In addition, cells overexpressing TrxR2 are more resistant to impairment of complex III in the mitochondrial respiratory chain upon both antimycin A and myxothiazol treatments, suggesting a complex III bypassing function of TrxR2. Furthermore, we show that cytochrome c is reduced by TrxR2 in vitro, not only by using NADPH as an electron donor but also by using NADH, pointing at TrxR2 as an important redox protein on complex III impairment. These findings may be valuable in understanding respiratory disorders in mitochondrial diseases.