The high resolution crystal structure of recombinant Crithidia fasciculata tryparedoxin-I.

Tryparedoxin-I is a recently discovered thiol-disulfide oxidoreductase involved in the regulation of oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata tryparedoxin-I in the oxidized state has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises residues 3 to 145 with 236 water molecules and has been refined using all data between a 19- and 1.4-A resolution to an R-factor and R-free of 19.1 and 22.3%, respectively. Despite sharing only about 20% sequence identity, tryparedoxin-I presents a five-stranded twisted beta-sheet and two elements of helical structure in the same type of fold as displayed by thioredoxin, the archetypal thiol-disulfide oxidoreductase. However, the relationship of secondary structure with the linear amino acid sequences is different for each protein, producing a distinctive topology. The beta-sheet core is extended in the trypanosomatid protein with an N-terminal beta-hairpin. There are also differences in the content and orientation of helical elements of secondary structure positioned at the surface of the proteins, which leads to different shapes and charge distributions between human thioredoxin and tryparedoxin-I. A right-handed redox-active disulfide is formed between Cys-40 and Cys-43 at the N-terminal region of a distorted alpha-helix (alpha1). Cys-40 is solvent-accessible, and Cys-43 is positioned in a hydrophilic cavity. Three C-H...O hydrogen bonds donated from two proline residues serve to stabilize the disulfide-carrying helix and support the correct alignment of active site residues. The accurate model for tryparedoxin-I allows for comparisons with the family of thiol-disulfide oxidoreductases and provides a template for the discovery or design of selective inhibitors of hydroperoxide metabolism in trypanosomes. Such inhibitors are sought as potential therapies against a range of human pathogens.     

Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif

Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an alpha helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.     

Heterodimer formation between thioredoxin f and fructose 1,6-bisphosphatase from spinach chloroplasts

Chloroplast fructose 1,6-bisphosphatase (FBPase) is activated by reduction of a regulatory disulfide through thioredoxin f (Trx f). In the course of this reduction a transient mixed disulfide is formed linking covalently Trx f with FBPase, which possesses three Cys on a loop structure, two of them forming the redox-active disulfide bridge. The goal of this study was to identify the Cys involved in the transient mixed disulfide. To stabilize this reaction intermediate, mutant proteins with modified active sites were used. We identified Cys-155 of the FBPase as the one engaged in the formation of the mixed disulfide intermediate with Cys-46 of Trx f.     

The structure of reduced tryparedoxin peroxidase reveals a decamer and insight into reactivity of 2Cys-peroxiredoxins

Tryparedoxin peroxidase (TryP) is a recently discovered 2Cys-peroxiredoxin involved in defence against oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata TryP, in the reduced state, has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises a decamer with 52 symmetry, ten chloride ions with 23 water molecules and has been refined, using data to 3.2 A resolution (1 A=0.1 nm), to an R-factor and R(free) of 27.3 and 28.6 %, respectively. Secondary structure topology places TryP along with tryparedoxin and glutathione peroxidase in a distinct subgroup of the thioredoxin super-family. The molecular details at the active site support ideas about the enzyme mechanism and comparisons with an oxidised 2Cys-peroxiredoxin reveal structural alterations induced by the change in oxidation state. These include a difference in quaternary structure from dimer (oxidised form) to decamer (reduced form). The 2Cys-peroxiredoxin assembly may prevent indiscriminate oligomerisation, localise ten peroxidase active sites and contribute to both the specificity of reduction by the redox partner tryparedoxin and attraction of peroxides into the active site.     

Plasmoredoxin, a novel redox-active protein unique for malarial parasites.

Thioredoxins are a group of small redox-active proteins involved in cellular redox regulatory processes as well as antioxidant defense. Thioredoxin, glutaredoxin, and tryparedoxin are members of the thioredoxin superfamily and share structural and functional characteristics. In the malarial parasite, Plasmodium falciparum, a functional thioredoxin and glutathione system have been demonstrated and are considered to be attractive targets for antimalarial drug development. Here we describe the identification and characterization of a novel 22 kDa redox-active protein in P. falciparum. As demonstrated by in silico sequence analyses, the protein, named plasmoredoxin (Plrx), is highly conserved but found exclusively in malarial parasites. It is a member of the thioredoxin superfamily but clusters separately from other members in a phylogenetic tree. We amplified the gene from a gametocyte cDNA library and overexpressed it in E. coli. The purified gene product can be reduced by glutathione but much faster by dithiols like thioredoxin, glutaredoxin, trypanothione and tryparedoxin. Reduced Plrx is active in an insulin-reduction assay and reduces glutathione disulfide with a rate constant of 640 m-1.s-1 at pH 6.9 and 25 degrees C; glutathione-dependent reduction of H2O2 and hydroxyethyl disulfide by Plrx is negligible. Furthermore, plasmoredoxin provides electrons for ribonucleotide reductase, the enzyme catalyzing the first step of DNA synthesis. As demonstrated by Western blotting, the protein is present in blood-stage forms of malarial parasites. Based on these results, plasmoredoxin offers the opportunity to improve diagnostic tools based on PCR or immunological reactions. It may also represent a specific target for antimalarial drug development and is of phylogenetic interest.     

Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli

The large subunit of ribonucleotide reductase from Escherichia coli contains redox-active cysteine residues. In separate experiments, five conserved and 2 nonconserved cysteine residues were substituted with alanines by oligonucleotide-directed mutagenesis. The activities of the mutant proteins were determined in the presence of three different reductants: thioredoxin, glutaredoxin, or dithiothreitol. The results indicate two different classes of redox-active cysteines in ribonucleotide reductase: 1) C-terminal Cys-754 and Cys-759 responsible for the interaction with thioredoxin and glutaredoxin; and 2) Cys-225 and Cys-439 located at the nucleotide-binding site. Our classification of redox-active cysteines differs from the location of the active site cysteines in E. coli ribonucleotide reductase suggested previously (Lin, A.-N. I., Ashley, G. W., and Stubbe, J. (1987) Biochemistry 26, 6905-6909).     

Oxidized and synchrotron cleaved structures of the disulfide redox center in the N-terminal domain of Salmonella typhimurium AhpF

The flavoprotein component (AhpF) of Salmonella typhimurium alkyl hydroperoxide reductase contains an N-terminal domain (NTD) with two contiguous thioredoxin folds but only one redox-active disulfide (within the sequence -Cys129-His-Asn-Cys132-). This active site is responsible for mediating the transfer of electrons from the thioredoxin reductase-like segment of AhpF to AhpC, the peroxiredoxin component of the two-protein peroxidase system. The previously reported crystal structure of AhpF possessed a reduced NTD active site, although fully oxidized protein was used for crystallization. To further investigate this active site, we crystallized an isolated recombinant NTD (rNTD); using diffraction data sets collected first at our in-house X-ray source and subsequently at a synchrotron, we showed that the active site disulfide bond (Cys129-Cys132) is oxidized in the native crystals but becomes reduced during synchrotron data collection. The NTD disulfide bond is apparently particularly sensitive to radiation cleavage compared with other protein disulfides. The two data sets provide the first view of an oxidized (disulfide) form of NTD and show that the changes in conformation upon reduction of the disulfide are localized and small. Furthermore, we report the apparent pKa of the active site thiol to be approximately 5.1, a relatively low pKa given its redox potential (approximately 265 mV) compared with most members of the thioredoxin family.     

Catalytic properties, thiol pK value, and redox potential of Trypanosoma brucei tryparedoxin

The dithiol protein tryparedoxin is a component of the unique trypanothione/trypanothione reductase metabolism of trypanosomatids and is involved in the parasite synthesis of deoxyribonucleotides and the detoxication of hydroperoxides. Tryparedoxin is a highly abundant protein in all life stages of Trypanosoma brucei, the causative agent of African sleeping sickness. As shown here, its functional properties are intermediate between those of classical thioredoxins and glutaredoxins. The redox potential of T. brucei tryparedoxin of -249 mV was determined by protein-protein redox equilibration with Escherichia coli thioredoxin. The trypanothione/tryparedoxin couple is probably the most significant factor determining the cytosolic redox potential of the parasites. The pK value of Cys(40), the first thiol in the WCPPC motif, is 7.2 as derived from the thiolate absorption at 240 nm and the rate of carboxymethylation. Alteration of the active site into that of thioredoxin (CGPC) did not affect the pK value. In contrast, in the mutant with the glutaredoxin motif (CPYC) the pK dropped to < or =4.0. The fact that the pK value of tryparedoxin coincides with the intracellular pH of the parasite may contribute to the reactivity of tryparedoxin in thiol disulfide exchange reactions.     

Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni

Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.     

Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis

AhpF, a homodimer of 57 kDa subunits, is a flavoenzyme which catalyzes the NADH-dependent reduction of redox-active disulfide bonds in the peroxidase AhpC, a member of the recently identified peroxiredoxin class of antioxidant enzymes. The structure of AhpF from Salmonella typhimurium at 2.0 A resolution, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210-521) is structurally like Escherichia coli thioredoxin reductase. In addition, AhpF has an N-terminal domain (residues 1-196) formed from two contiguous thioredoxin folds, but containing just a single redox-active disulfide (Cys129-Cys132). A flexible linker (residues 197-209) connects the domains, consistent with experiments showing that the N-terminal domain acts as an appended substrate, first being reduced by the C-terminal portion of AhpF, and subsequently reducing AhpC. Modeling studies imply that an intrasubunit electron transfer accounts for the reduction of the N-terminal domain in dimeric AhpF. Furthermore, comparing the N-terminal domain with protein disulfide oxidoreductase from Pyrococcus furiosis, we describe a new class of protein disulfide oxidoreductases based on a novel mirror-image active site arrangement, with a distinct carboxylate (Glu86) being functionally equivalent to the key acid (Asp26) of E. coli thioredoxin. A final fortuitous result is that the N-terminal redox center is reduced and provides a high-resolution view of the thiol-thiolate hydrogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.     

Acid-base catalysis in the mechanism of thioredoxin reductase from Drosophila melanogaster

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. Like other members of the pyridine nucleotide-disulfide oxidoreductase enzyme family, the enzyme from Drosophila melanogaster is a homodimer, and each catalytically active unit consists of three redox centers: FAD and an N-terminal Cys-57/Cys-62 redox-active disulfide from one monomer and a Cys-489'/Cys-490' C-terminal redox-active disulfide from the second monomer. Because dipteran insects such as D. melanogaster lack glutathione reductase, thioredoxin reductase (DmTrxR) is particularly important; in addition to its normal functions, it also reduces GSSG for antioxidant protection. DmTrxR, used as a model for the enzyme from the malaria vector, Anopheles gambiae, has been shown to cycle in catalysis between the two-electron and four-electron reduced states, EH2 and EH4 [Bauer, H. et al. (2003) J. Biol. Chem. 278, 33020-33028]. His-464' acts as an acid-base catalyst of the dithiol-disulfide interchange reactions required in catalysis. The H464'Q enzyme has only 2% of the wild-type activity, emphasizing the importance of this residue. The pH dependence of Vmax for wild-type DmTrxR has pKa values of 6.4 and 9.3 on the DmTrxR-DmTrx-2 complex, whereas H464'Q DmTrxR only has an observable pKa at 6.4, indicating that the pKa at pH 9.3 is contributed mainly by His-464'. The pKa at pH 6.4 has been assigned to Cys-57 and Cys-490'; the thiolate on Cys-490' is the nucleophile in the reduction of Trx. In contrast to wild-type DmTrxR, H464'Q DmTrxR does not stabilize a thiolate-FAD charge-transfer complex in the presence of excess NADPH. The rates of steps in both the reductive and the oxidative half-reactions are markedly diminished in H464'Q DmTrxR as compared to those of wild-type enzyme, indicating that His-464' is involved in both half-reactions.     

Glutathionylation of trypanosomal thiol redox proteins

Trypanosomatids, the causative agents of several tropical diseases, lack glutathione reductase and thioredoxin reductase but have a trypanothione reductase instead. The main low molecular weight thiols are trypanothione (N(1),N(8)-bis-(glutathionyl)spermidine) and glutathionyl-spermidine, but the parasites also contain free glutathione. To elucidate whether trypanosomes employ S-thiolation for regulatory or protection purposes, six recombinant parasite thiol redox proteins were studied by ESI-MS and MALDI-TOF-MS for their ability to form mixed disulfides with glutathione or glutathionylspermidine. Trypanosoma brucei mono-Cys-glutaredoxin 1 is specifically thiolated at Cys(181). Thiolation of this residue induced formation of an intramolecular disulfide bridge with the putative active site Cys(104). This contrasts with mono-Cys-glutaredoxins from other sources that have been reported to be glutathionylated at the active site cysteine. Both disulfide forms of the T. brucei protein were reduced by tryparedoxin and trypanothione, whereas glutathione cleaved only the protein disulfide. In the glutathione peroxidase-type tryparedoxin peroxidase III of T. brucei, either Cys(47) or Cys(95) became glutathionylated but not both residues in the same protein molecule. T. brucei thioredoxin contains a third cysteine (Cys(68)) in addition to the redox active dithiol/disulfide. Treatment of the reduced protein with GSSG caused glutathionylation of Cys(68), which did not affect its capacity to catalyze reduction of insulin disulfide. Reduced T. brucei tryparedoxin possesses only the redox active Cys(32)-Cys(35) couple, which upon reaction with GSSG formed a disulfide. Also glyoxalase II and Trypanosoma cruzi trypanothione reductase were not sensitive to thiolation at physiological GSSG concentrations.     

Structural basis for a distinct catalytic mechanism in Trypanosoma brucei tryparedoxin peroxidase

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys(47) and Cys(95) and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4A resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys(47), Gln(82), and Trp(137) do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys(107) against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys(97) and Lys(99) play a crucial role in the interaction with tryparedoxin.     

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.     

Characterization of two active site mutations of thioredoxin reductase from Escherichia coli

Thioredoxin reductase (TRR), a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, undergoes two sequential thiol-disulfide interchange reactions with thioredoxin during catalysis. In order to assess the catalytic role of each nascent thiol of the active site disulfide of thioredoxin reductase, the 2 cysteines (Cys-136 and Cys-139) forming this disulfide have been individually changed to serines by site-directed mutageneses of the cloned trxB gene of Escherichia coli. Spectral analyses of TRR(Ser-136,Cys-139) as a function of pH and ionic strength have revealed two pKa values associated with the epsilon 456, one of which increases from 7.0 to 8.3 as the ionic strength is increased, and a second at 4.4 which is seen only at high ionic strength. epsilon 458 of wild type TRR(Cys-136,Cys-139) and epsilon 453 of TRR(Cys-136,Ser-139) are pH-independent. A charge transfer complex (epsilon 530 = 1300 M-1 cm-1), unique to TRR(Ser-136,Cys-139), has been observed under conditions of high ammonium cation concentration (apparent Kd = 54 microM) at pH 7.6. These results suggest the assignment of Cys-139 as the FAD-interacting thiol in the reduction of thioredoxin by NADPH via thioredoxin reductase. If, as with other members of this enzyme family, the two distinct catalytic functions are each carried out by a different nascent thiol, then Cys-136 would perform the initial thiol-disulfide interchange with thioredoxin. Steady state kinetic analyses of the proteins have revealed turnover numbers of 10 and 50% of the value of the wild type enzyme for TRR(Ser-136,Cys-139) and TRR(Cys-136,Ser-139), respectively, and no changes in the apparent Km values of TR(S2) or NADPH. The finding of activity in the mutants indicates that the remaining thiol can carry out interchange with the disulfide of thioredoxin, and the resulting mixed disulfide can be reduced by NADPH via the flavin.     

Structural snapshots of yeast alkyl hydroperoxide reductase Ahp1 peroxiredoxin reveal a novel two-cysteine mechanism of electron transfer to eliminate reactive oxygen species

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that protect cells against reactive oxygen species and are involved in cellular signaling pathways. Alkyl hydroperoxide reductase Ahp1 belongs to the Prx5 subfamily and is a two-cysteine (2-Cys) Prx that forms an intermolecular disulfide bond. Enzymatic assays and bioinformatics enabled us to re-assign the peroxidatic cysteine (C_P) to Cys-62 and the resolving cysteine (C_R) to Cys-31 but not the previously reported Cys-120. Thus Ahp1 represents the first 2-Cys Prx with a peroxidatic cysteine after the resolving cysteine in the primary sequence. We also found the positive cooperativity of the substrate t-butyl hydroperoxide binding to Ahp1 homodimer at a Hill coefficient of ∼2, which enabled Ahp1 to eliminate hydroperoxide at much higher efficiency. To gain the structural insights into the catalytic cycle of Ahp1, we determined the crystal structures of Ahp1 in the oxidized, reduced, and Trx2-complexed forms at 2.40, 2.91, and 2.10 Å resolution, respectively. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing C_P and C_R. An intermolecular C_P-C_R disulfide bond crossing the A-type dimer interface distinguishes Ahp1 from other typical 2-Cys Prxs. The structure of the Ahp1-Trx2 complex showed for the first time how the electron transfers from thioredoxin to a peroxidase with a thioredoxin-like fold. In addition, site-directed mutagenesis in combination with enzymatic assays suggested that the peroxidase activity of Ahp1 would be altered upon the urmylation (covalently conjugated to ubiquitin-related modifier Urm1) of Lys-32.     

AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase.

A group of bacterial flavoproteins related to thioredoxin reductase contain an additional approximately 200-amino-acid domain including a redox-active disulfide center at their N-termini. These flavoproteins, designated NADH:peroxiredoxin oxidoreductases, catalyze the pyridine-nucleotide-dependent reduction of cysteine-based peroxidases (e.g. Salmonella typhimurium AhpC, a member of the peroxiredoxin family) which in turn reduce H2O2 or organic hydroperoxides. These enzymes catalyze rapid electron transfer (kcat > 165 s-1) through one tightly bound FAD and two redox-active disulfide centers, with the N-terminal-most disulfide center acting as a redox mediator between the thioredoxin-reductase-like part of these proteins and the peroxiredoxin substrates. A chimeric protein with the first 207 amino acids of S. typhimurium AhpF attached to the N-terminus of Escherichia coli thioredoxin reductase exhibits very high NADPH:peroxiredoxin oxidoreductase and thioredoxin reductase activities. Catalytic turnover by NADH:peroxiredoxin oxidoreductases may involve major domain rotations, analogous to those proposed for bacterial thioredoxin reductase, and cycling of these enzymes between two electron-reduced (EH2) and four electron-reduced (EH4) redox states.     

Peroxiredoxins

Present knowledge on peroxiredoxins is reviewed with special emphasis on catalytic principles, specificities and biological function. Peroxiredoxins are low efficiency peroxidases using thiols as reductants. They appear to be fairly promiscuous with respect to the hydroperoxide substrate; the specificities for the donor substrate vary considerably between the subfamilies, comprising GSH, thioredoxin, tryparedoxin and the analogous CXXC motifs in bacterial AhpF proteins. Peroxiredoxins are definitely responsible for antioxidant defense in bacteria (AhpC), yeast (thioredoxin peroxidase) and trypanosomatids (tryparedoxin peroxidase). They are considered to determine virulence of mycobacteria and trypanosomatids. In higher plants they are involved in balancing hydroperoxide production during photosynthesis. In higher animals peroxiredoxins appear to be involved in the redox-regulation of cellular signaling and differentiation, displaying in part opposite effects.     

A second class of peroxidases linked to the trypanothione metabolism

Trypanosoma brucei, the causative agent of African sleeping sickness, has three nearly identical genes encoding cysteine homologues of classical selenocysteine-containing glutathione peroxidases. The proteins are expressed in the mammalian and insect stages of the parasite. One of the genes, which contains a mitochondrial as well as a glycosomal targeting signal has been overexpressed. The recombinant T. brucei peroxidase has a high preference for the trypanothione/tryparedoxin couple as electron donor for the reduction of different hydroperoxides but accepts also T. brucei thioredoxin. The apparent rate constants k(2)' for the regeneration of the reduced enzyme are 2 x 10(5) m(-1) s(-1) with tryparedoxin and 5 x 10(3) m(-1) s(-1) with thioredoxin. No saturation kinetics was observed and the rate-limiting step of the overall reaction is reduction of the hydroperoxide. With glutathione, the peroxidase has marginal activity and reduction of the enzymes becomes limiting with a k(2)' value of 3 m (-1) s(-1). The T. brucei peroxidase, in contrast to the related Trypanosoma cruzi enzyme, also accepts hydrogen peroxide as substrate. The catalytic efficiency of the peroxidase studied here is comparable with that of the peroxiredoxin-like tryparedoxin peroxidases, which shows that trypanosomes possess two distinct peroxidase systems both dependent on the unique dithiol trypanothione.     

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.