Crucial effect of the first CXXC motif of human QSOX 1b on the activity to different substrates

Among all sulphhydryl oxidases involved in disulphide formation, quiescin-sulphhydryl oxidase (QSOX) is unique for its multidomain structure, protein thiol oxidation activity and highly efficient catalysis. In this study, site-directed mutagenesis and molecular modelling methods were integrated to investigate the structural and functional characteristics of QSOX, especially the importance of the three CXXC motifs. Site-directed mutagenesis suggested that the C449-C452 motif was essential for the activity of human QSOX 1b; the C70-C73 motif was fundamental in electron transfer from thiol-containing substrate including reduced proteins, DTT, GSH rather than the phosphine-based thiol reductant TCEP, to the C449-C452 motif; and the C509-C512 motif was not involved in electron transfer during disulphide formation. The different roles of the CXXC motifs indicated that there were discrepant electron transfer pathways for the oxidation of thiol-containing substrates and non-thiol disulphide reductants. Molecular modelling method was then used to draw a reasonable picture for the electron transfer process and to elucidate the mechanism of electron transfer when different substrates were oxidized, which will greatly enhance our understanding of the action mechanism of QSOX.     

Inter-domain redox communication in flavoenzymes of the quiescin/sulfhydryl oxidase family: role of a thioredoxin domain in disulfide bond formation

Flavoproteins of the quiescin/sulfhydryl oxidase (QSOX) family catalyze oxidation of peptide and protein thiols to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX family members contain several domains, including an N-terminal thioredoxin domain (Trx) and an FAD-binding-domain (ERV) toward the C-terminus. Partial proteolysis of avian QSOX leads to two fragments, designated 30 and 60 kDa from their apparent mobilities on SDS-PAGE. The 30 kDa fragment is a monomer under nondenaturing conditions and contains a Trx domain with a CxxC sequence typical of protein disulfide isomerase (WCGHC). This QSOX fragment is not detectably glycosylated, contains no detectable FAD, and shows undetectable sulfhydryl oxidase activity. In contrast, the 60 kDa fragment is a dimeric glycoprotein that binds FAD tightly and oxidizes dithiothreitol about 1000-fold slower than intact QSOX. Reduced RNase is not a significant substrate of the 60 kDa fragment. The redox behavior of the 60 kDa flavoprotein fragment is profoundly different from that of intact QSOX. Thus, dithionite or photochemical reduction of the 60 kDa fragment leads to two-electron reduction of the FAD without subsequent reduction of the other two CxxC motifs or the appearance of a thiolate to flavin charge-transfer complex. Further characterization of the fragments and insights gained from the crystal structure of yeast ERV2p (Gross, E., Sevier, C. S., Vala, A., Kaiser, C. A., and Fass, D. (2002) Nat. Struct. Biol. 9, 61-67) suggest that the flow of reducing equivalents in intact avian QSOX is dithiol substrate --> C80/83 --> C519/522 --> C459/462 --> FAD --> oxygen. The ancient fusion of thioredoxin domains to a catalytically more limited ERV domain has produced an efficient catalyst for the direct introduction of disulfide bonds into a wide range of proteins and peptides in multicellular organisms.     

Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.     

Oxidative protein folding in vitro: a study of the cooperation between quiescin-sulfhydryl oxidase and protein disulfide isomerase

The flavin-dependent quiescin-sulfhydryl oxidase (QSOX) inserts disulfide bridges into unfolded reduced proteins with the reduction of molecular oxygen to form hydrogen peroxide. This work investigates how QSOX and protein disulfide isomerase (PDI) cooperate in vitro to generate native pairings in two unfolded reduced proteins: ribonuclease A (RNase, four disulfide bonds and 105 disulfide isomers of the fully oxidized protein) and avian riboflavin binding protein (RfBP, nine disulfide bonds and more than 34 million corresponding disulfide pairings). Experiments combining avian or human QSOX with up to 200 muM avian or human reduced PDI show that the isomerase is not a significant substrate of QSOX. Both reduced RNase and RfBP can be efficiently refolded in an aerobic solution containing micromolar concentrations of reduced PDI and nanomolar levels of QSOX without any added oxidized PDI or glutathione redox buffer. Refolding of RfBP is followed continuously using the complete quenching of the fluorescence of free riboflavin that occurs on binding to apo-RfBP. The rate of refolding is half-maximal at 30 muM reduced PDI when the reduced client protein (1 muM) is used in the presence of 30 nM QSOX. The use of high concentrations of PDI, in considerable excess over the folding protein client, reflects the concentration prevailing in the lumen of the endoplasmic reticulum and allows the redox poise of these in vitro experiments to be set with oxidized and reduced PDI. In the absence of either QSOX or redox buffer, the fastest refolding of RfBP is accomplished with excess reduced PDI and just enough oxidized PDI to generate nine disulfides in the protein client. These in vitro experiments are discussed in terms of current models for oxidative folding in the endoplasmic reticulum.     

Going through the Barrier: COUPLED DISULFIDE EXCHANGE REACTIONS PROMOTE EFFICIENT CATALYSIS IN QUIESCIN SULFHYDRYL OXIDASE*

The quiescin sulfhydryl oxidase (QSOX) family of enzymes generates disulfide bonds in peptides and proteins with the reduction of oxygen to hydrogen peroxide. Determination of the potentials of the redox centers in Trypanosoma brucei QSOX provides a context for understanding catalysis by this facile oxidant of protein thiols. The CXXC motif of the thioredoxin domain is comparatively oxidizing (E′₀ of −144 mV), consistent with an ability to transfer disulfide bonds to a broad range of thiol substrates. In contrast, the proximal CXXC disulfide in the ERV (essential for respiration and vegetative growth) domain of TbQSOX is strongly reducing (E′₀ of −273 mV), representing a major apparent thermodynamic barrier to overall catalysis. Reduction of the oxidizing FAD cofactor (E′₀ of −153 mV) is followed by the strongly favorable reduction of molecular oxygen. The role of a mixed disulfide intermediate between thioredoxin and ERV domains was highlighted by rapid reaction studies in which the wild-type CGAC motif in the thioredoxin domain of TbQSOX was replaced by the more oxidizing CPHC or more reducing CGPC sequence. Mixed disulfide bond formation is accompanied by the generation of a charge transfer complex with the flavin cofactor. This provides thermodynamic coupling among the three redox centers of QSOX and avoids the strongly uphill mismatch between the formal potentials of the thioredoxin and ERV disulfides. This work identifies intriguing mechanistic parallels between the eukaryotic QSOX enzymes and the DsbA/B system catalyzing disulfide bond generation in the bacterial periplasm and suggests that the strategy of linked disulfide exchanges may be exploited in other catalysts of oxidative protein folding.     

Protein substrate discrimination in the quiescin sulfhydryl oxidase (QSOX) family

This work explores the substrate specificity of the quiescin sulfhydryl oxidase (QSOX) family of disulfide-generating flavoenzymes to provide enzymological context for investigation of the physiological roles of these facile catalysts of oxidative protein folding. QSOX enzymes are generally unable to form disulfide bonds within well-structured proteins. Use of a temperature-sensitive mutant of ubiquitin-conjugating enzyme 4 (Ubc4') as a model substrate shows that QSOX activity correlates with the unfolding of Ubc4' monitored by circular dichroism. Fusion of Ubc4' with the more stable glutathione-S-transferase domain demonstrates that QSOX can selectively introduce disulfides into the less stable domain of the fusion protein. In terms of intermolecular disulfide bond generation, QSOX is unable to cross-link well-folded globular proteins via their surface thiols. However, the construction of a septuple mutant of RNase A, retaining a single cysteine residue, demonstrates that flexible protein monomers can be directly coupled by the oxidase. Steady- and pre-steady-state kinetic experiments, combined with static fluorescence approaches, indicate that while QSOX is an efficient catalyst for disulfide bond formation between mobile elements of structure, it does not appear to have a significant binding site for unfolded proteins. These aspects of protein substrate discrimination by QSOX family members are rationalized in terms of the stringent steric requirements for disulfide exchange reactions.     

A flavin-dependent sulfhydryl oxidase in bovine milk

Both metal and flavin-dependent sulfhydryl oxidases catalyze the net generation of disulfide bonds with the reduction of oxygen to hydrogen peroxide. The first mammalian sulfhydryl oxidase to be described was an iron-dependent enzyme isolated from bovine milk whey (Janolino, V.G., and Swaisgood, H.E. (1975) J. Biol. Chem. 250, 2532-2537). This protein was reported to contain 0.5 atoms of iron per 89 kDa subunit and to be completely inhibited by ethylenediaminetetraacetate (EDTA). However the present work shows that a soluble 62 kDa FAD-linked and EDTA-insensitive sulfhydryl oxidase apparently constitutes the dominant disulfide bond-generating activity in skim milk. Unlike the metalloenzyme, the flavoprotein is not associated tightly with skim milk membranes. Sequencing of the purified bovine enzyme (>70% coverage) showed it to be a member of the Quiescin-sulfhydryl oxidase (QSOX) family. Consistent with its solubility, this bovine QSOX1 paralogue lacks the C-terminal transmembrane span of the long form of these proteins. Bovine milk QSOX1 is highly active toward reduced RNase and with the model substrate dithiothreitol. The significance of these new findings is discussed in relation to the earlier reports of metal-dependent sulfhydryl oxidases.     

Exploring the Smallest Active Fragment of HsQSOX1b and Finding a Highly Efficient Oxidative Engine

Human quiescin-sulfhydryl oxidase 1 isoform b (HsQSOX1b) is a highly efficient, multiple-domain enzyme that directly inserts disulfide bonds into client protein. However, previous studies have focused mainly on the catalytic activity of the whole protein rather than its domain structure. In this research, we dissected the structure and function of HsQSOX1b and explored its mechanism as a highly efficient sulfhydryl oxidase by analyzing the truncated variants. The results showed that the first HsQSOX1b thioredoxin domain was essential for thiol oxidase activity. The smallest active fragment (SAQ) was identified to consist of a helix-rich region (HRR) and an essential for respiration and viability/augmenter of liver regeneration (ERV/ALR) domain, which remained highly active to oxidize an artificial non-thiol substrate but not small molecular and protein thiols. Our study clearly demonstrated that SAQ is a highly efficient oxidative engine, which shows high efficiency in the de novo disulfide formation and oxygen reduction and that this more efficient oxidative engine is necessary for the highly efficient catalysis of QSOXs compared to Erv1 and Erv2. This study will help address the roles of different HsQSOX1b domains in de novo disulfide formation and encourage the engineering of more efficient QSOX variants for the in vitro folding of disulfide-containing proteins.     

Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase

The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter of liver regeneration, a sulfhydryl oxidase of the mitochondrial intermembrane space, and a larger protein containing the ERV/ALR domain, quiescin-sulfhydryl oxidase (QSOX). Dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4 electrons with a midpoint potential of -200 mV at pH 7.5. A charge-transfer complex between a proximal thiolate and the oxidized flavin is not observed in Erv2p consistent with a distribution of reducing equivalents over the flavin and distal disulfide redox centers. Upon coordination with Zn2+, full reduction of Erv2p requires 6 electrons. Zn2+ also strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine (TCEP) as the reducing substrate of the oxidase. In contrast to QSOX, Erv2p shows a comparatively low turnover with a range of small thiol substrates, with reduced Escherichia coli thioredoxin and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative protein folding.     

Role of conserved cysteine residues in hepatitis C virus glycoprotein e2 folding and function

Hepatitis C virus glycoprotein E2 contains 18 conserved cysteines predicted to form nine disulfide pairs. In this study, a comprehensive cysteine-alanine mutagenesis scan of all 18 cysteine residues was performed in E1E2-pseudotyped retroviruses (HCVpp) and recombinant E2 receptor-binding domain (E2 residues 384 to 661 [E2(661)]). All 18 cysteine residues were absolutely required for HCVpp entry competence. The phenotypes of individual cysteines and pairwise mutation of disulfides were largely the same for retrovirion-incorporated E2 and E2(661), suggesting their disulfide arrangements are similar. However, the contributions of each cysteine residue and the nine disulfides to E2 structure and function varied. Individual Cys-to-Ala mutations revealed discordant effects, where removal of one Cys within a pair had minimal effect on H53 recognition and CD81 binding (C486 and C569) while mutation of its partner abolished these functions (C494 and C564). Removal of disulfides at C581-C585 and C452-C459 significantly reduced the amount of E1 coprecipitated with E2, while all other disulfides were absolutely required for E1E2 heterodimerization. Remarkably, E2(661) tolerates the presence of four free cysteines, as simultaneous mutation of C452A, C486A, C569A, C581A, C585A, C597A, and C652A (M+C597A) retained wild-type CD81 binding. Thus, only one disulfide from each of the three predicted domains, C429-C552 (DI), C503-C508 (DII), and C607-C644 (DIII), is essential for the assembly of the E2(661) CD81-binding site. Furthermore, the yield of total monomeric E2 increased to 70% in M+C597A. These studies reveal the contribution of each cysteine residue and the nine disulfide pairs to E2 structure and function.     

Avian sulfhydryl oxidase is not a metalloenzyme: adventitious binding of divalent metal ions to the enzyme

Metal- and flavin-dependent sulfhydryl oxidases catalyze the generation of disulfide bonds with reduction of oxygen to hydrogen peroxide. The mammalian skin enzyme has been reported to be copper-dependent, but a recent protein sequence shows it belongs to the Quiescin/sulfhydryl oxidase (QSOX) flavoprotein family. This work demonstrates that avian QSOX is not a metalloenzyme, and that copper and zinc ions inhibit the oxidation of reduced pancreatic ribonuclease by the enzyme. Studies with Zn(2+), as a redox inactive surrogate for copper, show that one Zn(2+) binds to four-electron-reduced QSOX by diverting electrons away from the flavin and into two of the three redox active disulfide bridges in the enzyme. The resulting zinc complex is modestly air-stable, reverting to a spectrum of the native protein with a t(1/2) of 40 min, whereas the four-electron-reduced native QSOX is reoxidized in less than a second under comparable conditions. Using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), an alternate substrate of QSOX that binds Zn(2+) relatively weakly (unlike dithiothreitol), allows rapid inhibition of oxidase activity to be demonstrated at low micromolar metal levels. Zinc binding was followed by rapid-scanning spectrophotometry. Copper also binds the four-electron-reduced form of QSOX with a visible spectrum suggestive of active site occupancy. In addition to interactions with the reduced enzyme, dialysis experiments show that multiple copper and zinc ions can bind to the oxidized enzyme without the perturbation of the flavin spectrum seen earlier. These data suggest that a reinvestigation of the metal content of skin sulfhydryl oxidases is warranted. The redox-modulated binding of zinc to QSOX is considered in light of evidence for a role of zinc-thiolate interactions in redox signaling and zinc mobilization.     

Contribution of individual disulfide bonds to the oxidative folding of ribonuclease A

The eight cysteine residues of ribonuclease A form four disulfide bonds in the native protein. We have analyzed the folding of three double RNase A mutants (C65A/C72A, C58A/C110A, and C26A/C84A, lacking the C65-C72, C58-C110, and C26-C84 disulfide bonds, respectively) and two single mutants (C110A and C26A), in which a single cysteine is replaced with an alanine and the paired cysteine is present in the reduced form. The folding of these mutants was carried out in the presence of oxidized and reduced glutathione, which constitute the main redox agents present within the ER. The use of mass spectrometry in the analysis of the folding processes allowed us (i) to follow the formation of intermediates and thus the pathway of folding of the RNase A mutants, (ii) to quantitate the intermediates that formed, and (iii) to compare the rates of formation of intermediates. By comparison of the folding kinetics of the mutants with that of wild-type RNase A, the contribution of each disulfide bond to the folding process has been evaluated. In particular, we have found that the folding of the C65A/C72A mutant occurs on the same time scale as that of the wild-type protein, thus suggesting that the removal of the C65-C72 disulfide bond has no effect on the kinetics of RNase A folding. Conversely, the C58A/C110A and C26A/C84A mutants fold much more slowly than the wild-type protein. The removal of the C58-C110 and C26-C84 disulfide bonds has a dramatic effect on the kinetics of RNase A folding. Results described in this paper provide specific information about conformational folding events in the regions involving the mutated cysteine residues, thus contributing to a better understanding of the complex mechanism of oxidative folding.     

Mutations in domain a' of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A

Protein disulfide isomerase (PDI, EC 5.3.4.1), an enzyme and chaperone, catalyses disulfide bond formation and rearrangements in protein folding. It is also a subunit in two proteins, the enzyme collagen prolyl 4-hydroxylase and the microsomal triglyceride transfer protein. It consists of two catalytically active domains, a and a', and two inactive ones, b and b', all four domains having the thioredoxin fold. Domain b' contains the primary peptide binding site, but a' is also critical for several of the major PDI functions. Mass spectrometry was used here to follow the folding pathway of bovine pancreatic ribonuclease A (RNase A) in the presence of three PDI mutants, F449R, Delta455-457, and abb', and the individual domains a and a'. The first two mutants contained alterations in the last alpha helix of domain a', while the third lacked the entire domain a'. All mutants produced genuine, correctly folded RNase A, but the appearance rate of 50% of the product, as compared to wild-type PDI, was reduced 2.5-fold in the case of PDI Delta455-457, 7.5-fold to eightfold in the cases of PDI F449R and PDI abb', and over 15-fold in the cases of the individual domains a and a'. In addition, PDI F449R and PDI abb' affected the distribution of folding intermediates. Domains a and a' catalyzed the early steps in the folding but no disulfide rearrangements, and therefore the rate observed in the presence of these individual domains was similar to that of the spontaneous process.     

Adjacent cysteine residues as a redox switch.

Oxidation of adjacent cysteine residues into a cystine forms a strained eight-membered ring. This motif was tested as the basis for an enzyme with an artificial redox switch. Adjacent cysteine residues were introduced into two different structural contexts in ribonuclease A (RNase A) by site-directed mutagenesis to produce the A5C/A6C and S15C/S16C variants. Ala5 and Ala6 are located in an alpha-helix, whereas Ser15 and Ser16 are located in a surface loop. Only A5C/A6C RNase A had the desired property. The catalytic activity of this variant decreases by 70% upon oxidation. The new disulfide bond also decreases the conformational stability of the A5C/A6C variant. Reduction with dithiothreitol restores full enzymatic activity. Thus, the insertion of adjacent cysteine residues in a proper context can be used to modulate enzymatic activity.     

QSOX contains a pseudo-dimer of functional and degenerate sulfhydryl oxidase domains

Quiescin sulfhydryl oxidase (QSOX) catalyzes formation of disulfide bonds between cysteine residues in substrate proteins. Human QSOX1 is a multi-domain, monomeric enzyme containing a module related to the single-domain sulfhydryl oxidases of the Erv family. A partial QSOX1 crystal structure reveals a single-chain pseudo-dimer mimicking the quaternary structure of Erv enzymes. However, one pseudo-dimer “subunit” has lost its cofactor and catalytic activity. In QSOX evolution, a further concatenation to a member of the protein disulfide isomerase family resulted in an enzyme capable of both disulfide formation and efficient transfer to substrate proteins.     

Sulfur shuffle: modulating enzymatic activity by thiol-disulfide interchange

The facile modulation of biological processes is an important goal of biological chemists. Here, a general strategy is presented for controlling the catalytic activity of an enzyme. This strategy is demonstrated with ribonuclease A (RNase A), which catalyzes the cleavage of RNA. The side-chain amino group of Lys41 donates a hydrogen bond to a nonbridging oxygen in the transition state for RNA cleavage. Replacing Lys41 with a cysteine residue is known to decrease the value of k(cat)/K(m) by 10(5)-fold. Forming a mixed disulfide between the side chain of Cys41 of K41C RNase A and cysteamine replaces the amino group and increases k(cat)/K(m) by 10(3)-fold. This enzyme, which contains a mixed disulfide, is readily deactivated by dithiothreitol. Forming a mixed disulfide between the side chain of Cys41 and mercaptopropyl phosphate, which is designed to place a phosphoryl group in the active site, decreases activity by an additional 25-fold. This enzyme, which also contains a mixed disulfide, is reactivated in the presence of dithiothreitol and inorganic phosphate (which displaces the pendant phosphoryl group from the active site). An analogous control mechanism could be installed into the active site of virtually any enzyme by replacing an essential residue with a cysteine and elaborating the side chain of that cysteine into appropriate mixed disulfides.     

Essential cysteine residues for human RNase κ catalytic activity

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.     

Activation of class III ribonucleotide reductase by thioredoxin

Anaerobic ribonucleotide reductase provides facultative and obligate anaerobic microorganisms with the deoxyribonucleoside triphosphates used for DNA chain elongation and repair. In Escherichia coli, the dimeric alpha2 enzyme contains, in its active form, a glycyl radical essential for the reduction of the substrate. The introduction of the glycyl radical results from the reductive cleavage of S-adenosylmethionine catalyzed by the reduced (4Fe-4S) center of a small activating protein called beta. This activation reaction has long been known to have an absolute requirement for dithiothreitol. Here, we report that thioredoxin, along with NADPH and NADPH:thioredoxin oxidoreductase, efficiently replaces dithiothreitol and reduces an unsuspected critical disulfide bond probably located on the C terminus of the alpha protein. Activation of reduced alpha protein does not require dithiothreitol or thioredoxin anymore, and activation rates are much faster than previously reported. Thus, in E. coli, thioredoxin has very different roles for class I ribonucleotide reductase where it is required for the substrate turnover and class III ribonucleotide reductase where it acts only for the activation of the enzyme.     

Contrasting effects of various sulfhydryl reagents on the activity of lecithin: Cholesterol acyltransferase

Lecithin:cholesterol acyltransferase, E.C. 2.3.1.43, can be either stimulated or inhibited by reducing reagents containing free sulfhydryl groups. Mercaptoethanol and dithiothreitol stimulate enzyme activity while cysteine and reduced glutathione inhibit activity. The oxidizing disulfide reagent 2-pyridine disulfide has minimal effects on enzyme activity by itself but suppresses the stimulatory effect of mercaptoethanol.     

Generating disulfides with the Quiescin-sulfhydryl oxidases

The Quiescin-sulfhydryl oxidase (QSOX) family of flavoenzymes catalyzes the direct and facile insertion of disulfide bonds into unfolded reduced proteins with concomitant reduction of oxygen to hydrogen peroxide. This review discusses the chemical mechanism of these enzymes and the involvement of thioredoxin and flavin-binding domains in catalysis. The variability of CxxC motifs in the QSOX family is highlighted and attention is drawn to the steric factors that may promote efficient thiol/disulfide exchange during oxidative protein folding. The varied cellular location of these multi-domain sulfhydryl oxidases is reviewed and potential intracellular and extracellular roles are summarized. Finally, this review identifies important unresolved questions concerning this ancient family of sulfhydryl oxidases.