December 1, 1997Journal of Biological Chemistry413 citationsOpen Access

Redox Potentials of Glutaredoxins and Other Thiol-Disulfide Oxidoreductases of the Thioredoxin Superfamily Determined by Direct Protein-Protein Redox Equilibria

FÅFredrik Åslund KBKurt D. Berndt AHArne Holmgren

Key Points

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Abstract

Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX 1 X 2C). Standard state redox potential (E°′) values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E°′ resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E°′ values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coliglutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E°′ = −233 and −198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculatedE°′ values from a number of proteins ranging from −270 mV for E. coli Trx to −124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as −240 mV. Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH. Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX 1 X 2C). Standard state redox potential (E°′) values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E°′ resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E°′ values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coliglutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E°′ = −233 and −198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculatedE°′ values from a number of proteins ranging from −270 mV for E. coli Trx to −124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as −240 mV. Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH. Glutaredoxin (Grx1) 1The abbreviations used are: Grx1, E. coli glutaredoxin 1; Grx3, E. coli glutaredoxin-3; GSH, glutathione; GSSG, glutathione disulfide; DsbA, E. colidisulfide bond-promoting product of the gene dsbA; DsbATrx, DsbA with the active site changed into that of thioredoxin (Pro-33 replaced by Gly, and His-34 replaced by Pro); Δε, molar CD of the amide chromophore; E°′, standard state redox potential at 25 °C; EGSH°′, standard state redox potential of glutathione; ΔEAB°′, difference in standard state redox potentials between molecules A and B; EU°′, standard state redox potential of the unfolded form of the protein; f u , fraction of unfolded protein; GdnHCl, guanidine hydrochloride; ΔGNSS and ΔGUSS, the Gibbs energy of disulfide bond formation in the folded and unfolded state, respectively. ΔGH2O° andm G, intercept and slope of the linear extrapolation of unfolding Gibbs energy changes versusdenaturant concentration; K ox, equilibrium constant with GSH; K′, apparent concentration equilibrium constant; m N and m U, slopes of the pre- and post-transition region base line of the denaturation, respectively; PDI, protein disulfide isomerase; Trx,E. coli thioredoxin; TrxPDI, E. colithioredoxin with the active site changed into that of thioredoxin (Pro-34 replaced by His); HPLC, high performance liquid chromatography. was discovered as a GSH-dependent hydrogen donor for ribonucleotide reductase in Escherichia coli mutants lacking the first identified electron donor, thioredoxin (1Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2275-2279Crossref PubMed Scopus (364) Google Scholar). The presence of an additional hydrogen donor system for ribonucleotide reductase was postulated since a double mutant lacking both Grx1 and Trx was viable (2Russel M. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 990-994Crossref PubMed Scopus (48) Google Scholar). The search for this third hydrogen donor system resulted in the isolation of two additional glutaredoxins in E. coli, Grx2 and Grx3 (3Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google Scholar). The recent structural characterization of Grx3 showed that in addition to the 33% amino acid sequence identity with Grx1, the two proteins have highly conserved secondary structure elements and overall fold (4Åslund F. Nordstrand K. Berndt K.D. Nikkola M. Bergman T. Ponstingl H. Jörnvall H. Otting G. Holmgren A. J. Biol. Chem. 1996; 271: 6736-6745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). However, despite Grx1 and Grx3 being closely related 9-kDa redox proteins with identical active-site sequences (CPYC), Grx3 exhibits only a fraction of the activity of Grx1 as a reductant of ribonucleotide reductase (NrdAB and NrdEF; Refs. 3Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google Scholar and 5Jordan A. Pontis E. Atta M. Krook M. Gibert I. Barbé J. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12892-12896Crossref PubMed Scopus (98) Google Scholar) or of insulin disulfides (4Åslund F. Nordstrand K. Berndt K.D. Nikkola M. Bergman T. Ponstingl H. Jörnvall H. Otting G. Holmgren A. J. Biol. Chem. 1996; 271: 6736-6745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). One possible for be a difference in redox potential between the two and the the residues between the active site (CX 1 X as a for the standard state redox potential (E°′) of the thioredoxin superfamily of The first T. H. J. Biol. Chem. Full Text PDF PubMed Google G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) that redox potential of a protein be or reducing by the 1 and X for those of a protein redox potential was or respectively. the mutant TrxPDI, the active site of protein disulfide in a of mV in the this from that of G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The redox potential for the mutant mV and mV reducing the is from the = −270 mV determined for Trx T. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A similar of one of the of E°′ from to mV T. 1996; PubMed Scopus Google Scholar). A confirmed using a of mutants U. P. T. P. Full Text PDF PubMed Scopus Google that at for DsbA, a between the redox potential and the a of the active-site cysteine U. P. T. P. Full Text PDF PubMed Scopus Google as by the using molecules J. Chem. Scopus Google Scholar). the thioredoxin superfamily of oxidoreductases have redox potentials a the active-site sequences of oxidoreductases have similar redox potentials F. S. M. PubMed Scopus Google Scholar). This the that redox potentials of proteins are determined by in the folded state in a of the active site sequence present in a T. H. J. Biol. Chem. Full Text PDF PubMed Google G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google U. P. T. P. Full Text PDF PubMed Scopus Google Scholar). This is the with Grx1 and Grx3, which have identical active-site sequences only 33% sequence identity in their overall similar fold (4Åslund F. Nordstrand K. Berndt K.D. Nikkola M. Bergman T. Ponstingl H. Jörnvall H. Otting G. Holmgren A. J. Biol. Chem. 1996; 271: 6736-6745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). of the redox potentials of two glutaredoxins is in of their which one similar activity in reducing GSH-mixed disulfides (3Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google a that the disulfide form of the protein F. K. Holmgren A. PubMed Scopus Google and the large in the to protein ribonucleotide reductase or insulin (4Åslund F. Nordstrand K. Berndt K.D. Nikkola M. Bergman T. Ponstingl H. Jörnvall H. Otting G. Holmgren A. J. Biol. Chem. 1996; 271: 6736-6745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The standard state redox potential E°′ for a protein be calculated from the equilibrium constant of the redox reaction a with redox potential using the The used are glutathione/glutathione disulfide (GSH/GSSG) buffers or via an reductase Biol. Google Scholar). Standard state redox potentials the thioredoxin of thiol-disulfide oxidoreductases from E°′ = −124 A. PubMed Scopus Google M. J. Biol. PubMed Scopus Google Scholar) to −270 mV G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google to an overall equilibrium constant of the in the of an equilibrium constant is the the equilibrium constant is to one is to this of redox the of a for the of the standard state redox potential of a used redox standard to in the values for the standard state redox potential of glutathione from to mV J. Chem. Scopus Google Biol. Google J. S. PubMed Scopus Google J. Biol. Chem. Full Text PDF PubMed Google Scholar). as a of this in addition to in a thiol-disulfide reaction PubMed Scopus Google the redox potential for as protein disulfide = M. J. PubMed Scopus Google Scholar) to mV J. Holmgren A. PubMed Scopus Google a of a of in equilibrium have a method for the of redox potentials by direct protein-protein that a number of with used for redox have used with J. Chem. Scopus Google and proteins T. 1996; PubMed Scopus Google F. S. M. PubMed Scopus Google Biol. Google A. PubMed Scopus Google M. J. Biol. PubMed Scopus Google Scholar) in with or we that direct protein-protein be used to redox potential between coli Grx1 and Grx3, standard state redox = −198 and −233 mV were which were confirmed using a thermodynamic linkage between the stability of the protein and the stability of the disulfide bond the redox independent pair-wise of protein-protein redox we have an redox that is to the standard state of NADPH of E°′ = mV of which a of the thioredoxin superfamily of thiol-disulfide A mutant of E. coli Grx3 with replaced by from this K. F. A. and G. was for this The of the cysteine in the of this protein to form and as as the formation of a mixed disulfide with with This mutant identical activity as the and be to this as Grx3 to variable of of the (4Åslund F. Nordstrand K. Berndt K.D. Nikkola M. Bergman T. Ponstingl H. Jörnvall H. Otting G. Holmgren A. J. Biol. Chem. 1996; 271: 6736-6745Abstract Full Text Full Text PDF PubMed Scopus (59) Google which is a potential equilibrium constants using HPLC, as the reduced state of the was found to with the oxidized state of the To this we have used a of Grx3 that was found to be in the E. Holmgren A. PubMed Scopus Google Trx Holmgren A. PubMed Scopus Google and G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) were from this The DsbA mutant with the active-site sequence changed into that of was by and equilibrium reactions of two proteins in a and of 1 The reduced form of a protein was use by of protein for 1 at in the presence of by chromatography reactions between two proteins were by one of the two proteins in the reduced state and the in the oxidized state, the was to at and of were by the addition of of 1 m acid to a of by The oxidized and reduced forms of the proteins present in the were separated by reverse-phase a using a from acid in at a of at was at and The of oxidized and reduced forms of protein in the equilibrium was obtained from after of the identical were obtained from of at and and the was used in The of thiol-disulfide exchange is to be a reaction through a mixed disulfide The of the disulfide to be as it is standard The apparent concentration equilibrium for the disulfide exchange reaction between protein A and protein only the oxidized forms and reduced forms of the two proteins 1 The difference in redox potentials between the two ΔEAB°′, be obtained using the is the number of in the reaction = is constant and is the constant equilibria were by Grx1, Grx3, and at a concentration of protein in a and redox and 1 in 1 and m a a the folded forms of the proteins was in an identical The reactions were for at by acid and The concentration of in the redox was by of the = using both at the and at the of the equilibrium The of the oxidized and the reduced forms of protein were obtained from the in the the thiol-disulfide exchange reaction of the unfolded protein with glutathione the overall equilibrium be calculated the 1 m standard state redox potential of GSH, mV, the standard state redox potential of the state, EU°′, is obtained to unfolding of oxidized and reduced Grx1 and Grx3 was in 1 in the presence or of was by at as of were from an identical in as determined by PubMed Scopus Google Scholar) using a was allowed for which the was with the using and protein of and for Grx1 and Grx3, were after an The were to contain a of in of the and post-transition of the unfolding The CD in of were first to of molar CD of the amide using the is the is the molar concentration of protein. The were using to a general the as a of which the linear extrapolation method 1988; PubMed Scopus Google Scholar). and are the molar CD values of the and unfolded to and m N andm are the slopes of the pre- and post-transition region base The and ΔGH2O° are the values of the slope and of the linear extrapolation of unfolding Gibbs energy changes versusdenaturant of was to fraction of unfolded protein u with the f u = using the values of m andm obtained from The method of determining a redox potential using of as redox is in the of glutaredoxins to the presence of of the GSH-mixed disulfide which be to the glutaredoxins that have to contain a site for glutathione (4Åslund F. Nordstrand K. Berndt K.D. Nikkola M. Bergman T. Ponstingl H. Jörnvall H. Otting G. Holmgren A. J. Biol. Chem. 1996; 271: 6736-6745Abstract Full Text Full Text PDF PubMed Scopus (59) Google F. K. Holmgren A. PubMed Scopus Google Scholar). disulfides with Trx have and were used for the of the equilibrium constants and PubMed Scopus Google Scholar). found that the of the form of the glutaredoxins by the of the apparent of the equilibrium of this in the presence of the large of we for direct of determining redox in redox potential between two ΔEAB°′, be obtained by determining the of their oxidized and reduced forms at The of a method the of the thiol-disulfide exchange and the to and quantify equilibrium The two are by by reverse-phase and of the To that a state of redox equilibrium between the two proteins in was obtained in for we with protein that identical equilibrium constants were obtained of the redox state of the and or and reactions were to a after of at to that the reactions were at equilibrium at The of the oxidized and reduced proteins were obtained in separate by identical of protein. This identical molar for the reduced forms with the oxidized forms of as be at and Using this we determined a for = between Grx1 and Grx3 at to 3, Grx3 is mV Grx1, a that is at by that in the equilibrium concentration of is that of the is of The of the redox potential difference in this be by independently the pair-wise of Grx1 and Grx3 to a an of the pair-wise equilibria of Grx3 and Grx1, with This mutant was as it a redox potential for with the we = for the = for the to redox potential of = and mV, respectively. The difference between values the obtained from direct between Grx1 and this we comparisons to a of pair-wise equilibria between thiol-disulfide which resulted in an of their reducing capacities in of standard state redox potentials of the thioredoxin of thiol-disulfide from the of equilibrium constants between of redox active proteins in this are as The in redox potential between the proteins was calculated using the in redox potential between the folded state and the unfolded state, calculated from thermodynamic for redox potential determined by in the presence of thioredoxin reductase G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a , M. Sci. PubMed Scopus Google Scholar and , A. S. PubMed Scopus Google , J. Holmgren A. PubMed Scopus Google , , G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google f , of , Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google the of the equilibrium constant of thiol-disulfide between two proteins using this one standard state redox potentials for a of proteins through pair-wise to as as one of the an independently determined standard state redox The standard state redox potential of NADPH to be mV of Scholar) by and is in the The standard state redox potentials of E. coli Trx and TrxPDI, a thioredoxin system with was determined to be −270 and mV, G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of in 3, we have the between and Trx PubMed Scopus Google Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google 1 and which for an independent of the standard state redox potential of GSH. the of DsbATrx, we found that the equilibrium between and in a redox potential of mV, which with the mV obtained for in used = −240 a of −240 mV for the standard state redox potential of glutathione is with the equilibrium constants between proteins and To the Gibbs energy of disulfide bond formation in the folded state using the thermodynamic linkage one to the of the Gibbs energy of disulfide bond formation in the unfolded state and the of both the oxidized and reduced forms. One to is to that the only from the difference in of the unfolded state to disulfide bond This to be between and at K J. Biol. Chem. 1988; Full Text PDF PubMed Google J. Chem. Scopus Google Scholar). was using a that the equilibrium constant with in the presence of of to PubMed Scopus Google Scholar). This for the of the stability of the disulfide bond in the unfolded state by of a protein in redox buffers in the presence of a m the GSH-mixed disulfide forms of Grx1 and Grx3 were 1 Using = −240 mV, we found unfolded Grx1 and Grx3 to have identical redox potentials = and mV, unfolded E°′ = mV These are similar to the redox potentials for the state of DsbA, and for the of U. P. T. P. Full Text PDF PubMed Scopus Google A. PubMed Scopus Google A. S. PubMed Scopus Google Scholar). This that the of redox potentials for the unfolded state is as as found using the active-site region of of the thioredoxin superfamily F. S. M. PubMed Scopus Google Scholar). The of Grx1 and Grx3 in oxidized and reduced forms were by using and by CD at the oxidized and reduced forms of protein were to a reversible as by the of the molar after of in m into 1 The reduced form of Grx3 to be to the oxidized form the was found for Grx1, the oxidized form was the reduced form This difference to = mV Using the thermodynamic the redox potentials of the folded of Grx1 and Grx3 were determined to be E°′ = and mV, which is in with the values of E°′ = −233 and −198 mV obtained by the direct energy changes for unfolding of Grx1 and m of the denaturation, = = of of to with standard are of the denaturation, = = in a new of of to with standard are be that the stability of the oxidized form of Grx1 with the reduced form This is in to the for Grx1 by B. C. PubMed Scopus Google Scholar) the was using we have found that in buffers of as those used in their Grx1 the used the protein Grx1 confirmed in Scopus Google Scholar). addition to the we have using and and and are the of the The redox potentials in of the thioredoxin superfamily of thiol-disulfide oxidoreductases = −124 mV for E. coli DsbA U. P. T. P. Full Text PDF PubMed Scopus Google J. Chem. Scopus Google Scholar) to −270 mV for E. coli Trx T. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the thioredoxin a the of in is of The of the two residues between the active-site for T. H. J. Biol. Chem. Full Text PDF PubMed Google E. coli Trx G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google and DsbA U. P. T. P. Full Text PDF PubMed Scopus Google Scholar). the of DsbA, a linear between redox potential and the a of the of the active site G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a of the active site (CX 1 X is to the a of the and the stability of the reduced form of the protein to the oxidized in the of DsbA, the a of 1994; PubMed Scopus Google Scholar) is an for its highly The of the active-site as a in mixed disulfides of glutaredoxin U. PubMed Scopus Google and in this the of of GSH-mixed disulfide was by the a = of the active-site The method of pair-wise used for is of the equilibrium for the reversible thiol-disulfide exchange reaction between of redox active Standard state redox potentials are obtained through with or redox potential determined independently G. J. J. Pueyo C. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) via to NADPH = Using direct protein-protein we were to an E°′ of −233 for Grx1 and an E°′ of −198 mV for Using the thermodynamic linkage we obtained values for the redox potentials This an of the redox potential of the unfolded state of the EU°′, B. is that the Gibbs energy of active-site disulfide bond formation in the unfolded forms of Grx1 and Grx3, is = and for is = is the values = and J. Biol. Chem. 1988; Full Text PDF PubMed Google J. Chem. Scopus Google only an to the Gibbs energy of disulfide bond at K. for the is or is a The of = obtained for Trx Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) is in with this it is that a was found to be = mV, those Grx1, PDI, or thioredoxin reductase F. S. M. PubMed Scopus Google Scholar). To this one is to the slopes of the of The to with changes in the Sci. PubMed Scopus Google Scholar). both Grx1 and Grx3, the for the reduced form is for the oxidized an constant for the folded protein of redox state, this that the oxidized form of the protein m values obtained from oxidoreductases with the active-site sequence 1 X and mutants this with U. P. T. P. Full Text PDF PubMed Scopus Google E. PubMed Scopus Google J. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the oxidized form to be a in determining the redox potential of The method of determining redox potentials in this is and of present in a thiol-disulfide reaction in a with the redox this The between the equilibrium constant and the of the concentration to present in the of GSH. in determining the equilibrium constants for DsbA T. 1996; PubMed Scopus Google Scholar) and M. J. Biol. PubMed Scopus Google Scholar) have to be the of of the thiol-disulfide exchange reaction A. PubMed Scopus Google Scholar). This be in the presence of of in this new direct protein-protein which be at concentration of only oxidized and reduced of the thioredoxin it is that equilibrium was that proteins have an active-site A. Full Text Full Text PDF PubMed Scopus Google Full Text Full Text PDF PubMed Scopus Google Scholar). this new method to thiol-disulfide it be to equilibrium The redox potential difference between two proteins that have be obtained equilibrium constants between protein and a third protein have This the to of proteins that have A in E°′ as one from protein to from the independently determined standard state as the formation of a of redox potential and of thermodynamic errors be have found that using of the redox potential of to mV be determined be To for the of in redox of the proteins be be possible to a of the redox potential difference between two proteins the to the oxidized and reduced forms of one of the two that of the protein and is the addition of the determined redox potentials for Grx1 and Grx3 as as a DsbA active-site mutant in this an of proteins with redox potentials that as for the of the redox potentials of A of the of in redox potentials is for −240 mV for the 1 m standard state of GSH. This of −240 mV is by the equilibrium constant to as K of m to = that determined for the between Trx and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This is in to the K of using = Biol. Google which is in the One of the difference in redox potential is that from a thermodynamic of Grx1 is a disulfide reductant This to Grx3 only activity of Grx1 as a hydrogen donor for ribonucleotide reductase (3Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google F. Nordstrand K. Berndt K.D. Nikkola M. Bergman T. Ponstingl H. Jörnvall H. Otting G. Holmgren A. J. Biol. Chem. 1996; 271: 6736-6745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The redox potential difference between Grx1 and Grx3 the of Grx1 to protein disulfide However, Grx1 is a reductant of ribonucleotide reductase (3Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) Trx K m despite the redox potential of the structural in the protein-protein to the of the reaction in the postulated in thiol-disulfide exchange reactions A. Full Text Full Text PDF PubMed Scopus Google Scholar). of proteins by determining both equilibrium constants and a and this new method in

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