Human Mitochondrial Glutaredoxin Reduces S-Glutathionylated Proteins with High Affinity Accepting Electrons from Either Glutathione or Thioredoxin Reductase

Abstract

Glutaredoxins catalyze glutathione-dependent thiol disulfide oxidoreductions via a GSH-binding site and active cysteines. Recently a second human glutaredoxin (Grx2), which is targeted to either mitochondria or the nucleus, was cloned. Grx2 contains the active site sequence CSYC, which is different from the conserved CPYC motif present in the cytosolic Grx1. Here we have compared the activity of Grx2 and Grx1 using glutathionylated substrates and active site mutants. The kinetic studies showed that Grx2 catalyzes the reduction of glutathionylated substrates with a lower rate but higher affinity compared with Grx1, resulting in almost identical catalytic efficiencies (kcat/Km). Permutation of the active site motifs of Grx1 and Grx2 revealed that the CSYC sequence of Grx2 is a prerequisite for its high affinity toward glutathionylated proteins, which comes at the price of lower kcat. Furthermore Grx2 was a substrate for NADPH and thioredoxin reductase, which efficiently reduced both the active site disulfide and the GSH-glutaredoxin intermediate formed in the reduction of glutathionylated substrates. Using this novel electron donor pathway, Grx2 reduced low molecular weight disulfides such as CoA but with particular high efficiency glutathionylated substrates including GSSG. These results suggest an important role for Grx2 in protection and recovery from oxidative stress. Glutaredoxins catalyze glutathione-dependent thiol disulfide oxidoreductions via a GSH-binding site and active cysteines. Recently a second human glutaredoxin (Grx2), which is targeted to either mitochondria or the nucleus, was cloned. Grx2 contains the active site sequence CSYC, which is different from the conserved CPYC motif present in the cytosolic Grx1. Here we have compared the activity of Grx2 and Grx1 using glutathionylated substrates and active site mutants. The kinetic studies showed that Grx2 catalyzes the reduction of glutathionylated substrates with a lower rate but higher affinity compared with Grx1, resulting in almost identical catalytic efficiencies (kcat/Km). Permutation of the active site motifs of Grx1 and Grx2 revealed that the CSYC sequence of Grx2 is a prerequisite for its high affinity toward glutathionylated proteins, which comes at the price of lower kcat. Furthermore Grx2 was a substrate for NADPH and thioredoxin reductase, which efficiently reduced both the active site disulfide and the GSH-glutaredoxin intermediate formed in the reduction of glutathionylated substrates. Using this novel electron donor pathway, Grx2 reduced low molecular weight disulfides such as CoA but with particular high efficiency glutathionylated substrates including GSSG. These results suggest an important role for Grx2 in protection and recovery from oxidative stress. Thiol groups are central to most redox-sensitive processes in the cell, and their redox state controls cellular processes like growth, differentiation, and apoptosis. The intracellular thiol homeostasis is maintained by the thioredoxin and glutaredoxin systems, which both utilize reducing equivalents from NADPH to reduce protein and low molecular weight disulfides (1Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 2Arner E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2041) Google Scholar). The thioredoxin system is composed of thioredoxin reductase (TrxR), 1The abbreviations used are: TrxR, thioredoxin reductase; Trx, thioredoxin; Grx, glutaredoxin; BSA, bovine serum albumin; BSA-SG, glutathionylated bovine serum albumin; RNase, ribonuclease A; RNase-SG, glutathionylated ribonuclease A; β-ME-SG, glutathionylated β-mercaptoethanol; HED, hydroxyethyl disulfide; Cys-SG, l-cysteinyl-glutathione disulfide; CoA, reduced coenzyme A; CoA-SG, glutathionylated coenzyme A. in mammalian cells a selenocysteine-containing, dimeric nicotinamide nucleotide disulfide oxidoreductase (3Zhong L. Arner E.S. Ljung J. Aslund F. Holmgren A. J. Biol. Chem. 1998; 273: 8581-8591Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), which reduces thioredoxin (Trx) (Reaction 1), the major protein disulfide reductase with a large number of functions in mammalian cells (4Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Trx-S2+NADPH+H+→TrxRTrx-(SH)2+NADP+(Eq. 1) The glutaredoxin system consists of glutathione reductase, a dimeric enzyme with similarities to TrxR, which catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH), which in turn reduces glutaredoxin (Grx) (Reactions 2 and 3) (5Luthman M. Eriksson S. Holmgren A. Thelander L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2158-2162Crossref PubMed Scopus (96) Google Scholar). Reduced glutaredoxin acts as a disulfide reductase but is active in particular in the reduction of S-glutathionylated substrates (6Gravina S.A. Mieyal J.J. Biochemistry. 1993; 32: 3368-3376Crossref PubMed Scopus (277) Google Scholar, 7Holmgren A. Åslund F. Methods Enzymol. 1995; 252: 283-292Crossref PubMed Scopus (299) Google Scholar). GSSG+NADPH+H+→GR2GSH+NADP+(Eq. 1) Grx-S2+2GSH→Grx-(SH)2+GSSG(Eq. 1) Thioredoxins and glutaredoxins share a similar three-dimensional structure, known as the thioredoxin fold (8Martin J.L. Structure. 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar), and both use two redox-active cysteines in a conserved CXXC active site motif to reduce protein disulfides and low molecular weight disulfides. These reactions are initiated by a nucleophilic attack of the N-terminal CXXC thiolate on the disulfide substrate, resulting in a mixed disulfide intermediate, which subsequently is reduced by the C-terminal CXXC cysteine, releasing the reduced substrate (4Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Grx-catalyzed reduction of S-glutathionylated substrates, also called glutathione-mixed disulfides, proceeds by a monothiol pathway, requiring only the N-terminal active site cysteine (Reaction 4) (9Bushweller J.H. Åslund F. Wüthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (204) Google Scholar). In these reactions a glutathione-glutaredoxin intermediate is formed that is reduced by a second molecule of GSH. RS-SG+GSH→GrxRSH+GSSG(Eq. 1) Complete Trx and the Grx systems in mammalian cells are present both in the cytosol and the mitochondria (10Pedrajas J.R. Kosmidou E. Miranda-Vizuete A. Gustafsson J.A. Wright A.P. Spyrou G. J. Biol. Chem. 1999; 274: 6366-6373Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 11Damdimopoulos A.E. Miranda-Vizuete A. Pelto-Huikko M. Gustafsson J.A. Spyrou G. J. Biol. Chem. 2002; 277: 33249-33257Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 12Lundberg M. Johansson C. Chandra J. Enoksson M. Jacobsson G. Ljung J. Johansson M. Holmgren A. J. Biol. Chem. 2001; 276: 26269-26275Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 13Gladyshev V.N. Liu A. Novoselov S.V. Krysan K. Sun Q.A. Kryukov V.M. Kryukov G.V. Lou M.F. J. Biol. Chem. 2001; 276: 30374-30380Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Whereas the cytosolic TrxR and Trx display high similarity to their mitochondrial counterparts (14Rigobello M.P. Callegaro M.T. Barzon E. Benetti M. Bindoli A. Free Radic. Biol. Med. 1998; 24: 370-376Crossref PubMed Scopus (121) Google Scholar, 15Watabe S. Makino Y. Ogawa K. Hiroi T. Yamamoto Y. Takahashi S.Y. Eur. J. Biochem. 1999; 264: 74-84Crossref PubMed Scopus (69) Google Scholar, 16Miranda-Vizuete A. Damdimopoulos A.E. Pedrajas J.R. Gustafsson J.A. Spyrou G. Eur. J. Biochem. 1999; 261: 405-412Crossref PubMed Scopus (150) Google Scholar, 17Miranda-Vizuete A. Damdimopoulos A.E. Spyrou G. Antioxid. Redox Signal. 2000; 2: 801-810Crossref PubMed Scopus (122) Google Scholar), there are surprisingly large differences between the cytosolic and mitochondrial glutaredoxins M. Johansson C. Chandra J. Enoksson M. Jacobsson G. Ljung J. Johansson M. Holmgren A. J. Biol. 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Full Text PDF PubMed Google Scholar). was to a of 2 BSA, glutathione of glutaredoxin was to the and was to the The in at was using a was as of NADPH using a of was as the of of The for glutathione was with the using and Grx1, or and substrate toward glutathionylated substrates using either HED, RNase-SG, or in a of BSA, and glutathione The was by the of either or glutaredoxin to the and an of to the Grx activity was from the in at at substrate and the and by using the Grx2 as for reduction of Grx2 by TrxR was in a BSA, and The was by of either bovine or bovine The of NADPH was from the at The was compared with the reduction of Grx2 by in the as TrxR with and glutathione The and of TrxR for Grx2 in a of BSA, and to glutathione-mixed Grx2 was to the and was to the The was initiated by of both to the and the and the activity was from the in at The and by using the In Grx2 or was reduced by TrxR in a BSA, and The was by of as substrate, and activity was from the in at of Grx2 Using from of low molecular weight disulfides by of was in a of BSA, and Grx2 was to the and was to the or was to both and was from the in at of glutathione-mixed disulfides was by the using TrxR, and CoA-SG, Cys-SG, or GSSG. of by Grx1 and a lower activity compared with Grx1 M. Johansson C. Chandra J. Enoksson M. Jacobsson G. Ljung J. Johansson M. Holmgren A. J. Biol. Chem. 2001; 276: 26269-26275Abstract Full Text Full Text PDF PubMed Scopus (266) Google as in the glutaredoxin activity with as a substrate M. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In this glutaredoxin activity is as the of NADPH at in a system with and glutathione reductase; and in the to disulfide an for M. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Grx catalyzes the reduction of by which is the reactions are at a high with have the kinetic of Grx1 and Grx2 using two glutathionylated proteins, and BSA-SG, as as as substrates in the of M. Holmgren A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The reactions the for both Grx1 and Grx2 with a rate at is in with the with the the reduction of the intermediate by a second molecule of (6Gravina S.A. Mieyal J.J. Biochemistry. 1993; 32: 3368-3376Crossref PubMed Scopus (277) Google Scholar). The kinetic at to reduce the in the of The and using lower The results of these in Grx2 the reduction of these substrates with a lower rate Grx1, the of Grx2 higher resulting in higher catalytic efficiencies (kcat/Km). The efficiency was for by and Grx2 showed almost the affinity for and disulfides that and both lower for HED, an of the of These results that Grx2 is for glutathione-mixed disulfides and in particular S-glutathionylated of glutathionylated substrates by human glutaredoxins and active site in a The for by the of in the was higher for Grx2 compared with Grx1 but in the as the for which are in the A. J. Biol. Chem. 1979; Full Text PDF PubMed Google Scholar, M. M. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. Åslund F. Spyrou G. T. Holmgren A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google reduction of the mixed disulfide formed between glutathione and hydroxyethyl in a of of the of the to in the active of Grx2 and Grx1 1), we an active site of their active also the C-terminal active site of Grx2 by a to and The the active site in Grx1, the activity of Grx2 in the The activity was of higher compared with the enzyme but lower compared with Grx1. The lower affinity for both and but higher compared with the protein the of Grx1, the active site in showed almost the low activity of and catalytic as Grx2 revealed affinity and for and compared with Grx1. These that a to in the active site of Grx2 is the major for the affinity for glutathionylated substrates as as for the kcat. of the C-terminal active site cysteine in Grx2 with a the activity in the by this showed higher in the reduction of glutathionylated substrates, the in in an lower catalytic efficiency compared with the These results that similar to Grx1, only the active site thiol to the to reduce glutathionylated substrates. The for was lower for this compared with the enzyme also similar to for monothiol of Grx1 J. Biol. Chem. Full Text PDF PubMed Google Scholar, Y. S. S. Mieyal J.J. J. Biochemistry. 1998; PubMed Scopus Google Scholar). Grx2 a for differences between Grx1 and Grx2 in and catalytic to Grx2 is substrate for thioredoxin like contains a disulfide in the active site and we the of NADPH in (Reaction 1) both the cytosolic and the mitochondrial of mammalian TrxR to reduce human Grx2 human Grx1 was a substrate for either of the TrxR results (5Luthman M. Eriksson S. Holmgren A. Thelander L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2158-2162Crossref PubMed Scopus (96) Google Scholar). The reduction of Grx2 by TrxR was also to the reduction of The reduction of by Grx2 in this that Grx2 used reducing equivalents from NADPH via TrxR to reduce glutathionylated substrates. the of this and the of TrxR for Grx2 was to and the was to of the of TrxR for the substrate Trx M. Holmgren A. Biochemistry. PubMed Scopus Google of Grx2 by TrxR to the reduction of The kinetic of TrxR for human Grx2 at in a of BSA, Grx2 was to the as and the was initiated by the of both to the and was from the in the of two the catalytic of the reduction of Grx2 by TrxR we used the monothiol active site in a with either or was also a substrate for TrxR, higher activity compared with Grx2 these results that TrxR reduce both the active site disulfide in human Grx2 and the mixed disulfide formed between glutathione and Grx2 reactions and for reduction of the and the glutathione-mixed disulfide in Grx2 by of glutathione-mixed disulfides by Grx2 results in a intermediate 1), which subsequently reduced by the C-terminal active site cysteine, releasing glutathione The disulfide formed in the active site of Grx2 is by the in the active site of TrxR activity of the to the that also the intermediate by the TrxR The intermediate between Grx2 and TrxR formed in both reactions and is reduced by the cysteine in the active site of TrxR reduced TrxR is reduced by NADPH of of electron via TrxR to substrates for Grx2 in a The reduction of Grx2 by TrxR was to the reduction of the CoA-SG, and as as the disulfide the of second rate which Grx2 reduced both the and the second rate for reactions the glutathione-mixed disulfides was almost higher These results that reduced Grx2 reduce low molecular weight disulfides as the to reduce S-glutathionylated substrates via a GSH-binding site is the of a also that Grx2 use reducing equivalents from TrxR to efficiently reduce a important system in of low molecular weight disulfides by Grx2 using from TrxR and molecular weight rate in a The major differences in between cytosolic Grx1 and mitochondrial Grx2 are also in their catalytic as revealed in this mitochondrial Grx2 a high affinity for S-glutathionylated substrates, and this is to a large on the of the in the CPYC active site sequence by a was the that Grx2 is a substrate for thioredoxin reductase and acts as a to from both and as as from GSH. is a of a cellular glutaredoxin to this studies have that in E. or mammalian cells there is these results in the of the of the Grx2 in mitochondria or the results that Grx2 reduced glutathionylated substrates with lower Grx1 and E. glutaredoxins J. 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Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), an system that of and reduction of to the redox these results suggest an important role for Grx2 in protection and recovery from oxidative stress. In Grx2 a of via the mitochondrial are to Åslund for and for