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. 1997 Oct;6(10):2188–2195. doi: 10.1002/pro.5560061013

Evidence for two conformational states of thioredoxin reductase from Escherichia coli: use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation.

S B Mulrooney 1, C H Williams Jr 1
PMCID: PMC2143557  PMID: 9336841

Abstract

Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two-stranded beta sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox-active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66 degrees relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816), placing either redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active-site mutant. The flavin fluorescence of this mutant is only 7% that of wild-type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active-site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5-fold increase in fluorescence. Titration of the PMA-treated mutant with the nonreducing NADP(H) analogue, 3-aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild-type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA-treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.

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Selected References

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  1. Arscott L. D., Thorpe C., Williams C. H., Jr Glutathione reductase from yeast. Differential reactivity of the nascent thiols in two-electron reduced enzyme and properties of a monoalkylated derivative. Biochemistry. 1981 Mar 17;20(6):1513–1520. doi: 10.1021/bi00509a016. [DOI] [PubMed] [Google Scholar]
  2. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  3. Evans D. R., Lipscomb W. N. The modification of the catalytic chain sulfhydryl group of aspartate transcarbamylase with mercurinitrophenols. J Biol Chem. 1979 Nov 10;254(21):10679–10685. [PubMed] [Google Scholar]
  4. Ghisla S., Massey V., Lhoste J. M., Mayhew S. G. Fluorescence and optical characteristics of reduced flavines and flavoproteins. Biochemistry. 1974 Jan 29;13(3):589–597. doi: 10.1021/bi00700a029. [DOI] [PubMed] [Google Scholar]
  5. Henrich B., Plapp R. Use of the lysis gene of bacteriophage phi X174 for the construction of a positive selection vector. Gene. 1986;42(3):345–349. doi: 10.1016/0378-1119(86)90239-8. [DOI] [PubMed] [Google Scholar]
  6. Holmgren A. Thioredoxin and glutaredoxin systems. J Biol Chem. 1989 Aug 25;264(24):13963–13966. [PubMed] [Google Scholar]
  7. Hopkins N., Williams C. H., Jr Characterization of lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. Biochemistry. 1995 Sep 19;34(37):11757–11765. doi: 10.1021/bi00037a013. [DOI] [PubMed] [Google Scholar]
  8. Karplus P. A., Schulz G. E. Refined structure of glutathione reductase at 1.54 A resolution. J Mol Biol. 1987 Jun 5;195(3):701–729. doi: 10.1016/0022-2836(87)90191-4. [DOI] [PubMed] [Google Scholar]
  9. Kuriyan J., Krishna T. S., Wong L., Guenther B., Pahler A., Williams C. H., Jr, Model P. Convergent evolution of similar function in two structurally divergent enzymes. Nature. 1991 Jul 11;352(6331):172–174. doi: 10.1038/352172a0. [DOI] [PubMed] [Google Scholar]
  10. Lennon B. W., Williams C. H., Jr Effect of pyridine nucleotide on the oxidative half-reaction of Escherichia coli thioredoxin reductase. Biochemistry. 1995 Mar 21;34(11):3670–3677. doi: 10.1021/bi00011a023. [DOI] [PubMed] [Google Scholar]
  11. Lennon B. W., Williams C. H., Jr Reductive half-reaction of thioredoxin reductase from Escherichia coli. Biochemistry. 1997 Aug 5;36(31):9464–9477. doi: 10.1021/bi970307j. [DOI] [PubMed] [Google Scholar]
  12. MacKenzie R. E., Föry W., McCormick D. B. Flavinyl peptides. II. Intramolecular interactions in flavinyl aromatic amino acid peptides. Biochemistry. 1969 May;8(5):1839–1844. doi: 10.1021/bi00833a009. [DOI] [PubMed] [Google Scholar]
  13. Mattevi A., Schierbeek A. J., Hol W. G. Refined crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. A comparison with the structure of glutathione reductase. J Mol Biol. 1991 Aug 20;220(4):975–994. doi: 10.1016/0022-2836(91)90367-f. [DOI] [PubMed] [Google Scholar]
  14. Mulrooney S. B. Application of a single-plasmid vector for mutagenesis and high-level expression of thioredoxin reductase and its use to examine flavin cofactor incorporation. Protein Expr Purif. 1997 Apr;9(3):372–378. doi: 10.1006/prep.1996.0698. [DOI] [PubMed] [Google Scholar]
  15. O'Donnell M. E., Williams C. H., Jr Reaction of both active site thiols of reduced thioredoxin reductase with N-ethylmaleimide. Biochemistry. 1985 Dec 17;24(26):7617–7621. doi: 10.1021/bi00347a018. [DOI] [PubMed] [Google Scholar]
  16. Prongay A. J., Engelke D. R., Williams C. H., Jr Characterization of two active site mutations of thioredoxin reductase from Escherichia coli. J Biol Chem. 1989 Feb 15;264(5):2656–2664. [PubMed] [Google Scholar]
  17. Prongay A. J., Williams C. H., Jr Evidence for direct interaction between cysteine 138 and the flavin in thioredoxin reductase. A study using flavin analogs. J Biol Chem. 1990 Nov 5;265(31):18968–18975. [PubMed] [Google Scholar]
  18. Prongay A. J., Williams C. H., Jr Oxidation-reduction properties of Escherichia coli thioredoxin reductase altered at each active site cysteine residue. J Biol Chem. 1992 Dec 15;267(35):25181–25188. [PubMed] [Google Scholar]
  19. Russel M., Model P. The role of thioredoxin in filamentous phage assembly. Construction, isolation, and characterization of mutant thioredoxins. J Biol Chem. 1986 Nov 15;261(32):14997–15005. [PubMed] [Google Scholar]
  20. TU S. C. Spectral characterization of a fluorescent nicotinamide adenine dinucleotide analog: 3-aminopyridine adenine dinucleotide. Arch Biochem Biophys. 1981 May;208(2):487–494. doi: 10.1016/0003-9861(81)90535-x. [DOI] [PubMed] [Google Scholar]
  21. Thelander L. Thioredoxin reductase. Characterization of a homogenous preparation from Escherichia coli B. J Biol Chem. 1967 Mar 10;242(5):852–859. [PubMed] [Google Scholar]
  22. WEBER G. Fluorescence of riboflavin and flavin-adenine dinucleotide. Biochem J. 1950 Jun-Jul;47(1):114–121. doi: 10.1042/bj0470114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Waksman G., Krishna T. S., Williams C. H., Jr, Kuriyan J. Crystal structure of Escherichia coli thioredoxin reductase refined at 2 A resolution. Implications for a large conformational change during catalysis. J Mol Biol. 1994 Feb 25;236(3):800–816. [PubMed] [Google Scholar]
  24. Wang P. F., Veine D. M., Ahn S. H., Williams C. H., Jr A stable mixed disulfide between thioredoxin reductase and its substrate, thioredoxin: preparation and characterization. Biochemistry. 1996 Apr 16;35(15):4812–4819. doi: 10.1021/bi9526793. [DOI] [PubMed] [Google Scholar]
  25. Williams C. H., Jr Mechanism and structure of thioredoxin reductase from Escherichia coli. FASEB J. 1995 Oct;9(13):1267–1276. doi: 10.1096/fasebj.9.13.7557016. [DOI] [PubMed] [Google Scholar]
  26. Wu F. Y., McCormick D. B. The fluorescence quenching of aromatic amino acid and flavin portions of flavinyl peptides. Biochim Biophys Acta. 1971 Feb 16;229(2):440–443. doi: 10.1016/0005-2795(71)90204-2. [DOI] [PubMed] [Google Scholar]
  27. Yasukawa T., Kanei-Ishii C., Maekawa T., Fujimoto J., Yamamoto T., Ishii S. Increase of solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin. J Biol Chem. 1995 Oct 27;270(43):25328–25331. doi: 10.1074/jbc.270.43.25328. [DOI] [PubMed] [Google Scholar]

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