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High-frequency EPR and ENDOR: Time-domain spectroscopy of ribonucleotide reductase

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Abstract

This paper discusses time-domain electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) experiments aimed at elucidating the enzymatic mechanism of ribonucleotide reductase (RNR), the enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides. The article begins with a discussion of the current state of the art of instrumentation for high-frequency EPR and ENDOR and some suggestions as to future developments. We then provide an introduction to the chemistry of RNR and a discussion of the high-field EPR and ENDOR spectra of the tyrosyl radical (Y⋅) in the R2 subunit of class I RNR. Finally, we describe two examples illustrating the use of high-frequency EPR and ENDOR to elucidate the enzymatic mechanism of RNR. EPR and ENDOR have played an important role for these studies since the mechanism involves several different radical intermediates. These intermediates are all present in low concentrations relative to the Y⋅ concentration and they possess similarg-values. Spectral overlap, therefore, has been a problem with X-band EPR. At high frequencies the spectra are resolved to the point that individual powder lineshapes are discernible. In addition, we describe our approach, on the basis of differential relaxation, to suppress the spectrum of the dominant Y⋅. High-frequency EPR and ENDOR therefore has permitted us to determine the structure of several radical intermediates which in turn have contributed to the understanding of the enzymatic mechanism of RNR.

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References

  1. Lebedev Y.S. in: Modern Pulsed and Continuous-Wave Electron Spin Resonance (Kevan L., Bowman M.K., eds.), pp. 365–404. New York: InterScience 1990.

    Google Scholar 

  2. Allgeier J., Disselhorst J.A.J.M., Weber R.T., Wenckebach W.T., Schmidt J. in: Modern Pulsed and Continuous-Wave Electron Spin Resonance (Kevan L., Bowman M.K., eds.), pp. 267–283. New York: InterScience 1990.

    Google Scholar 

  3. Prisner T.F.: Adv. Mag. Opt. Res.20, 245–283 (1997)

    Article  Google Scholar 

  4. Un S., Bryant J., Griffin R.G.: J. Magn. Reson.101, 92–94 (1993)

    Article  Google Scholar 

  5. Prisner T.F., Un S., Griffin R.G.: Isr. J. Chem.32, 357–363 (1992)

    Google Scholar 

  6. Becerra L.R., Gerfen G.J., Bellew B.F., Bryant J.A., Hall D.A., Inati S.J., Weber R.T., Un S., Prisner T.F., McDermott A.E., Fishbein K.W., Kreischer K.E., Temkin R.J., Singel D.J., Griffin R.G.: J. Magn. Reson.117, 28–40 (1995)

    Article  Google Scholar 

  7. Bennati M., Farrar C.T., Bryant J.A., Inati S.J., Weis V., Gerfen G.J., Riggs-Gelasco P., Stubbe J., Griffin R.G.: J. Magn. Reson.138, 232–243 (1999)

    Article  ADS  Google Scholar 

  8. Farrar C.T., Hall D.A., Gerfen G.J., Inati S.J., Griffin R.G.: J. Chem. Phys.114, 4922 (2001)

    Article  ADS  Google Scholar 

  9. Burghaus O., Rohrer M., Götzinger T., Plato M., Möbius K.: Meas. Sci. Technol.3, 765–774 (1992)

    Article  ADS  Google Scholar 

  10. Disselhorst J.A.J.M., van der Meer H., Poluektov O.G., Schmidt J.: J. Magn. Reson.116, 183–188 (1995)

    Google Scholar 

  11. Weis V., Bennati M., Rosay M., Bryant J.A., Griffin R.G.: J. Magn. Reson.140, 293–299 (1999)

    Article  ADS  Google Scholar 

  12. Gruber K., Forrer J., Schweiger A., Gunthard H.H.: J. Phys. E Sci. Instrum.7, 569 (1973)

    Article  ADS  Google Scholar 

  13. Weis V., Bennati M., Rosay M., Griffin R.G.: J. Chem. Phys.113, 6802 (2000)

    ADS  Google Scholar 

  14. Felch K.L., Danly B.G., Jory H.R., Kreischer K.E., Lawson W., Levush B., Temkin R.J. in: Proceedings of the IEEE, pp. 752 (May 1999).

  15. Danly B.G., Blank M., Calame J.P., Levush B., Nguyen K.T., Pershing D.E., Parker R.K., Felch K.L., James B.G., Borchard P., Cahalan P., Chu T.S., Jory H.R., Hargreaves T.A., True R.B., Lawson W.G., Antonsen T.M.J.: IEEE Trans. Plasma Sci.28, 713 (2000)

    Article  ADS  Google Scholar 

  16. Blank M., Danly B.G., Levush B., Latham P.E., Pershing D.E.: Phys. Rev. Lett.79, 4485 (1997)

    Article  ADS  Google Scholar 

  17. Sirigiri J.R., Kreischer K.E., Shapiro M.A., Temkin R.J.: A Novel W-band Quasioptical Gyro-TWT, 25th International Conference on Millimeter Waves IEEE, Piscataway, NJ 2000.

  18. Stubbe J.: Proc. Natl. Acad. Sci. USA95, 2723 (1998)

    Article  ADS  Google Scholar 

  19. Stubbe J., van der Donk W.A.: Chem. Rev.98, 705–762 (1998)

    Article  Google Scholar 

  20. Reichard P.: Science260, 1773–1777 (1993)

    Article  ADS  Google Scholar 

  21. Sjöberg B.-M.: Struct. Bonding (Berlin)88, 139–173 (1997)

    Google Scholar 

  22. Stubbe J.: J. Biol. Chem.265, 5329–5332 (1990)

    Google Scholar 

  23. Uhlin U., Eklund H.: Nature370, 533 (1994)

    Article  ADS  Google Scholar 

  24. Nordlund P., Sjöberg B., Eklund H.: Nature345, 593–598 (1990)

    Article  ADS  Google Scholar 

  25. Sjöberg B.-M.: Structure2, 793 (1994)

    Article  Google Scholar 

  26. Atkin C.L., Thelander L., Reichard P., Lang G.: J. Biol. Chem.248, 7464 (1973)

    Google Scholar 

  27. Tong W.H., Chen S., Lloyd S.G., Edmondson D.E., Huynh B.H., Stubbe J.: J. Am. Chem. Soc.118, 2107 (1996)

    Article  Google Scholar 

  28. Burdi D., Sturgeon B.E., Tong W.H., Stubbe J., Hoffman B.M.: J. Am. Chem. Soc.118, 281–282 (1996)

    Article  Google Scholar 

  29. Sturgeon B.E., Burdi D., Chen S., Huynh B., Edmondson D.E., Stubbe J., Hoffman B.M.: J. Am. Chem. Soc.118, 7551–7557 (1996)

    Article  Google Scholar 

  30. Petersson L., Gräslund A., Ehrenberg A., Sjöberg B.-M., Reichard P.: J. Biol. Chem.255, 6706 (1980)

    Google Scholar 

  31. Sjöberg B.-M., Reichard P., Gräslund A., Ehrenberg A.: J. Biol. Chem.252, 536 (1977)

    Google Scholar 

  32. Sjöberg B.-M., Reichard P., Gräslund A., Ehrenberg A.: J. Biol. Chem.253, 6863 (1978)

    Google Scholar 

  33. Gerfen G.J., Bellew B.F., Un S. Jr., J.M.B. Stubbe J., Griffin R.G., Singel D.J.: J. Am. Chem. Soc.115, 6420–6421 (1993)

    Article  Google Scholar 

  34. Allard P., Barra A.L., Andersson K.K., Schmidt P.P., Atta M., Gräslund A.: J. Am. Chem. Soc.118, 895 (1996)

    Article  Google Scholar 

  35. Schmidt P.P., Andersson K.K., Barra A.L., Thelander L., Gräslund A.: J. Biol. Chem.271, 23615 (1996)

    Article  Google Scholar 

  36. Sauge-Merle S., Laulhere J.P., Coves J., Le Pape L., Menage S., Fontecave M.: J. Biol. Inorg. Chem.2, 586 (1997)

    Article  Google Scholar 

  37. van Dam P.J., Willems J., Schmidt P.P., Pötsch S., Barra A., Hagen W.R., Hoffman B.M., Andersson K.K., Gräslund A.: J. Am. Chem. Soc.120, 5080–5085 (1998)

    Article  Google Scholar 

  38. Liu A., Barra A., Rubin H., Lu G., Gräslund A.: J. Am. Chem. Soc.122, 1974 (2000)

    Article  Google Scholar 

  39. Bar G., Bennati M., Nguyen H., Je J., Stubbe J., Griffin R.: J. Am. Chem. Soc.123, 3569 (2001)

    Article  Google Scholar 

  40. Kauppi B., Nielsen B., Ramaswamy S., Kjoller Larsen I., Thelander M., Thelander L., Eklund H.: J. Mol. Biol.262, 706 (1996)

    Article  Google Scholar 

  41. Farrar C.T., Force D.A., Gerfen G.J., Britt R.D., Griffin R.G.: J. Phys. Chem. B101, 6634–6641 (1997)

    Article  Google Scholar 

  42. Himo F., Gräslund A., Eriksson L.A.: Biophys. J.72, 1556–1567 (1997)

    Article  Google Scholar 

  43. Sahlin M., Petersson L., Gräslund A., Ehrenberg A., Sjöberg B.-M., Thelander L.: Biochemistry26, 5541 (1987)

    Article  Google Scholar 

  44. Hoganson C.W., Babcock G.T.: Biochemistry31, 11874–11880 (1992)

    Article  Google Scholar 

  45. Bender C.J., Sahlin M., Babcock G.T., Barry B.A., Chandrashekar T.K., Salowe S.P., Stubbe J., Lindstrom B., Petersson L., Ehrenberg A., Sjoeberg B.M.: J. Am. Chem. Soc.111, 8076 (1989)

    Article  Google Scholar 

  46. Hoganson C.W., Sahlin M., Sjoeberg B.M., Babcock G.T.: J. Am. Chem. Soc.118, 4672 (1996)

    Article  Google Scholar 

  47. Hirsh D.J., Beck W.F., Lynch J.B., Que L., Brudvig G.W.: J. Am. Chem. Soc.114, 7475 (1992)

    Article  Google Scholar 

  48. Galli C., Atta M., Andersson K.K., Gräslund A., Brudvig G.: J. Am. Chem. Soc.117, 740 (1995)

    Article  Google Scholar 

  49. Lawrence C.C., Bennati M., Obias H.V., Bar G., Griffin R.G., Stubbe J.: Proc. Natl. Acad. Sci. USA96, 8979–8984 (1999)

    Article  ADS  Google Scholar 

  50. Stubbe J., van der Donk W.: Chem. Biol.2, 793 (1995)

    Article  Google Scholar 

  51. Thelander L., Larsson B., Hobbs J., Eckstein F.: J. Biol. Chem.251, 1398 (1976)

    Google Scholar 

  52. Coves J., de Fallois L.L.H., Pape L.L., Decout J., Fontecave M.: Biochemistry35, 8595–8602 (1996)

    Article  Google Scholar 

  53. Gerfen G.J., van der Donk W.A., Yu G., McCarthy J.R., Matthews D.P., Jarvi E.T., Farrar C., Griffin R.G., Stubbe J.: J. Am. Chem. Soc.120, 3824 (1998)

    Article  Google Scholar 

  54. van der Donk W., Yu G., Perez L., Sanchez R.J., Stubbe J.: J. Am. Chem. Soc.37, 6419 (1998)

    Google Scholar 

  55. Salowe S.P., Bollinger J.M., Ator M., Stubbe J., McCracken J., Peisach J., Samano M.C., Robins M.J.: Biochemistry32, 12749 (1993)

    Article  Google Scholar 

  56. van der Donk W., Stubbe J., Gerfen G., Bellew B., Griffin R.: J. Am. Chem. Soc.117, 8908 (1995)

    Article  Google Scholar 

  57. Sjöberg B.-M., Gräslund A., Eckstein F.: J. Biol. Chem.258, 8060 (1983)

    Google Scholar 

  58. Ator M., Salowe S.P., Stubbe J., Emptage M.H., Robins M.J.: J. Am. Chem. Soc.106, 1886 (1984)

    Article  Google Scholar 

  59. van der Donk W., Yu G., Silva D.J., Stubbe J., McCarthy J.R., Jarvi E.T., Matthews D.P., Resvick R.J., Wagner E.: Biochemistry35, 8381 (1996)

    Article  Google Scholar 

  60. Persson A.L., Eriksson M., Katterele B., Pötsch S., Sahlin M., Sjöberg B.: J. Biol. Chem.272, 31533–31541 (1997)

    Article  Google Scholar 

  61. Persson A.L., Sahlin M., Sjöberg B.: J. Biol. Chem.273, 31016–31020 (1998)

    Article  Google Scholar 

  62. Kurita Y., Gordy W.: J. Chem. Phys.34, 282 (1961)

    Article  ADS  Google Scholar 

  63. Hadley J.H., Gordy W.: Proc. Natl. Acad. Sci. USA72, 3486 (1974)

    Article  ADS  Google Scholar 

  64. Nelson D.J., Petersen R.L., Symons M.C.R.: J. Chem. Soc. Perkin Trans.2, 2005 (1977)

    Google Scholar 

  65. Rao D.N.R., Symons M.C.R., Stephenson J.M.: J. Chem. Soc. Perkin Trans.2, 727 (1983)

    Google Scholar 

  66. Engström M., Vahtras O., Agren H.: Chem. Phys. Lett.328, 483 (2000)

    Article  ADS  Google Scholar 

  67. Himo F., Siegbahn P.E.M.: J. Phys. Chem.104, 7502 (2000)

    Google Scholar 

  68. Akasaka K.: J. Chem. Phys.43, 1182 (1965)

    Article  ADS  Google Scholar 

  69. Kou W.W.H., Box H.C.: J. Chem. Phys.64, 3060 (1976)

    Article  ADS  Google Scholar 

  70. Parast C.V., Wong K.K., Kozarich J.W., Peisach J., Magliozzo R.S.: Biochemistry34, 5712 (1995)

    Article  Google Scholar 

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Authors and Affiliations

  1. Center for Magnetic Resonance, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    M. Bennati & R. G. Griffin

  2. Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    M. Bennati & R. G. Griffin

  3. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    J. Stubbe

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  1. M. Bennati
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  2. J. Stubbe
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Bennati, M., Stubbe, J. & Griffin, R.G. High-frequency EPR and ENDOR: Time-domain spectroscopy of ribonucleotide reductase. Appl. Magn. Reson. 21, 389–410 (2001). https://doi.org/10.1007/BF03162416

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