Thiyl radicals in ribonucleotide reductases

Science

Volumn 271, Issue 5248, 1996, Pages 477-481

  Licht, Stuart ^a   Gerfen, Gary J ^b   Stubbe, Joanne ^a  

^a MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

^b MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

COBAMAMIDE; CYSTEINE; DEOXYADENOSINE; NUCLEOTIDE; RIBONUCLEOTIDE REDUCTASE;

ARTICLE; CATALYSIS; ELECTRON SPIN RESONANCE; ENZYME MECHANISM; ENZYME STRUCTURE; LACTOBACILLUS; NONHUMAN; PRIORITY JOURNAL; REDUCTION;

ESCHERICHIA COLI; LACTOBACILLUS; LACTOBACILLUS LEICHMANNII;

EID: 0030028971     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5248.477     Document Type: Article

References (40)

1. J Stubbe, Annu Rev. Biochem. 58, 257 (1989).
2. A. F. V. Wagner, M. Frey, F. A Neugebauer, W. Schaefer, J. Knappe, Proc Natl. Acad. Sci. U.S A. 89, 996 (1992).
3. R Karthein, R. Dietz, W Nastainczyk, H.H. Ruf, Eur. J. Biochem. 171, 313 (1988).
4. S -T. Kim, A Sancar, C. Essenmacher, G. T. Babcock, Proc Natl. Acad Sci. U.S.A. 90, 8023 (1993).
5. B -M. Sjoberg, P. Reichard, A Gräslund, A. Ehrenberg, J Biol. Chem 252, 536 (1977).
6. L. Thelander, ibid 249, 4858 (1974).
7. J. Stubbe, Adv Enzymol Related Areas Mol. Biol. 63, 349 (1990).
8. _, J. Biol. Chem. 265, 5329 (1990)
9. P. Reichard, Science 260, 1773 (1993).
10. Y. Tamao and R L Blakley, Biochemistry 12, 24 (1973)
11. W. H. Orme-Johnson, H Beinert, R L Blakley, J Biol Chem 249, 2338 (1974).
12. P Reichard, ibid 268, 8383 (1993)
13. A Willing, H Follmann, G. Auling, Eur. J. Biochem. 175, 167 (1988).
14. S. Booker, S. Licht, J Broderick, J. Stubbe, Biochemistry 33, 12679 (1994).
15. S. Booker and J. Stubbe, Proc. Natl. Acad. Sci. U.S.A 90, 8352 (1993).
16. Abbreviations for the amino acid residues are: A, Ala; C, Cys; E, Glu; G, Gly; I, Ile; L, Leu; N, Asn; P, Pro; S, Ser; and T, Thr.
17. R. H. Abeles and W. S. Beck, J. Biol. Chem. 242, 3589 (1967).
18. H. P. C. Hogenkamp, R. K. Ghambeer, C. Brownson, R. L. Blakley, E. Vitols, ibid. 243, 799 (1968).
19. G. W. Ashley, G. Harris, J. Stubbe, ibid. 261, 3958 (1986).
20. 5 cpm/ μmol), 0.2 mM NADPH, 65 μM TR, 0 5 μM TRR These reaction mixtures were incubated in dim light at 37°C. For the detailed kinetics of the exchange reaction and its requirements, see S Booker, thesis, Massachusetts Institute of Technology (1994).
21. 525 at 37°C.
22. A Kin-Tek Instruments model RQF-3 was used.
23. 2. The samples were maintained at 100 K. For recording at temperatures below 100 K, an Oxford Instruments ESR 900 continuous-flow liquid helium cryostat was used.
24. J A Hamilton et al , Biochemistry 10, 347 (1971).
25. ∥N = 18 G, is very different from the spectrum of the intermediate.
26. J. E Wertz and J R. Bolton, Electron Spin Resonance (Chapman & Hall, New York, 1986), p. 498 (table C), pp. 249-250.
27. 2H]AdoCbl was prepared by the procedure of Hogenkamp et al. (18) and charactenzed by electrospray mass spectrometry. The isotopic enrichment was >95 percent
28. 2]cysteine into RIPR was carried out by the procedure of Hibler et al. (28). Gas chromatography-mass spectrometry analysis of the iodoacetamide-modified cysteine derivatized with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (29) revealed that 60 percent of the cysteine in RTPR was dideuterated and the remaining cysteine was diprotonated.
29. D. W. Hibler et al., Methods Enzymol. 177, 74 (1989).
30. T. P. Mawhinney, R. S. R. Robinett, A. Atalay, M. A. Madson, J. Chromatogr. 358, 231 (1986)
31. 2H]cysteine (Figs. 3B and 4B) could also indicate a thiolate ligated to cob(II)-alamin. Two experiments have shown this not to be the case, (i) If this formulation were correct, an additional EPR-active species should be detected, because an amino acid residue would have to be oxidized in order to reduce a thiyl radical to a thiolate, No such species was observed, (II) If the signal was due to a kinetically competent intermediate, then warming the sample under anaerobic conditions should result in re-formation of the carbon-cobalt bond, generating AdoCbl. However, if the observed signal is the result of a quenching artifact, then cob(II)-alamin, which is stable under anaerobic conditions, should still be present and readily detected by EPR spectroscopy. When this experiment was performed, the spectrum revealed no cob(II)alamin.
32. B. Malmström, B. Reinhammar, T. Vänngârd, Biochim Biophys Acta 205, 48 (1970). For the formula used to calculate spin concentrations, see (25), p. 463
33. 4, a dilution factor, or "packing factor," of 0.7 was used in the calculation of spin concentration for comparison with stopped-flow UV-vis spectroscopic results
34. G. J. Gerfen, S Licht, J.-P. Willems, B. M Hoffmann, J. Stubbe, in preparation.
35. H. Cheng, W. M. Westler, B. Xia, B.-H. Oh, J. L. Markley, Arch. Biochem. Biophys. 316, 619 (1995).
36. G. N. Sando, R. L. Blakley, H P C Hogenkamp, P. J Hoffmann, J. Biol. Chem. 250, 8774 (1975).
37. U Uhlin and H. Eklund, Nature 370, 533 (1994).
38. C W. Hoganson and G T Babcock, in Metal Ions in Biological Systems, H. Sigel and A. Sigel, Eds. (Dekker, New York, 1994), p. 77.
39. A-L. Tsai, R. J. Kulmacz, G Palmer, J. Biol. Chem. 250, 1053 (1995).
40. 3H]AdoCbl, J Simms and S Gardner for design and construction of the nitrogen cooling system, J. Kozarich and C Parast for providing E coli JM15, and members of the Stubbe group for helpful discussions.