The [4fe-4s]1+ cluster of pyruvate formate-lyase activating enzyme generates the glycyl radical on pyruvate formate-lyase: EPR-detected single turnover [17]

Journal of the American Chemical Society

Volumn 122, Issue 34, 2000, Pages 8331-8332

  Henshaw, Timothy F ^a   Cheek, Jennifer ^a   Broderick, Joan B ^a  

^a MICHIGAN STATE UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

FORMIC ACID DERIVATIVE; GLYCINE; IRON COMPLEX; LYASE; PYRUVIC ACID DERIVATIVE; RADICAL; SULFUR DERIVATIVE;

CATALYSIS; CHEMICAL REACTION; ELECTRON SPIN RESONANCE; ENZYME ACTIVITY; ENZYME MECHANISM; LETTER; PHOTOCHEMISTRY;

EID: 0034734328     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja002012q     Document Type: Letter

References (29)

1. Knappe, J.; Elbert, S.; Frey, M.; Wagner, A. F. V. Biochem. Soc. Trans. 1993, 21, 731.
2. (b) Wong, K. K.; Kozarich, J. W. Met. Ions Biol. Syst. 1994, 30, 279.
3. (a) Ollagnier, S.; Mulliez, E.; Schmidt, P. P.; Eliasson, R,; Gaillard, J.; Deronzier, C.; Bergman, T.; Gräslund, A.; Reichard. P.; Fontecave, M. J. Biol. Chem. 1997, 272, 24216.
4. (b) Ollagnier, S.; Meier, C.; Mulliez, E.; Gaillard, J.; Schuenemann, V.; Trautwein, A.; Mattioli, T.; Lutz, M.; Fontecave, M. J. Am. Chem. Soc. 1999, 121, 6344.
5. Sanyal, I.; Cohen, G.; Flint, D. H. Biochemistry 1994, 33, 3625.
6. Duin, E. C.; Lafferty, M. E.; Crouse, B. R.; Allen, R. M.; Sanyal, I.; Flint, D. H.; Johnson, M. K. Biochemistry 1997, 36, 11811.
7. Busby, R. W.; Schelvis, J. P. M.; Yu, D. S.; Babcock, G. T.; Marletta, M. A. J. Am. Chem. Soc. 1999, 121, 4706.
8. Ollagnier-de Choudens, S.; Fontecave, M. FEBS Lett. 1999, 453, 25.
9. (a) Lieder, K.; Booker, S.; Ruzicka, F. J.; Beinert, H.; Reed, G. H.; Frey, P. A. Biochemistry 1998, 37, 2578.
10. (b) Petrovich, R. M.; Ruzicka, F. J.; Reed, G. H.; Frey, P. A. Biochemistry 1992, 31, 10774.
11. Frey, M.; Rothe, M.; Wagner, A. F. V.; Knappe, J. J. Biol. Chem. 1994, 269, 12432.
12. Moss, M. L.; Frey, P. A. J. Biol. Chem. 1990, 265, 18112.
13. Magnusson, O. T.; Reed, G. H.; Frey, P. A. J. Am. Chem. Soc. 1999, 121, 9764.
14. Broderick, J. B.; Duderstadt, R. E.; Fernandez, D. C.; Wojtuszewski, K.; Henshaw, T. F.; Johnson, M. K. J. Am. Chem. Soc. 1997, 119, 7396.
15. Külzer, R.; Pils, T.; Kappl, R.; Hüttermann, J.; Knappe, J. J. Biol. Chem 1998, 273, 4897.
16. Broderick, J. B.; Henshaw, T. F.; Cheek, J.; Wojtuszewski, K.; Trojan, M. R.; McGhan, R.; Smith, S. R.; Kopf, A.; Kibbey, M.; Broderick, W. E. Biochem. Biophys. Res. Commun 2000, 269, 451.
17. 12 U/mg for assays in the absence of added iron).
18. 13 except that I mM DTT was included in all buffers. Samples were prepared in an anaerobic chamber at 0 °C using 200 μM PFL-AE (2.65 Fe/monomer) in 50 mM Hepes, 50 mM Tris (pH 7.4). 5′-Deazariboflavin was added in the dark to a final concentration of 100 μM. The samples were illuminated by a 500 W halogen lamp for the indicated times, followed by addition of AdoMet to 2 mM. In the dark, each sample was split into two, and to one an equal volume of PFL solution (200 μM PFL, 20 mM oxamate (allosteric effector), 100 μM 5-deazariboflavin) was added. Samples were stored frozen in the dark until EPR spectra were recorded.
19. + cluster (not shown).
20. 1+ signals are 0.5 × the actual spin quantitations calculated for these spectra. This was done to correct for the difference in protein concentration in the -PFL and +PFL samples.
21. 1+ signals are 0.5 × the actual spin quantitations calculated for these spectra. This was done to correct for the difference in protein concentration in the -PFL and +PFL samples.
22. + spectra reported here have g = 2.01, 1.89, and 1.88.
23. 15 (20) The g value (2.007) and splitting (17 g) of the gly• EPR signal are similar to values previously reported: Wagner, A. F. V.; Frey, M.; Neugebauer, F. A.; Schäfer, W.; Knappe, J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 996.
24. Data not shown. Further characterization of the cluster product of turnover by Mössbauer spectroscopy and other techniques is underway, as are more extensive studies of the redox cycling of the cluster.
25. Knappe, J.; Neugebauer, F. A.; Blaschkowski, H. P.; Gänzler, M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1332.
26. Frey, P. A. Chem. Rev. 1990, 90, 1343.
27. Frey, P. A. FASEB J. 1993, 7, 662.
28. Stubbe, J.; van der Donk, W. A. Chem. Rev. 1998, 98, 705.
29. Beinert, H. J. Biol. Inorg. Chem. 2000, 5, 2.