Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
Science Career Fair

Site Tools

  • AAAS
  • Subscribe
  • Feedback

Site Search

Search Advanced

Science 19 July 1991:
Vol. 253. no. 5017, pp. 292 - 298
DOI: 10.1126/science.1650033
Prev | Table of Contents | Next

Articles

Science, Vol 253, Issue 5017, 292-298
Copyright © 1991 by American Association for the Advancement of Science

articles

Mechanism of assembly of the tyrosyl radical-dinuclear iron cluster cofactor of ribonucleotide reductase

JM Bollinger Jr, DE Edmondson, BH Huynh, J Filley, Norton JR, and J Stubbe

Department of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge 02139.

Incubation of the apoB2 subunit of Escherichia coli ribonucleotide reductase with Fe2+ and O2 produces native B2, which contains the tyrosyl radical-dinuclear iron cluster cofactor required for nucleotide reduction. The chemical mechanism of this reconstitution reaction was investigated by stopped-flow absorption spectroscopy and by rapid freeze-quench EPR (electron paramagnetic resonance) spectroscopy. Two novel intermediates have been detected in the reaction. The first exhibits a broad absorption band centered at 565 nanometers. Based on known model chemistry, this intermediate is proposed to be a mu-peroxodiferric complex. The second intermediate exhibits a broad absorption band centered at 360 nanometers and a sharp, isotropic EPR signal with g = 2.00. When the reaction is carried out with 57Fe2+, this EPR signal is broadened, demonstrating that the intermediate is an iron-coupled radical. Variation of the ratio of Fe2+ to B2 in the reaction and comparison of the rates of formation and decay of the intermediates to the rate of formation of the tyrosyl radical (.Y122) suggest that both intermediates can generate .Y122. This conclusion is supported by the fact that both intermediates exhibit an increased lifetime in a mutant B2 subunit (B2-Y122F) lacking the oxidizable Y122. Based on these kinetic and spectroscopic data, a mechanism for the reaction is proposed. Unlike reactions catalyzed by heme-iron peroxidases, oxygenases, and model complexes, the reconstitution reaction appears not to involve high-valent iron intermediates.


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Orientation of the Tyrosyl Radical in Salmonella typhimurium Class Ib Ribonucleotide Reductase Determined by High Field EPR of R2F Single Crystals.
M. Galander, M. Uppsten, U. Uhlin, and F. Lendzian (2006)
J. Biol. Chem. 281, 31743-31752
   Abstract »    Full Text »    PDF »
The Involvement of Arg265 of Mouse Ribonucleotide Reductase R2 Protein in Proton Transfer and Catalysis.
A. J. Narvaez, N. Voevodskaya, L. Thelander, and A. Graslund (2006)
J. Biol. Chem. 281, 26022-26028
   Abstract »    Full Text »    PDF »
High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase.
V. P. Denysenkov, T. F. Prisner, J. Stubbe, and M. Bennati (2006)
PNAS 103, 13386-13390
   Abstract »    Full Text »    PDF »
Ribonucleotide Reductase Modularity: ATYPICAL DUPLICATION OF THE ATP-CONE DOMAIN IN PSEUDOMONAS AERUGINOSA.
E. Torrents, M. Westman, M. Sahlin, and B.-M. Sjoberg (2006)
J. Biol. Chem. 281, 25287-25296
   Abstract »    Full Text »    PDF »
Chlamydial ribonucleotide reductase: Tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster.
N. Voevodskaya, A.-J. Narvaez, V. Domkin, E. Torrents, L. Thelander, and A. Graslund (2006)
PNAS 103, 9850-9854
   Abstract »    Full Text »    PDF »
Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.
G. Xing, Y. Diao, L. M. Hoffart, E. W. Barr, K. S. Prabhu, R. J. Arner, C. C. Reddy, C. Krebs, and J. M. Bollinger Jr. (2006)
PNAS 103, 6130-6135
   Abstract »    Full Text »    PDF »
Structures of eukaryotic ribonucleotide reductase I provide insights into dNTP regulation..
H. Xu, C. Faber, T. Uchiki, J. W. Fairman, J. Racca, and C. Dealwis (2006)
PNAS 103, 4022-4027
   Abstract »    Full Text »    PDF »
A dityrosyl-diiron radical cofactor center is essential for human ribonucleotide reductases.
B. Zhou, J. Shao, L. Su, Y.-C. Yuan, C. Qi, J. Shih, B. Xi, B. Chu, and Y. Yen (2005)
Mol. Cancer Ther. 4, 1830-1836
   Abstract »    Full Text »    PDF »
Mutations in the R2 Subunit of Ribonucleotide Reductase That Confer Resistance to Hydroxyurea.
J. L. Sneeden and L. A. Loeb (2004)
J. Biol. Chem. 279, 40723-40728
   Abstract »    Full Text »    PDF »
The Radical Site in Chlamydial Ribonucleotide Reductase Defines a New R2 Subclass.
M. Hogbom, P. Stenmark, N. Voevodskaya, G. McClarty, A. Graslund, and P. Nordlund (2004)
Science 305, 245-248
   Abstract »    Full Text »    PDF »
Redox Studies of Subunit Interactivity in Aerobic Ribonucleotide Reductase from Escherichia coli.
T. Zlateva, L. Quaroni, L. Que, and M. T. Stankovich (2004)
J. Biol. Chem. 279, 18742-18747
   Abstract »    Full Text »    PDF »
Why multiple small subunits (Y2 and Y4) for yeast ribonucleotide reductase? Toward understanding the role of Y4.
J. Ge, D. L. Perlstein, H.-H. Nguyen, G. Bar, R. G. Griffin, and J. Stubbe (2001)
PNAS 98, 10067-10072
   Abstract »    Full Text »    PDF »
Proton and metal ion-dependent assembly of a model diiron protein.
A. Pasternak, J. Kaplan, J. D. Lear, and W. F. Degrado (2001)
Protein Sci. 10, 958-969
   Abstract »    Full Text »
Purification of ribonucleotide reductase subunits Y1, Y2, Y3, and Y4 from yeast: Y4 plays a key role in diiron cluster assembly.
H.-H. T. Nguyen, J. Ge, D. L. Perlstein, and J. Stubbe (1999)
PNAS 96, 12339-12344
   Abstract »    Full Text »    PDF »
High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5'-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase.
C. C. Lawrence, M. Bennati, H. V. Obias, G. Bar, R. G. Griffin, and J. Stubbe (1999)
PNAS 96, 8979-8984
   Abstract »    Full Text »    PDF »
The Iron-Oxygen Reconstitution Reaction in Protein R2-Tyr-177 Mutants of Mouse Ribonucleotide Reductase. EPR AND ELECTRON NUCLEAR DOUBLE RESONANCE STUDIES ON A NEW TRANSIENT TRYPTOPHAN RADICAL.
S. Potsch, F. Lendzian, R. Ingemarson, A. Hornberg, L. Thelander, W. Lubitz, G. Lassmann, and A. Graslund (1999)
J. Biol. Chem. 274, 17696-17704
   Abstract »    Full Text »    PDF »
Kinetic Evidence That a Radical Transfer Pathway in Protein R2 of Mouse Ribonucleotide Reductase Is Involved in Generation of the Tyrosyl Free Radical.
P. P. Schmidt, U. Rova, B. Katterle, L. Thelander, and A. Graslund (1998)
J. Biol. Chem. 273, 21463-21472
   Abstract »    Full Text »    PDF »
Antiviral Activity of a Selective Ribonucleotide Reductase Inhibitor against Acyclovir-Resistant Herpes Simplex Virus Type 1 In Vivo.
J. Duan, M. Liuzzi, W. Paris, M. Lambert, C. Lawetz, N. Moss, J. Jaramillo, J. Gauthier, R. Déziel, and M. G. Cordingley (1998)
Antimicrob. Agents Chemother. 42, 1629-1635
   Abstract »    Full Text »
Activation of the Anaerobic Ribonucleotide Reductase from Escherichia coli. THE ESSENTIAL ROLE OF THE IRON-SULFUR CENTER FOR S-ADENOSYLMETHIONINE REDUCTION.
S. Ollagnier, E. Mulliez, P. P. Schmidt, R. Eliasson, J. Gaillard, C. Deronzier, T. Bergman, A. Graslund, P. Reichard, and M. Fontecave (1997)
J. Biol. Chem. 272, 24216-24223
   Abstract »    Full Text »    PDF »
Kinetics of Transient Radicals in Escherichia coli Ribonucleotide Reductase. FORMATION OF A NEW TYROSYL RADICAL IN MUTANT PROTEIN R2.
B. Katterle, M. Sahlin, P. P. Schmidt, S. Potsch, D. T. Logan, A. Graslund, and B.-M. Sjoberg (1997)
J. Biol. Chem. 272, 10414-10421
   Abstract »    Full Text »    PDF »
Ribonucleotide reductase in the archaeon Pyrococcus furiosus: A critical enzyme in the evolution of DNA genomes?.
J. Riera, F. T. Robb, R. Weiss, and M. Fontecave (1997)
PNAS 94, 475-478
   Abstract »    Full Text »    PDF »
Transient Free Radicals in Iron/Oxygen Reconstitution of Mutant Protein R2 Y122F.
M. Sahlin, Gün. Lassmann, S. Pötsch, B.-M. Sjöberg, and A. Gräslund (1995)
J. Biol. Chem. 270, 12361-12372
   Abstract »    Full Text »    PDF »
Residues Important for Radical Stability in Ribonucleotide Reductase from Escherichia coli.
M. Ormö, K. Regnström, Z. Wang, L. Que Jr., M. Sahlin, and B.-M. Sjöberg (1995)
J. Biol. Chem. 270, 6570-6576
   Abstract »    Full Text »    PDF »
Metalloenzymes, structural motifs, and inorganic models.
K. Karlin (1993)
Science 261, 701-708
   Abstract »    PDF »
From RNA to DNA, why so many ribonucleotide reductases?.
P Reichard (1993)
Science 260, 1773-1777
   Abstract »    PDF »
Oxygen activation at the diiron center of ribonucleotide reductase.
L Que Jr (1991)
Science 253, 273-274
   PDF »
Ribonucleotide Reductase, a Possible Agent in Deoxyribonucleotide Pool Asymmetries Induced by Hypoxia.
K. Chimploy, M. L. Tassotto, and C. K. Mathews (2000)
J. Biol. Chem. 275, 39267-39271
   Abstract »    Full Text »    PDF »
Restoring Proper Radical Generation by Azide Binding to the Iron Site of the E238A Mutant R2 Protein of Ribonucleotide Reductase from Escherichia coli.
M. Assarsson, M. E. Andersson, M. Hogbom, B. O. Persson, M. Sahlin, A.-L. Barra, B.-M. Sjoberg, P. Nordlund, and A. Graslund (2001)
J. Biol. Chem. 276, 26852-26859
   Abstract »    Full Text »    PDF »


ADVERTISEMENT
Click Me!

ADVERTISEMENT
Click Me!

To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)