Spectroscopic Studies of the Iron and Manganese Reconstituted Tyrosyl Radical in Bacillus Cereus Ribonucleotide Reductase R2 Protein

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g 1-value of 2.0090 for the tyrosyl radical was extracted. This g 1-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν7a = 1500 cm−1) was found to be insensitive to deuterium-oxide exchange. Additionally, the 18O-sensitive Fe-O-Fe symmetric stretching (483 cm−1) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g 1-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053–33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.

[1]  P. Nordlund,et al.  HF-EPR, Raman, UV/VIS Light Spectroscopic, and DFT Studies of the Ribonucleotide Reductase R2 Tyrosyl Radical from Epstein-Barr Virus , 2011, PloS one.

[2]  B. Sjöberg,et al.  NrdH-Redoxin Protein Mediates High Enzyme Activity in Manganese-reconstituted Ribonucleotide Reductase from Bacillus anthracis , 2011, The Journal of Biological Chemistry.

[3]  J. Stubbe,et al.  Bacillus subtilis class Ib ribonucleotide reductase is a dimanganese(III)-tyrosyl radical enzyme. , 2011, Biochemistry.

[4]  P. Nordlund,et al.  Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization , 2011, Nature Structural &Molecular Biology.

[5]  A. Rosenzweig,et al.  Structural Basis for Activation of Class Ib Ribonucleotide Reductase , 2010, Science.

[6]  B. Sjöberg,et al.  High‐resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states – an unusual flavodoxin , 2010, The FEBS journal.

[7]  W. Lubitz,et al.  A tyrosyl-dimanganese coupled spin system is the native metalloradical cofactor of the R2F subunit of the ribonucleotide reductase of Corynebacterium ammoniagenes. , 2010, Journal of the American Chemical Society.

[8]  Å. Røhr,et al.  Tracking flavin conformations in protein crystal structures with Raman spectroscopy and QM/MM calculations. , 2010, Angewandte Chemie.

[9]  J. Stubbe,et al.  An active dimanganese(III)-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase. , 2010, Biochemistry.

[10]  B. Sjöberg,et al.  RNRdb, a curated database of the universal enzyme family ribonucleotide reductase, reveals a high level of misannotation in sequences deposited to Genbank , 2009, BMC Genomics.

[11]  D. Svistunenko,et al.  Tyrosyl radicals in proteins: a comparison of empirical and density functional calculated EPR parameters. , 2009, Physical chemistry chemical physics : PCCP.

[12]  J. Stubbe,et al.  Redox-linked structural changes in ribonucleotide reductase. , 2009, Journal of the American Chemical Society.

[13]  Helen Piontkivska,et al.  Cross-species mapping of bidirectional promoters enables prediction of unannotated 5' UTRs and identification of species-specific transcripts , 2009, BMC Genomics.

[14]  E. Solomon,et al.  Circular dichroism and magnetic circular dichroism studies of the biferrous site of the class Ib ribonucleotide reductase from Bacillus cereus: comparison to the class Ia enzymes. , 2008, Biochemistry.

[15]  R. Kaur,et al.  Modulation of the ligand-field anisotropy in a series of ferric low-spin cytochrome c mutants derived from Pseudomonas aeruginosa cytochrome c-551 and Nitrosomonas europaea cytochrome c-552: a nuclear magnetic resonance and electron paramagnetic resonance study. , 2008, Journal of the American Chemical Society.

[16]  Joseph A Cotruvo,et al.  NrdI, a flavodoxin involved in maintenance of the diferric-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase , 2008, Proceedings of the National Academy of Sciences.

[17]  A. Gräslund,et al.  High catalytic activity achieved with a mixed manganese–iron site in protein R2 of Chlamydia ribonucleotide reductase , 2007, FEBS letters.

[18]  C. Krebs,et al.  A manganese(IV)/iron(IV) intermediate in assembly of the manganese(IV)/iron(III) cofactor of Chlamydia trachomatis ribonucleotide reductase. , 2007, Biochemistry.

[19]  G. Brudvig,et al.  Measuring distances in proteins by saturation-recovery EPR , 2007, Nature Protocols.

[20]  C. Krebs,et al.  The active form of Chlamydia trachomatis ribonucleotide reductase R2 protein contains a heterodinuclear Mn(IV)/Fe(III) cluster with S = 1 ground state. , 2007, Journal of the American Chemical Society.

[21]  C. Krebs,et al.  A Manganese(IV)/Iron(III) Cofactor in Chlamydia trachomatis Ribonucleotide Reductase , 2007, Science.

[22]  U. Uhlin,et al.  Orientation of the Tyrosyl Radical in Salmonella typhimurium Class Ib Ribonucleotide Reductase Determined by High Field EPR of R2F Single Crystals* , 2006, Journal of Biological Chemistry.

[23]  M. Vodnala,et al.  Enzymatically Active Mammalian Ribonucleotide Reductase Exists Primarily as an α6β2 Octamer* , 2006, Journal of Biological Chemistry.

[24]  Y. Yen,et al.  Ribonucleotide reductase inhibitors and future drug design. , 2006, Current cancer drug targets.

[25]  M. Vodnala,et al.  Enzymatically active mammalian ribonucleotide reductase exists primarily as an alpha6beta2 octamer. , 2006, The Journal of biological chemistry.

[26]  B. Sjöberg,et al.  Efficient growth inhibition of Bacillus anthracis by knocking out the ribonucleotide reductase tyrosyl radical. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[27]  S. Un.  The g‐values and hyperfine coupling of amino acid radicals in proteins: comparison of experimental measurements with ab initio calculations , 2005, Magnetic resonance in chemistry : MRC.

[28]  V. Gallicchio.  Ribonucleotide reductase: target therapy for human disease , 2005 .

[29]  S. Scherer,et al.  Bacillus cereus, the causative agent of an emetic type of food-borne illness. , 2004, Molecular nutrition & food research.

[30]  S. Karlsen,et al.  Crystal Structural Studies of Changes in the Native Dinuclear Iron Center of Ribonucleotide Reductase Protein R2 from Mouse* , 2004, Journal of Biological Chemistry.

[31]  P. Nordlund,et al.  The Radical Site in Chlamydial Ribonucleotide Reductase Defines a New R2 Subclass , 2004, Science.

[32]  U. Uhlin,et al.  Crystal structure of the biologically active form of class Ib ribonucleotide reductase small subunit from Mycobacterium tuberculosis , 2004, FEBS letters.

[33]  C. Cooper,et al.  A new method of identifying the site of tyrosyl radicals in proteins. , 2004, Biophysical journal.

[34]  M. Kolberg,et al.  Structure, function, and mechanism of ribonucleotide reductases. , 2004, Biochimica et biophysica acta.

[35]  A. Barra,et al.  The use of Very High Frequency EPR (VHF-EPR) in Studies of Radicals and Metal Sites in Proteins and Small Inorganic Models , 2004 .

[36]  P. Nordlund,et al.  Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-Å x-ray data , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  A. Palmer,et al.  Examples of high-frequency EPR studies in bioinorganic chemistry , 2003, JBIC Journal of Biological Inorganic Chemistry.

[38]  A. Barra,et al.  The use of high field/frequency EPR in studies of radical and metal sites in proteins and small inorganic models. , 2002, Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy.

[39]  B. Sjöberg,et al.  Crystal structure of the di-iron/radical protein of ribonucleotide reductase from Corynebacterium ammoniagenes. , 2002, Biochemistry.

[40]  U. Uhlin,et al.  Structure and function of the radical enzyme ribonucleotide reductase. , 2001, Progress in biophysics and molecular biology.

[41]  M. Fontecave,et al.  Sensitivity of tyrosyl radical g-values to changes in protein structure: a high-field EPR study of mutants of ribonucleotide reductase. , 2001, Journal of the American Chemical Society.

[42]  B. Sjöberg,et al.  The Active Form of the R2F Protein of Class Ib Ribonucleotide Reductase from Corynebacterium ammoniagenes Is a Diferric Protein* , 2000, The Journal of Biological Chemistry.

[43]  Richard L. McCreery,et al.  Raman Spectroscopy for Chemical Analysis , 2000 .

[44]  A. Rutherford,et al.  Orientation of the tyrosyl D, pheophytin anion, and semiquinone Q(A)(*)(-) radicals in photosystem II determined by high-field electron paramagnetic resonance. , 2000, Biochemistry.

[45]  A. Gräslund,et al.  Resonance Raman Evidence for a Hydrogen-Bonded Oxo Bridge in the R2 Protein of Ribonucleotide Reductase from Mouse , 1999 .

[46]  A. Barra,et al.  The tyrosyl free radical of recombinant ribonucleotide reductase from Mycobacterium tuberculosis is located in a rigid hydrophobic pocket. , 1998, Biochemistry.

[47]  M. Eriksson,et al.  Structure of Salmonella typhimurium nrdF ribonucleotide reductase in its oxidized and reduced forms. , 1998, Biochemistry.

[48]  W. Hagen,et al.  High-Frequency EPR and Pulsed Q-Band ENDOR Studies on the Origin of the Hydrogen Bond in Tyrosyl Radicals of Ribonucleotide Reductase R2 Proteins from Mouse and Herpes Simplex Virus Type 1 , 1998 .

[49]  P. Reichard,et al.  Ribonucleotide reductases. , 1998, Annual review of biochemistry.

[50]  A. Gräslund,et al.  EPR study of the mixed-valent diiron sites in mouse and herpes simplex virus ribonucleotide reductases. Effect of the tyrosyl radical on structure and reactivity of the diferric center. , 1997, Biochemistry.

[51]  F. Himo,et al.  Density functional calculations on model tyrosyl radicals. , 1997, Biophysical journal.

[52]  B. Sjöberg.  Ribonucleotide reductases — a group of enzymes with different metallosites and a similar reaction mechanism , 1997 .

[53]  A. Barra,et al.  High Field EPR Studies of Mouse Ribonucleotide Reductase Indicate Hydrogen Bonding of the Tyrosyl Radical* , 1996, The Journal of Biological Chemistry.

[54]  M. Sahlin,et al.  Electron Magnetic Resonance of the Tyrosyl Radical in Ribonucleotide Reductase from Escherichia coli , 1996 .

[55]  G. Brudvig,et al.  Effects of dipole-dipole interactions on microwave progressive power saturation of radicals in proteins. , 1996, Journal of magnetic resonance. Series B.

[56]  G. Nocentini,et al.  Ribonucleotide reductase inhibitors: new strategies for cancer chemotherapy. , 1996, Critical reviews in oncology/hematology.

[57]  J. Stubbe,et al.  Mechanism of Assembly of the Diferric Cluster−Tyrosyl Radical Cofactor of Escherichia coli Ribonucleotide Reductase from the Diferrous Form of the R2 Subunit , 1996 .

[58]  A. Barra,et al.  CHARACTERIZATION OF A NEW TYROSYL FREE RADICAL IN SALMONELLA TYPHIMURIUM RIBONUCLEOTIDE REDUCTASE WITH EPR AT 9.45 AND 245 GHZ , 1996 .

[59]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

[60]  U. Rova,et al.  Evidence by site-directed mutagenesis supports long-range electron transfer in mouse ribonucleotide reductase. , 1995, Biochemistry.

[61]  G. Brudvig,et al.  VARIATIONS OF THE DIFERRIC EXCHANGE COUPLING IN THE R2 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE FROM FOUR SPECIES AS DETERMINED BY SATURATION-RECOVERY EPR SPECTROSCOPY , 1995 .

[62]  A. Gräslund,et al.  Diiron–Oxygen Proteins , 1995 .

[63]  J. Bollinger,et al.  Use of rapid kinetics methods to study the assembly of the diferric-tyrosyl radical cofactor of E. coli ribonucleotide reductase. , 1995, Methods in enzymology.

[64]  E. Montserrat.  [The never ending story]. , 1995, Medicina clinica.

[65]  M. Atta,et al.  EPR Studies of Mixed-Valent [FeIIFeIII] Clusters formed in the R2 Subunit of Ribonucleotide Reductase from Mouse or Herpes Simplex Virus: Mild Chemical Reduction of the Diferric Centers , 1994 .

[66]  D. Singel,et al.  High-frequency (139.5 GHz) EPR spectroscopy of the tyrosyl radical in Escherichia coli ribonucleotide reductase , 1993 .

[67]  P. Reichard,et al.  From RNA to DNA, why so many ribonucleotide reductases? , 1993, Science.

[68]  B. Sjöberg,et al.  Resonance Raman spectroscopy of ribonucleotide reductase. Evidence for a deprotonated tyrosyl radical and photochemistry of the binuclear iron center. , 1989, Biochemistry.

[69]  H. Follmann,et al.  Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme. , 1988, European journal of biochemistry.

[70]  B. Sjöberg,et al.  Magnetic interaction between the tyrosyl free radical and the antiferromagnetically coupled iron center in ribonucleotide reductase. , 1987, Biochemistry.

[71]  B. Sjöberg,et al.  Ribonucleotide Reductase , 2020, Allosteric Enzymes.

[72]  B. Sjöberg,et al.  The tyrosine free radical in ribonucleotide reductase from Escherichia coli. , 1978, The Journal of biological chemistry.

[73]  Sanat K. Dhar,et al.  Metal Ions in Biological Systems , 1973, Advances in Experimental Medicine and Biology.

[74]  T. Castner.  Saturation of the Paramagnetic Resonance of a V Center , 1959 .

[75]  A. M. Portis,et al.  Electronic Structure of F Centers: Saturation of the Electron Spin Resonance , 1953 .

[76]  E. Bright Wilson,et al.  The Normal Modes and Frequencies of Vibration of the Regular Plane Hexagon Model of the Benzene Molecule , 1934 .