Niels Højmark Andersen

• Andersen, Niels Højmark; Klæboe, Peter; Nielsen, Claus Jørgen; Shumberger, Brendan M. & Guirgis, Gamil A. (2019). The Raman and infrared spectra, ab initio calculations and spectral assignments of 1,1-dicyclopropyl-2,2-dimethylethene (c-C3H5)2C]C(CH3)2. Journal of Molecular Structure.  ISSN 0022-2860.  1195, s 528- 541 . doi: 10.1016/j.molstruc.2019.05.108
• Zhong, Xin; Andersen, Niels Højmark; Dabrowski, Marcin & Jamtveit, Bjørn (2019). Zircon and quartz inclusions in garnet used for complementary Raman thermobarometry: application to the Holsnøy eclogite, Bergen Arcs, Western Norway. Contributions to Mineralogy and Petrology.  ISSN 0010-7999.  174(6) . doi: 10.1007/s00410-019-1584-4
• Lerum, Hans Vigeland; Andersen, Niels Højmark; Eriksen, Dag Øistein; Hansen, Eddy Walther; Petersen, Dirk; Wibetoe, Grethe & Omtvedt, Jon Petter (2018). Study of Cadmium Extraction with Aliquat 336 from Highly Saline Solutions. Journal of Solution Chemistry.  ISSN 0095-9782.  47(8), s 1395- 1417 . doi: 10.1007/s10953-018-0795-z
• Platania, Elena; Streeton, Noëlle Lynn Wenger; Kutzke, Hartmut; Karlsson, Arne; Uggerud, Einar & Andersen, Niels Højmark (2018). Infrared, Raman and Computational Study of a Crystalline Mononuclear Copper Complex of relevance to the Pigment Verdigris. Vibrational Spectroscopy.  ISSN 0924-2031.  97, s 66- 74 . doi: 10.1016/j.vibspec.2018.05.004
• Zaltariov, Mirela F.; Hammerstad, Marta; Arabshahi, Homayon John; Jovanovic, Katarina; Richter, Klaus W.; Cazacu, Maria; Shova, Sergiu; Balan, Mihaela; Andersen, Niels Højmark; Radulovic, Sinisa; Reynisson, Jóhannes; Andersson, Karl Kristoffer & Arion, Vladimir B (2017). New Iminodiacetate–Thiosemicarbazone Hybrids and Their Copper(II) Complexes Are Potential Ribonucleotide Reductase R2 Inhibitors with High Antiproliferative Activity. Inorganic Chemistry.  ISSN 0020-1669.  56, s 3532- 3549 . doi: 10.1021/acs.inorgchem.6b03178 Full text in Research Archive.
• Samarasingha, Pushpaka; Andersen, Niels Højmark; Sørby, Magnus Helgerud; Kumar, Susmit; Nilsen, Ola & Fjellvåg, Helmer (2016). Neutron diffraction and Raman analysis of LiMn1.5Ni0.5O4 spinel type oxides for use as lithium ion battery cathode and their capacity enhancements. Solid State Ionics.  ISSN 0167-2738.  284, s 28- 36 . doi: 10.1016/j.ssi.2015.11.018
• Wallington, Timothy J.; Pivesso, Bruno Pasquini; Lira, Alane Moura; Anderson, James E.; Nielsen, Claus Jørgen; Andersen, Niels Højmark & Hodnebrog, Øivind (2016). CH3Cl, CH2Cl2, CHCl3, and CCl4: Infrared spectra, radiative efficiencies, and global warming potentials. Journal of Quantitative Spectroscopy and Radiative Transfer.  ISSN 0022-4073.  174, s 56- 64 . doi: 10.1016/j.jqsrt.2016.01.029 Show summary

  Infrared spectra for the title compounds were measured experimentally in 700 Torr of air at 295 K and systematically modeled in B3LYP, M06-2X and MP2 calculations employing various basis sets. Calibrated infrared spectra over the wavenumber range 600–3500 cm−1 are reported and combined with literature data to provide spectra for use in experimental studies and radiative transfer calculations. Integrated absorption cross sections are (units of cm−1 molecule−1): CH3Cl, 660–780 cm−1, (3.89±0.19)×10−18; CH2Cl2, 650-800 cm−1, (2.16±0.11)×10−17; CHCl3, 720–810 cm−1, (4.08±0.20)×10−17; and CCl4, 730–825 cm−1, (6.30±0.31)×10−17. CH3Cl, CH2Cl2, CHCl3, and CCl4 have radiative efficiencies of 0.004, 0.028, 0.070, and 0.174 W m−2 ppb−1 and global warming potentials (100 year horizon) of 5, 8, 15, and 1775, respectively. Quantum chemistry calculations generally predict larger band intensities than the experimental values. The best agreement with experiments is obtained in MP2(Full) calculations employing basis sets of at least triple-zeta quality augmented by diffuse functions. The B3LYP functional is found ill-suited for calculating vibrational frequencies and infrared intensities of halocarbons.

• Bailey, Louise R.; Proudfoot, Gary; Mackenzie, Brodie; Andersen, Niels Højmark; Karlsson, Arne & Ulyashin, Alexander (2015). High rate amorphous and crystalline silicon formation by pulsed DC magnetron sputtering deposition for photovoltaics. Physica Status Solidi (a) applications and materials science.  ISSN 1862-6300.  212(1), s 42- 46 . doi: 10.1002/pssa.201431768 Show summary

  Two methods of pulsed DC magnetron sputtering deposition have been used to form high rate, hydrogen-free crystalline silicon layers. The first method is in situ crystalline silicon deposition. The second method is high rate amorphous silicon deposition followed by an anneal to induce crystallization. Over 20 μm thick crystalline silicon can be formed on wafers up to 200 mm round or 156 mm square. Two vacuum deposition systems were used for substrate cleaning and deposition. The crystallinity of silicon layers was analyzed by ellipsometry and Raman spectroscopy. Almost fully crystalline silicon is deposited in situ at table temperatures greater and equal to 650 °C. In situ crystalline silicon has been deposited at 40 nm/min and amorphous silicon can be deposited at over 400 nm/min subject to power density limitations for the silicon target. Up to 20 μm thick amorphous silicon deposited at room temperature is fully crystallized by annealing in vacuum on a 1000 °C table for 2 h. This work demonstrates that sputtering offers significant potential for depositing the absorber layer in silicon based photovoltaics.

• Nielsen, Claus Jørgen; Klæboe, Peter; Andersen, Niels Højmark; Guirgis, Gamil A.; Hickman, Daniel V. & Morris, Tamia B. (2015). Infrared and Raman spectra, DFT-calculations and spectral assignments of 1,1,3,3,5,5-hexafluoro-1,3,5-trisilacyclohexane. Journal of Molecular Structure.  ISSN 0022-2860.  1099, s 399- 406 . doi: 10.1016/j.molstruc.2015.05.065
• Sjåstad, Anja Olafsen; Andersen, Niels Højmark; Vajeeston, Ponniah; Karthikeyan, Jayakumar; Arstad, Bjørnar; Karlsson, Arne & Fjellvåg, Helmer (2015). On the Thermal Stability and Structures of Layered Double Hydroxides Mg1-xAlx(OH)2(NO3)x·mH2O (0.18 ≤ x ≤ 0.38). European Journal of Inorganic Chemistry.  ISSN 1434-1948.  2015(10), s 1775- 1788 . doi: 10.1002/ejic.201403232 Show summary

  Nitrate-based layered double hydroxides, Mg1–xAlx(OH)2(NO3)x·mH2O, have been investigated to settle issues relating to stability towards humidity, dehydration and staging, nitrate group orientation, Al migration and crystal structure. Raman and IR spectroscopy together with modelling methods proved that flat-lying water and nitrate become tilted towards [001] at higher nitrate concentrations, accompanied by expansion along the c-axis. The distribution of the molecular tilts is greatest for x = 0.25, which explains the extraordinary broad diffraction peaks. The unit cell volume was modified for all samples when subjected to moist air, most strongly for x = 0.25. On heating, in situ synchrotron diffraction data showed continuous changes along the c-axis during dehydration of samples with x = 0.20 and 0.33. For x = 0.25, an intermediate phase appeared. Dehydroxylation was initiated at the OH groups bonded to the cations MgAl2, Mg2Al and Mg3 (in said order), whereas complete nitrate loss occurred at higher temperatures for higher Al contents. Some aluminium simultaneously moved to tetrahedral sites, which, according to 1H–27Al CP NMR spectroscopy, lack protons in their near neighbourhood. In contrast, octahedral aluminium remains surrounded by protons during decomposition. For the metastable oxide product, neutron diffraction indicated that 2/3 of aluminium reside in tetrahedral interstices. This oxide is defective, probably mimicking wüstite rather than a regular rock salt structure.

• Andersen, Niels Højmark; Klæboe, Peter; Nielsen, Claus Jørgen; Guirgis, Gamil A.; Overby, Jason S. & Askarian, Sahand M. (2014). Infrared and Raman spectra, DFT calculations and spectral assignments of 1,3,5-trisilacyclohexane. Journal of Molecular Structure.  ISSN 0022-2860.  1076, s 419- 425 . doi: 10.1016/j.molstruc.2014.07.077
• Hammerstad, Marta; Røhr, Åsmund Kjendseth; Andersen, Niels Højmark; Gräslund, Astrid; Högbom, Martin & Andersson, K. Kristoffer (2014). The Class Ib Ribonucleotide Reductase from Mycobacterium tuberculosis has Two Active R2F Subunits. Journal of Biological Inorganic Chemistry.  ISSN 0949-8257.  19(6), s 893- 902 . doi: 10.1007/s00775-014-1121-x
• Nagayoshi, Hiroshi; Diplas, Spyridon; Walmsley, John; Andersen, Niels Højmark; Karlsson, Arne; Graff, Joachim Seland; Chirvony, Vladimir; Pastor, Juan M. & Ulyashin, Alexander (2014). One-step formation of nanostructures on silicon surfaces using pure hydrogen-radical-initiated reactions. Physica Status Solidi (a) applications and materials science.  ISSN 1862-6300.  211(1), s 231- 238 . doi: 10.1002/pssa.201330220 Show summary

  One-step formation of silicon nanowires, sheets, and texture surface on a silicon substrate has been achieved using hydrogen-radical etching reactions. Metallic tungsten and for comparison purposes a tungsten hot wire, were used as catalysts for the hydrogen-molecular cracking. It was shown that a variety of surface structures on silicon such as inverted pyramid texture, V-groove texture, dense silicon nanowire growth over texture, and nanosheet structure can be obtained by controlling the process conditions. The obtained results suggested that the formation of nanotungsten silicide particle is an essential prerequisite to obtain these structures. The particles work as an etching mask against hydrogen-radical etching, as well as a catalyst for vapor–solid–solid (VSS) growth. SEM, TEM, micro-RAMAN, and XPS were used for the analysis of the hydrogen-radical-treated Si samples. The Si nanowires growth model, as well as the texturing mechanism initiated by hydrogen-radical treatment of Si surface in the presence of tungsten nanoparticle is discussed. It is concluded that the proposed acid-free method, which is based on a modification of Si surfaces only by hydrogen radicals, can be considered as a “green” technology approach, which can be used for the costeffective fabrication of silicon nanostructures, which can be considered as a base for several types of advanced devices in the future.

• Arachchige, Gumunu L. Samarakoon; Andersen, Niels Højmark; Perinu, Cristina & Jens, Klaus-Joachim (2013). Equilibria of MEA, DEA and AMP with Bicarbonate and Carbamate: a Raman Study. Energy Procedia.  ISSN 1876-6102.  37, s 2002- 2010 . doi: 10.1016/j.egypro.2013.06.080
• Can, Mehmet; Krucinska, Jolanta; Zoppellaro, Giorgio; Andersen, Niels Højmark; Wedekind, Joseph E.; Hersleth, Hans-Petter; Andersson, K. Kristoffer & Bren, Kara L (2013). Structural Characterization of Nitrosomonas europaea Cytochrome c-552 Variants with Marked Differences in Electronic Structure. ChemBioChem.  ISSN 1439-4227.  14(14), s 1828- 1838 . doi: 10.1002/cbic.201300118 Show summary

  Hemes carry out diverse functions in biology as a result of the influence of the polypeptide surrounding the heme on heme reactivity. The crucial link between heme protein structure and reactivity is the electronic structure of the heme, which is modulated by the polypeptide surrounding the heme. Here, Nitrosomonas europaea cytochrome c-552 (Ne c-552) variants with the same His/Met axial ligand set but different EPR spectra are characterized structurally to aid the understanding of how molecular structure determines heme electronic structure. Light absorption and resonance Raman spectroscopy on the protein crystals is performed along with structure determination. The structures solved are of Ne c-552, which displays a “HALS,” or highly anisotropic axial low-spin EPR spectrum, and the deletion mutant Ne N64, which has has a rhombic EPR spectrum. Two X-ray crystal structures of wild-type Ne c-552 are reported; one is of the protein isolated from N. europaea cells (Ne c-552n, 2.35-Å resolution) and the other is of recombinant protein expressed in E. coli (Ne c-552r, 1.63-Å resolution). Ne N64 crystallized in two different space groups, and two structures are reported (monoclinic, 2.1-Å resolution; hexagonal, 2.3-Å resolution). Comparison of the structures of the wild-type and mutant proteins reveals that heme ruffling is increased in the mutant; increased ruffling is predicted to yield a more rhombic EPR spectrum. The 2.35-Å Ne c-552n structure displays 18 molecules in the asymmetric unit; analysis of heme axial Met conformations is consistent with the population of multiple axial Met configurations, although the “S” configuration, pointing toward pyrrole A, appears to be preferred, and is the conformation observed in the higher-resolution Ne c-552r structure. In Ne N6 is in the “R” configuration, pointing toward pyrrole B. Finally, the mutation is shown to yield a more hydrophobic heme pocket and expel waters from near the axial Met. These structures reveal that residue 64 plays multiple roles in regulating the axial ligand orientation and the interaction of water with the heme. These results support the hypothesis that more highly ruffled hemes are associated with rhombic EPR signals in cytochromes c with His/Met axial ligation.

• Tomter, Ane Berg; Zoppellaro, Giorgio; Andersen, Niels Højmark; Hersleth, Hans-Petter; Hammerstad, Marta; Røhr, Åsmund Kjendseth; Sandvik, Guro Katrine; Strand, Kari Røren; Nilsson, Göran Erik; Bell, Caleb B.; Barra, Anne-Laure; Blasco, Emmanuelle; Le Pape, Laurent; Solomon, Edward I. & Andersson, K. Kristoffer (2013). Ribonucleotide reductase class I with different radical generating clusters. Coordination chemistry reviews.  ISSN 0010-8545.  257(1), s 3- 26 . doi: 10.1016/j.ccr.2012.05.021 Show summary

  Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to their corresponding deoxyribonucleotides. They are formed through a radical-induced reduction of ribonucleotides. Three classes of RNR generate the catalytically active site thiyl radical using different co-factors: a tyrosyl-radical in most cases (class I), homolytic cleavage of deoxyadenosyl-cobalamin (class II), or a glycyl-radical (class III), respectively. Class I RNR has a larger subunit R1/R1E containing the active site and a smaller subunit R2/R2F with (the thiyl-generating power from) a tyrosyl radical or an oxidized iron-manganese cluster and is reviewed herein. Class I is divided into subclasses, Ia (tyrosyl-radical and di-iron-oxygen cluster), Ib (tyrosyl-radical and di-manganese-oxygen cluster) and Ic (an iron-manganese cluster). Presented here is an overview of recent developments in the understanding of class I RNR: metal-ion cluster identities, novel 3D structures, magnetic-optical properties, and reaction mechanisms. It became clear in the last years that the primitive bacterial RNR sources can utilize different metal-ion clusters to fulfil function. Within class Ia that includes members from eukaryotes (mammalians, fish) and some viruses species, the presence of hydrogen bonding interactions from water at different distances with the tyrosyl-radical site can occur. This demonstrates a large versatility in the mechanism to form the thiyl radical.

• Andersen, Christian Brix Folsted; Torvund-Jensen, Morten; Nielsen, Marianne Jensby; Pinto de Oliveira, Cristiano Luis; Hersleth, Hans-Petter; Andersen, Niels Højmark; Pedersen, Jan Skov; Andersen, Gregers Rom & Moestrup, Søren Kragh (2012). Structure of the haptoglobin-haemoglobin complex. Nature.  ISSN 0028-0836.  489, s 456- 459 . doi: 10.1038/nature11369 Show summary

  Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies1, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin–haemoglobin complex, which represents a virtually irreversible non-covalent protein–protein interaction2. Here we present the crystal structure of the dimeric porcine haptoglobin–haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected β-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and β-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αβ dimer is highly overlapping with the interface between the two αβ dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin–haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin–haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin–haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin–haemoglobin for CD163 (ref. 4).

• Tomter, Ane Berg; Zoppellaro, Giorgio; Bell, Caleb B.; Barra, Anne-Laure; Andersen, Niels Højmark; Solomon, Edward I. & Andersson, K. Kristoffer (2012). Spectroscopic Studies of the Iron- and Manganese Reconstituted Tyrosyl Radical in Bacillus cereus Ribonucleotide Reductase R2 Protein. PLOS ONE.  ISSN 1932-6203. . doi: 10.1371/journal.pone.0033436 Full text in Research Archive. Show summary

  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 ribonucleotide reduction active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. WIn this work, we studied the R2F subunit of B. cereus reconstituted with both iron and or alternatively with manganese ions, then subsequently reacted with molecular . oxygen to generate tyrosyl-radicals. The catalytically active tyrosyl radical forms were probed by EPR, and resonance Raman spectroscopy. The X-band spectrum of the RNR R2F tyrosyl radical reconstituted with iron was slightly different from that reconstituted with manganese.T However, the two similar X-band EPR spectra of both did not change significantly over the temperature range examined (4-50 K). From the 285 GHz EPR spectrum of the iron form, a g1-value of 2.0090 for the tyrosyl radical was extracted. This g1-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, 7a = 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 bond cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g1-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 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) results indicate that both the manganese and iron reconstituted RNR R2F could beare functional and the manganese form might be very importantmost significant, as it has 8 times higher activity.

• Andersen, Niels Højmark; Zoppellaro, Giorgio; Bubacco, L; Casella, L & Andersson, K. Kristoffer (2011). Raman, UV/vis and CD Spectroscopic Studies of Dodecameric Oxy-Hemocyanin from Carcinus aestuarii. Chemistry Letters.  ISSN 0366-7022.  40(12), s 1360- 1362 . doi: 10.1246/cl.2011.1360 Show summary

  The stable dodecameric oxy-form of Carcinus aestuarii hemocyanin (Cae oxy-Hc) has been probed by resonance Raman, UV/Vis and CD spectroscopy. A clear signal emerged at 746 cm–1 (Raman) sensitive to isotopic exchange with 18O2 (−40 cm–1), thus confirming the presence of a μ-η2:η2 peroxide coordinated to the cupric centres. The optical fingerprints of Cae oxy-Hc show that symmetry breaking exists at the Cu(II)-O22–Cu(II) core and exciton coupling effects are present in the visible region of the spectra.

• Andersen, Niels Højmark; Zoppellaro, Giorgio; Bubacco, L; Casella, L & Andersson, K. Kristoffer (2011). Symmetry Breaking at the Cu2O2 Core in Oxy-Hemocyanin from Carcinus Aestuarii - a combined DFT, CD, UV/vis and Raman study. Journal of Biological Inorganic Chemistry.  ISSN 0949-8257.  16, s 493- 493 . doi: 10.1007/s00775-011-0862-z Show summary

  Symmetry Breaking at the Cu2O2 Core in Oxy-Hemocyanin from Carcinus Aestuarii - a combined DFT, CD, UV/vis and Raman study N. H. Andersen1; G. Zoppellaro2; L. Bubacco3; L. Casella4; K. Andersson2 1. Department of Chemistry, University of Oslo, Oslo, Norway. 2. Department of Molecular Biosciences, University of Oslo, Oslo, Norway. 3. Department of Biology, University of Padova, Padova, Italy. 4. Department of Gereral Chemistry, University of Pavia, Pavia, Italy. Carcinus Aestuarii (Cae) Hemocyanin (Hc) is present as dodecamers consisting of about 75kDa subunits containing 2 Copper atoms each.1,2,3,4 These, labeled CuA and CuB, are both ligated by three histidines and are slightly different regarding structure and surroundings. The holohedrized symmetry of the His6Cu2O2 site, based on the only published crystal structure of oxy-Hc from the arthropod Limulus Polyphemus, is close to C2h. The EPR-silent blue dicopper peroxide site is antiferromagnetically coupled. This causes DFT modelling of this kind of metalcentres more intriguing.5 However, by optimizing a C2h symmetric model compound, we vere able to simulate a peroxide streching frequency that is close to the observed resonance Raman spectrum that shows a frequency of 746 cm–1, that downshifts to 706 cm–1 by oxygen-18 exchange. This also compares well with oxy-Hc from L. Polyphemus that has a μ-η2:η2 Cu2O2 core, but is significantly lower than that of C. Magister.6 We also examined the effect of various kinds of symmetry breaking on the CD spectrum. [1] P. Dolashka-Angelova, R. Hristova, S. Stoeva and W. Voelter Spectrochim. Acta A 1999, 55, 2927-2934. [2] E.Pidcock, H.V. Obias, M. Abe, H.-C. Liang, K.D. Karlin and E.I. Solomon J. Am. Chem. Soc. 1999, 121, 1299-1308. [3] S. Hirota, T. Kawahara, M. Beltramini, P.D. Muro, R.S. Magliozzo, J. Peisach, L.S. Powers, N. Tanaka, S. Nagao and L. Bubacco J. Biol. Chem 2010, accepted, Doi 10.1074/jbc.M803433200. [4] S. Hirota, N. Tanaka, I. Mičetić, P.D. Muro, S. Nagao, H. Kitagishi, K. Kano, R.S. Magliozzo, J. Peisach, M. Beltramini and L. Bubacco J. Biol. Chem 2010, accepted, Doi 10.1074/jbc.M109.076067. [5] J.-P. Piquemal and J. Pilme, J. Mol. Struct.: THEOCHEM 2006, 764, 77–86. [6] J. Ling. L.P. Nestor, R.S. Czernuszwicz, T.G. Spiro, R. Fraczkiewicz, K.D. Sharma, T.M. Loehr and J. Sanders-Loehr J. Am. Chem. Soc. 1994, 116, 7682-7691.

• Tomter, Ane Berg; Zoppellaro, Giorgio; Schmitzberger, Florian; Andersen, Niels Højmark; Barra, Anne-Laure; Engman, Henrik; Nordlund, Pär & Andersson, K. Kristoffer (2011). HF-EPR, Raman, UV/VIS Light Spectroscopic, and DFT Studies of the Ribonucleotide Reductase R2 Tyrosyl Radical from Epstein-BarrVirus. PLOS ONE.  ISSN 1932-6203.  6(9) . doi: 10.1371/journal.pone.0025022 Show summary

  Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase small subunit (RNR R2) is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. RNR R2 generates a stable tyrosyl radical required for enzymatic turnover. Here, the electronic and magnetic properties of the tyrosyl radical in EBV R2 have been determined by X-band and high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy recorded at cryogenic temperatures. The radical exhibits an unusually low g1-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results a relatively high C-O stretching frequency associated with the phenoxyl radical (at 1508 cm-1) is observed with resonance Raman spectroscopy. In contrast to mouse R2, EBV R2 does not show a deuterium shift in the resonance Raman spectra. Thus, the presence of a water molecule as a hydrogen bond donor moiety could not be identified unequivocally. Theoretical simulations showed that a water molecule placed at a distance of 2.6 Å from the tyrosyl-oxygen does not result in a detectable deuterium shift in the calculated Raman spectra. UV/VIS light spectroscopic studies with metal chelators and tyrosyl radical scavengers are consistent with a more accessible dimetal binding/radical site and a lower affinity for Fe2+ in EBV R2 than in Escherichia coli R2. Comparison with previous studies of RNR R2s from mouse, bacteria, and herpes viruses, demonstrates that finely tuned electronic properties of the radical exist within the same RNR R2 Ia class.

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• Andersen, Niels Højmark; Hammerstad, Marta; Zaltariov, Mirela F.; Rapta, Peter; Arion, Vladimir B. & Hersleth, Hans-Petter (2019). Interaction of Thiosemicarbazones with the Ribonucleotide Reductase R2 subunit Studied by Resonance Raman Spectroscopy.
• Kendall-Price, Sophie; Evans, Rhiannon M; Rowbotham, Jack S; Reeve, Holly A; Carr, Stephen B; Frogley, Mark D; Cinque, Gianfelice; Andersen, Niels Højmark; Hersleth, Hans-Petter; Vincent, Kylie Alison & Ash, Philip A (2019). Generating Single Metalloprotein Crystals in Well-Defined Redox States – Electrochemical Microspectroscopy As a Tool for Mechanistic Studies. Meeting Abstracts - The Electrochemical Society [ECS].  ISSN 1091-8213.  9, s 425- 425
• Andersen, Niels Højmark; Hammerstad, Marta; Zaltariov, Mirela F.; Arion, Vladimir B. & Hersleth, Hans-Petter (2018). Interaction of Thiosemicarbazones with Ribonucleotide Reductase studied by Resonance Raman Spectroscopy.
• Lofstad, Marie; Gudim, Ingvild; Kjendseth, Åsmund Røhr; Andersen, Niels Højmark; van Beek, Wouter; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2017). Combining Protein X-ray crystallography and in situ single-crystal UV-Vis and Raman spectroscopy to grasph the true structure of haem- and flavoproteins.
• Zaltariov, Mirela-Fernanda; Hammerstad, Marta; Arabshahi, Homayon John; Jovanović, Katarina; Richter, Klaus W.; Cazacu, Maria; Shova, Sergiu; Balan, Mihaela; Radulović, Siniša; Andersen, Niels Højmark; Reynisson, Jóhannes; Andersson, K. Kristoffer & Arion, Vladimir B (2017). New iminodiacetate-thiosemicarbazone hybrids and their copper(II) complexes are potential mR2 RNR inhibitors with high antiproliferative activity. NBS-nytt.  ISSN 0801-3535.  s 104- 104
• Hammerstad, Marta; Kjendseth, Åsmund Røhr; Andersen, Niels Højmark; Graslund, Astrid; Högbom, Martin & Andersson, K. Kristoffer (2016). The Class Ib Ribonucleotide Reductase from Mycobacterium Tuberculosis has Two Active R2f Subunits.
• Zoppellaro, Giorgio; Andersson, K. Kristoffer; Andersen, Niels Højmark & Zboril, Radek (2016). The non-innocent nature of graphene oxide as organic platform for biomedical applications and its reactivity towards metal-based anticancer drugs.
• Andersen, Niels Højmark; Karlsson, Arne; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2014). In Crystallo Resonance Raman Spectroscopic Disclosure of the Ferryl Stretching Mode in Myoglobin. Journal of Biological Inorganic Chemistry.  ISSN 0949-8257.  19, s S339- S339
• Brogaard, Rasmus Yding; Ganjkhanlou, Yadolah; Andersen, Niels Højmark; Bleken, Bjørn Tore Lønstad; Giordanino, Filippo; Svelle, Stian; Berlier, G; Olsbye, Unni & Bordiga, Silvia (2014). Transition metal-doped zeolite catalysts for ethene oligomerization: characterization by in situ spectroscopy and DFT calculations.
• Hammerstad, Marta; Røhr, Åsmund Kjendseth; Andersen, Niels Højmark; Graslund, Astrid; Högbom, Martin & Andersson, K. Kristoffer (2014). The Class Ib Ribonucleotide Reductase from Mycobacterium tuberculosis has two Active R2F Subunits. NBS-nytt.  ISSN 0801-3535.  s 85- 85
• Andersen, Niels Højmark; Hersleth, Hans-Petter; Karlsson, Arne & Andersson, K. Kristoffer (2013). In Crystallo Resonance Raman Spectroscopic Disclosure of the Ferryl Stretching Mode in Myoglobin (poster presentation absent due to personal resons).
• Can, Mehmet; Krucinska, Juliusz; Zoppellaro, Giorgio; Andersen, Niels Højmark; Wedekind, Joseph E.; Andersson, K. Kristoffer & Bren, Kara L (2013). Angewandte Spotlights of paper: Structural Characterization of Nitrosomonas europaea Cytochrome c-552 Variants with Marked Differences in Electronic Structure, in ChemBioChem. Angewandte Chemie International Edition.  ISSN 1433-7851.  52, s 10684- 10686
• Hammerstad, Marta; Tomter, Ane Berg; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Zoppellaro, Giorgio; Andersen, Niels Højmark; Sandvik, Guro Katrine; Nilsson, Göran Erik; Barra, Anne-Laure; Högbom, Martin; Graslund, Astrid & Andersson, K. Kristoffer (2013). Studies of the tyrosyl radicals and metal clusters in R2 of class Ia and Ib ribonucleotide reductase.
• Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Lofstad, Marie; Hammerstad, Marta; Skråmo, Silje; Andersen, Niels Højmark; Van Beek, Wouter; Can, Mehmet; Zhao, Xiangbo; Pompidor, Guillaume; Magliozzo, Richard S.; Bren, Kara L & Andersson, K. Kristoffer (2013). Using in situ single-crystal UV-vis and Raman spectroscopy to study the effect of X-ray radiation damage on the crystal structures of haem and flavoproteins.
• Tomter, Ane Berg; Zoppellaro, Giorgio; Hammerstad, Marta; Barra, Anne-Laure; Sandvik, Guro Katrine; Andersen, Niels Højmark; Røhr, Åsmund Kjendseth; Bergan, Jonas; Ellefsen, Stian; Nilsson, Göran Erik; Bell, Caleb B.; Schmitzberger, Florian; Nordlund, Pär; Solomon, Edward I. & Andersson, K. Kristoffer (2013). Spectroscopic and DFT studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase. Show summary

  Spectroscopic and dft studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase Ane B. Tomter1; Giorgio Zoppellaro1; Marta Hammerstad1; Niels H. Andersen1;Anne-Laure Barra2; Guro K. Sandvik1; Åsmund K. Roehr1; Jonas Bergan1; Stian Ellefsen1; Göran E. Nilsson1;Caleb B. Bell III 3; Florian Schmitzberger4; Pär Nordlund4; Edward I. Solomon4; and K. Kristoffer Andersson1* 1 Department of Molecular Biosciences, University of Oslo, NO-0316 Oslo (Norway), 2 Grenoble High Magnetic Field Laboratory, CNRS, Grenoble ,France 3Departmen of Chemistry, Stanford University, Stanford, CA ,USA 4Department of Med. Biochem. & Biophys., Karolinska Institutet, Stockholm, Sweden k.k.andersson@imbv.uio.no The Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to the deoxyribonucleotides. Class I RNR is oxygen dependent and consists of two non-identical homodimeric subunits, named R1 and R2. The small subunit R2 carries a stable tyrosyl radical which is necessary for enzymatic activity [1]. We have studied the reduced state of the class Ib RNR R2F enzyme from Bacillus cereus, an opportunistic pathogen causing food poisoning, by CD, MCD, and VTVH-MCD spectroscopy [2]. Different tyrosyl radicals were analysed by electron paramagnetic resonance (EPR/HF-EPR) [3] and resonance Raman (rRaman) spectroscopy also of RNR from an anoxia tolerant vertebrate (crucian carp) and a virus. We have published structures of mouse R2 protein and integer spin EPR of di-nuclear Co and Fe clusters [4]. In the B. cereus R2, the rRaman fingerprints, and g-tensor values of tyrosyl radical is similar those featured by E. coli R2. In other R2s differences can be seen especially in the hyperfine A-tensor and g-values values, which indicate a different rotational conformation of the phenoxyl ring and presence of sometime hydrogen bonds to phenyl-oxygens. We can also observe a tyrosyl-radical interacting with a di-manganese cluster in B. cereus R2F with help of NrdI [5,6], similar to other class Ib R2F [6,7]. The manganase R2F [8, 9] has higher specific activity than iron form with natural reductant. It seems we can obtain tyrosyl-radical interacting magnetically with other metal ions as well like Co(II). The results will be compared to the studies of E. coli R2 (class Ia and Ib), mouse R2 [4], Epstein Barr Virus R2 [10], carp R2 [11,12] and p53R2 [11,12, 13]. References. [1] K.K. Andersson (Ed.), Ribonucleotide reductase, Nova Science Publishers, Inc. Hauppauge, NY, USA, ISBN: 978-1-60456-199-9, 2008; [2] A.B. Tomter, et al., Biochemistry 2008; 47, 11300; [3] K.K. Andersson, et al., J. Biol. Inorg. Chem. 2003, 8, 23; [4] K.R. Strand, et al., J. Biol. Chem., 2002, 277, 34229; 2004, 279, 46794; . Biochemistry 2003, 42, 12223; [5] Å.K. Røhr et al., Angew. Chem. Int. Ed.. 2010, 49, 2324; [6] J.A. Cortruvo and J. Stubbe, Biochemistry 2010, 49, 1297; [7] Cox et al., J. Am. Chem. Soc. 2010, 132, 11197; [8] A.B. Tomter, et al. PLoS ONE 2012, 7, e33436; [9] Crona et al, J. Biol. Chem. 2011, 286, 33053; [10] A.B. Tomter, et al., PloS ONE 2011, 6, e25022; [11] A.B. Tomter, et al. Coordin. Chem. Rev. 2013, 257, 3-26 , doi: 10.1016/j.ccr.2012.05.021; [12] G.K. Sandvik, et al. PLoS ONE. 2012, 8, e42784; [13] Wei et al., Biochemistry 2006; 45; 14043

• Tomter, Ane Berg; Zoppellaro, Giorgio; Hersleth, Hans-Petter; Andersen, Niels Højmark; Barra, Anne-Laure; Sandvik, Guro Katrine; Røhr, Åsmund Kjendseth; Nilsson, Göran Erik; Bell, Caleb B.; Schmitzberger, Florian; Nordlund, Pär; Solomon, Edward I. & Andersson, K. Kristoffer (2013). Spectroscopic, structural and DFT studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase. NBS-nytt.  ISSN 0801-3535.  1, s 74- 74 Show summary

  Abstract M53 Spectroscopic, structural and DFT studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase Ane B. Tomter1, Giorgio Zoppellaro1, Marta Hammerstad1, Hans-Petter Hersleth1, Niels H. Andersen1, Anne-Laure Barra2, Guro K. Sandvik1, Åsmund Kjendseth Røhr1, Göran E. Nilsson1, Caleb B. Bell III3, Florian Schmitzberger4, Pär Nordlund4, Edward I. Solomon4 and K. Kristoffer Andersson1 1Department of Biosciences, University of Oslo, NO-0316 Oslo, Norway 2Grenoble High Magnetic Field Laboratory, CNRS, Grenoble, France 3Department of Chemistry, Stanford University, Stanford, CA , USA 4Department of Med. Biochem. & Biophys., Karolinska Institutet, Stockholm, Sweden The Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to the deoxyribonucleotides. Class I RNR is oxygen dependent and consists of two non-identical subunits. The small class I subunit R2 carries a stable tyrosyl radical which is necessary for enzymatic activity. Different tyrosyl radicals were analysed by electron paramagnetic resonance (EPR/HF-EPR/DFT) and resonance Raman (rRaman) spectroscopy also of RNR from an anoxia tolerant vertebrate (crucian carp) and a virus. In the B. cereus R2, the rRaman fingerprints, and g-tensor values of tyrosyl radical is similar those featured by E. coli R2. The results will be compared to the studies of mouse R2, Epstein Barr Virus R2, carp R2 and p53R2. We can also observe a tyrosyl-radical magnetically interacting with di-manganese/di-cobalt clusters in B. cereus R2F with help of NrdI, similar to other class Ib R2F. A.B. Tomter, et al., Coordin. Chem. Rev. 2013.

• Andersen, Niels Højmark; Zoppellaro, Giorgio; Tomter, Ane Berg; Barra, Anne-Laure & Andersson, K. Kristoffer (2012). Combined quantum chemical and resonance Raman studies on ribonucleotide reductase di-metal-clusters and tyrosyl radicals.
• Tomter, Ane Berg; Zoppellaro, Giorgio; Hammerstad, Marta; Barra, Anne-Laure; Sandvik, Guro Katrine; Andersen, Niels Højmark; Røhr, Åsmund Kjendseth; Nilsson, Göran Erik; Nordlund, Pär; Solomon, Edward I. & Andersson, K. Kristoffer (2012). Spectroscopic and dft studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase. Show summary

  Spectroscopic and dft studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase Ane B. Tomter; Giorgio Zoppellaro; Marta Hammerstad, Anne-Laure Barra; Guro K. Sandvik; Niels H. Andersen; Åsmund K. Roehr; Jonas Bergan; Stian Ellefsen; Göran E. Nilsson; Caleb B. Bell III; Florian Schmitzberger; Pär Nordlund; Edward I. Solomon; and K. Kristoffer Andersson Department of Molecular Biosciences, University of Oslo, NO-0316 Oslo (Norway), Grenoble High Magnetic Field Laboratory, CNRS, Grenoble ,France Departmen of Chemistry, Stanford University, Stanford, CA ,USA Department of Med. Biochem. and Biophys., Karolinska Institutet, Stockholm, Sweden The Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to the deoxyribonucleotides. Class I RNR is oxygen dependent and consists of two non-identical homodimeric subunits, named R1 and R2. The small subunit R2 carries a stable tyrosyl radical which is necessary for enzymatic activity [1]. We have studied the reduced state of the class Ib RNR R2F enzyme from Bacillus cereus, an opportunistic pathogen causing food poisoning, by CD, MCD, and VTVH-MCD spectroscopy [2]. Different tyrosyl radicals were analysed by electron paramagnetic resonance (EPR/HF-EPR) [3] and resonance Raman (rRaman) spectroscopy also of RNR from an anoxia tolerant vertebrate (crucian carp) and a virus. We have published structures of mouse R2 protein and integer spin EPR of di-nuclear Co and Fe clusters [4]. In the B. cereus R2, the rRaman fingerprints, and g-tensor values of tyrosyl radical is similar those featured by E. coli R2. In other R2s differences can be seen especially in the hyperfine A-tensor and g-values values, which indicate a different rotational conformation of the phenoxyl ring and presence of sometime hydrogen bonds to phenyl-oxygens. We can also observe a tyrosyl-radical interacting with a di-manganese cluster in B. cereus R2F with help of NrdI [5,6], similar to other class Ib R2F [6,7]. The manganase R2F [8, 9] has higher specific activity than iron form with natural reductant. It seems we can obtain tyrosyl-radical interacting magnetically with other metal ions. The results will be compared to the studies of E. coli R2 (class Ia and Ib), mouse R2 [4], Epstein Barr Virus R2 [10], carp R2 [11,12] and p53R2 [11,12]. References. [1] K.K. Andersson (Ed.), Ribonucleotide reductase, Nova Science Publishers, Inc. Hauppauge, NY, USA, ISBN: 978-1-60456-199-9, 2008; [2] A.B. Tomter, et al., Biochemistry 2008; 47, 11300; [3] K.K. Andersson, et al., J. Biol. Inorg. Chem. 2003, 8, 23; [4] K.R. Strand, et al., J. Biol. Chem., 2002, 277, 34229; 2004, 279, 46794; [5] Å.K. Røhr et al., Angew. Chem. Int. Ed.. 2010, 49, 2324; [6] J.A. Cortruvo, J. Stubbe, Biochemistry 2010, 49, 1297; [7] Cox et al., J. Am. Chem. Soc. 2010, 132, 11197; [8] A.B. Tomter, et al. PLoS ONE 2012, 7(3), e33436; [9] Crona et al, J. Biol. Chem. 2011, 286, 33053; [10] A.B. Tomter, et al., PloS ONE 2011, 6 (9), e25022; [11] A.B. Tomter, et al. Coordin. Chem. Rev. 2012, (in press); [12] G.K. Sandvik, et al. PLoS ONE. 2012, (in press)

• Andersen, Niels Højmark; Zoppellaro, Giorgio; Bubacco, L; Casella, L & Andersson, K. Kristoffer (2011). Raman Spectroscopic Evidence for a μ-η2:η2 Type Cu2O2 Core in Dodecameric Oxy-Hemocyanin from Carcinus Aestuarii. Show summary

  Raman Spectroscopic Evidence for a μ-η2:η2 Type Cu2O2 Core in Dodecameric Oxy-Hemocyanin from Carcinus Aestuarii N.H. Andersen1, G. Zoppellaro1, L. Bubacco2, L. Casella3 and K.K. Andersson1 1 Department of Molecular Biosciences, University of Oslo, Norway 2 Department of Biology, University of Padova, Italy 3 Department of Gereral Chemistry, University of Pavia, Italy E-mail: n.h.andersen@imbv.uio.no Hemocyanins (Hc) are oxygen carrier and storage proteins found in molluscs and arthropods.1 Arthropod Hc’s differ in being composed of subunits with a molecular mass in the range of 65-90 kDa, whereas molluscan Hc’s consist of much larger subunits. In case of Carcinus Aestuarii (Cae) (previously referred as Carcinus mediterraneus), Hc is present as dodecamers consisting of about 75kDa subunits containing 2 Copper atoms each.2 Calcium ions, L-Lactate3 and sugar moieties all play important structural roles for the stability of dodecamers over hexamers and other aggregation forms. The dodecamer Hc aggregate, that also exhibits Bohr effect, provides a complicated allosteric system for binding and release of molecular dioxygen. However, the Oxy-Hc structure is less sensitive to pH changes than the Cu(I)Cu(I) deoxy-Hc form.4,5 The two copper ions, each ligated by three histidines, are labeled CuA and CuB due to minor differences in structure and access to the solvent. The overall symmetry of the holohedrized His6Cu2O2 site, based on the only published crystal structure of oxy-Hc from the arthropod Limulus Polyphemus, is close to C2h if dihedral angles of the His-imidazole planes are not taken into account. This is different from the tyrosinases where the dicopper site symmetry is closer to C2v.1 In oxy form (oxy-Hc) the dioxygen is bound in the μ-η2:η2 mode as peroxide in a EPR-silent blue Cu(II)-O22–Cu(II) dicopper site. The resonance Raman spectrum shows a peroxide streching frequency at 746 cm–1, that downshifts to 706 cm–1 by oxygen-18 exchange. This compares well with oxy-Hc from L. Polyphemus that is known to have a μ-η2:η2 Cu2O2 core with peroxide characteristics, but is significantly lower than that of C. Magister.6 Source Type Organism O-O streching frequency Arthropod Carsinus Aestuarii 746 cm–1 Arthropod Cancer Magister 752 cm–1 Arthropod Limulus Polyphemus 744 cm–1 Mollusc Octopus Vulgaris 749 cm–1 Mollusc Busycon Canaliculatum 749 cm–1 [1] E.Pidcock, H.V. Obias, M. Abe, H.-C. Liang, K.D. Karlin and E.I. Solomon J. Am. Chem. Soc. 1999, 121, 1299-1308. [2] P. Dolashka-Angelova, R. Hristova, S. Stoeva and W. Voelter Spectrochim. Acta A 1999, 55, 2927-2934. [3] S. Hirota, T. Kawahara, M. Beltramini, P.D. Muro, R.S. Magliozzo, J. Peisach, L.S. Powers, N. Tanaka, S. Nagao and L. Bubacco J. Biol. Chem 2010, accepted, Doi 10.1074/jbc.M803433200. [4] S. Hirota, N. Tanaka, I. Mičetić, P.D. Muro, S. Nagao, H. Kitagishi, K. Kano, R.S. Magliozzo, J. Peisach, M. Beltramini and L. Bubacco J. Biol. Chem 2010, accepted, Doi 10.1074/jbc.M109.076067. [5] S. Hirota, T. Kawahara, M. Beltramini, P.D. Muro, R.S. Magliozzo, J. Peisach, L.S. Powers, N. Tanaka, S. Nagao and L. Bubacco J. Biol. Chem 2008, 283, 31941-31948. [6] J. Ling. L.P. Nestor, R.S. Czernuszwicz, T.G. Spiro, R. Fraczkiewicz, K.D. Sharma, T.M. Loehr and J. Sanders-Loehr J. Am. Chem. Soc. 1994, 116, 7682-7691.

• Andersen, Niels Højmark; Zoppellaro, Giorgio; Bubacco, L; Casella, L & Andersson, K. Kristoffer (2011). Symmetry Breaking at the Cu2O2 Core in Oxy-Hemocyanin from Carcinus Aestuarii - a combined DFT, CD, UV/vis and Raman study. Show summary

  Symmetry Breaking at the Cu2O2 Core in Oxy-Hemocyanin from Carcinus Aestuarii - a combined DFT, CD, UV/vis and Raman study N. H. Andersen1; G. Zoppellaro2; L. Bubacco3; L. Casella4; K. Andersson2 1. Department of Chemistry, University of Oslo, Oslo, Norway. 2. Department of Molecular Biosciences, University of Oslo, Oslo, Norway. 3. Department of Biology, University of Padova, Padova, Italy. 4. Department of Gereral Chemistry, University of Pavia, Pavia, Italy. Carcinus Aestuarii (Cae) Hemocyanin (Hc) is present as dodecamers consisting of about 75kDa subunits containing 2 Copper atoms each.1,2,3,4 These, labeled CuA and CuB, are both ligated by three histidines and are slightly different regarding structure and surroundings. The holohedrized symmetry of the His6Cu2O2 site, based on the only published crystal structure of oxy-Hc from the arthropod Limulus Polyphemus, is close to C2h. The EPR-silent blue dicopper peroxide site is antiferromagnetically coupled. This causes DFT modelling of this kind of metalcentres more intriguing.5 However, by optimizing a C2h symmetric model compound, we vere able to simulate a peroxide streching frequency that is close to the observed resonance Raman spectrum that shows a frequency of 746 cm–1, that downshifts to 706 cm–1 by oxygen-18 exchange. This also compares well with oxy-Hc from L. Polyphemus that has a μ-η2:η2 Cu2O2 core, but is significantly lower than that of C. Magister.6 We also examined the effect of various kinds of symmetry breaking on the CD spectrum. [1] P. Dolashka-Angelova, R. Hristova, S. Stoeva and W. Voelter Spectrochim. Acta A 1999, 55, 2927-2934. [2] E.Pidcock, H.V. Obias, M. Abe, H.-C. Liang, K.D. Karlin and E.I. Solomon J. Am. Chem. Soc. 1999, 121, 1299-1308. [3] S. Hirota, T. Kawahara, M. Beltramini, P.D. Muro, R.S. Magliozzo, J. Peisach, L.S. Powers, N. Tanaka, S. Nagao and L. Bubacco J. Biol. Chem 2010, accepted, Doi 10.1074/jbc.M803433200. [4] S. Hirota, N. Tanaka, I. Mičetić, P.D. Muro, S. Nagao, H. Kitagishi, K. Kano, R.S. Magliozzo, J. Peisach, M. Beltramini and L. Bubacco J. Biol. Chem 2010, accepted, Doi 10.1074/jbc.M109.076067. [5] J.-P. Piquemal and J. Pilme, J. Mol. Struct.: THEOCHEM 2006, 764, 77–86. [6] J. Ling. L.P. Nestor, R.S. Czernuszwicz, T.G. Spiro, R. Fraczkiewicz, K.D. Sharma, T.M. Loehr and J. Sanders-Loehr J. Am. Chem. Soc. 1994, 116, 7682-7691.

• Andersson, K. Kristoffer; Tomter, Ane Berg; Zoppellaro, Giorgio; Barra, Anne-Laure; Sandvik, Guro Katrine; Andersen, Niels Højmark; Røhr, Åsmund Kjendseth; Bergan, Jonas; Ellefsen, Stian; Bell, Caleb B.; Schmitzberger, Florian; Nordlund, Pär; Solomon, Edward I. & Nilsson, Göran Erik (2011). Spectroscopic and DFT Studies of the Tyrosyl Radicals and the Binuclear Metal-ion Sites in R2F/R2 subunits of Ribonucleotide Reductase. Journal of Biological Inorganic Chemistry.  ISSN 0949-8257.  16, s S417- S417 . doi: 10.1007/s00775-011-0862-z Show summary

  Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to the deoxyribonucleotides. Class I RNR is oxygen dependent and consists of two non-identical homodimeric subunits, named R1 and R2. The small subunit R2 carries a stable tyrosyl radical which is necessary for enzymatic activity (1). We have studied the reduced state of the class Ib RNR R2F enzyme from Bacillus cereus, an opportunistic pathogen causing food poisoning, by CD, MCD, and VTVH-MCD spectroscopy (2). Different tyrosyl radicals were analysed by electron paramagnetic resonance (EPR/HF-EPR) (3) and resonance Raman (rRaman) spectroscopy also of RNR from an anoxia tolerant vertebrate (crucian carp) and a virus. We have published structures of mouse R2 protein and integer spin EPR of di-nuclear Co and Fe clusters (4). In the B. cereus R2, the rRaman fingerprints, and g-tensor values of tyrosyl radical is similar those featured by E. coli R2. In other R2s differences can be seen especially in the hyperfine A-tensor and g-values values, which indicate a different rotational conformation of the phenoxyl ring and presence of sometime hydrogen bonds to phenyl-oxygens. We can also observe a radical in the manganese substituted B. cereus R2F with help of NrdI (5), similar to other class Ib R2F (6), it seems we can obtain radical with other metal ions. The results will be compared to the studies of E. coli R2 (class Ia and Ib), mouse R2, and p53R2. (1) M. Kolberg et al, Biochim. Biophys. Acta 2004, 1699, 1; K.K. Andersson (Ed.) Ribonucleotide reductase 2008, Nova Science, New York. (2) A.B. Tomter, et al. Biochemistry 2008, 47, 11300 (3) K.K. Andersson & A.-L. Barra Spectrochim. Acta (A) Mol. Biomol. Spectrosc. 2002, 58, 1101; K.K. Andersson, et a., J. Biol. Inorg. Chem. 2003, 8, 23; A.-L. Barra et al., In Very High Frequency (VHR) ESR/EPR. Biol. Magn. Resonance 2004, 22, 145 (4) K.R. Strand et al., J. Biol. Chem., 2002, 277, 34229; K.R. Strand et al., J. Biol. Chem., 2004, 279, 46794 (5) Å.K. Røhr et al., Angew. Chem. Int. Ed.. 2010, 49, 2324 (6) J.A. Cortruvo & J. Stubbe, Biochemistry 2010, 49, 1297

• Hersleth, Hans-Petter; Andersen, Niels Højmark & Andersson, K. Kristoffer (2011). Grasping the Nature of the Myoglobin Compound II state by combining X-Ray Diffraction and in-situ Light Absorption and Raman Spectroscopy. Show summary

  Myoglobin is the protein responsible for oxygen storage, but is also believed to have several other functions e.g. scavenging of reactive oxygen species. In the reaction with peroxides myoglobin propagates through similar intermediates as the peroxidases, catalases and oxygenases. The nature of the compound II intermediate in haem proteins, as being either a FeIV-OH or FeIV=O state, has long been debated [1]. A potential problem with the results from the crystal structures is that the X-rays used for solving the structures can lead to radiation-induced reduction of the redox site [2]. We have used in-situ (online) UV-vis and Raman spectroscopy to study how different oxidation states of myoglobin are influenced by X-rays during crystallographic data collection. The spectroscopic changes have been monitored as a function of X-ray exposure (dose absorbed), and show that the different redox-states in myoglobin vary in how fast they are influenced by the X-rays (e.g. ferric Fe3+ myoglobin is faster reduced than ferryl FeIV=O myoglobin) [3]. The higher oxidation states are not reduced to normal ferrous Fe2+ or ferric Fe3+ states, but end up in some intermediate state. We have from these studies been able to estimate a lifedose (how much X-rays you can use before the crystal structure is too much influenced by the X-rays) for each of the states. By combining this with in-situ UV-vis light absorption and Raman spectroscopy we have been able to grasp the nature of the compound II state in myoglobin. We have also been able to use radiation damage to generate the otherwise unstable and unattainable peroxymyoglobin state by cryoradiolytic reduction of an oxymyoglobin equivalent [4]. References [1] H.-P. Hersleth, et al., J. Biol. Chem. 2007, 282, 23372-23386. [2] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Chem. Biodiv. 2008, 5, 2067-2089. [3] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011, In press. DOI: 10.1016/j.bbapap.2010.07.019 [4] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Biochem. J. 2008, 412, 257-264. Fig Crystal structure of the myoglobin compound II and the single-crystal UV-vis and Raman spectra showing the radiation-induced changes.

• Hersleth, Hans-Petter; Andersen, Niels Højmark & Andersson, K. Kristoffer (2011). Grasping the Nature of the Myoglobin Compound II state by combining X-Ray Diffraction and in-situ Light Absorption and Raman Spectroscopy. Journal of Biological Inorganic Chemistry.  ISSN 0949-8257.  16, s S481- S481 . doi: 10.1007/s00775-011-0862-z Show summary

  Myoglobin is the protein responsible for oxygen storage, but is also believed to have several other functions e.g. scavenging of reactive oxygen species. In the reaction with peroxides myoglobin propagates through similar intermediates as the peroxidases, catalases and oxygenases. The nature of the compound II intermediate in haem proteins, as being either a FeIV-OH or FeIV=O state, has long been debated [1]. A potential problem with the results from the crystal structures is that the X-rays used for solving the structures can lead to radiation-induced reduction of the redox site [2]. We have used in-situ (online) UV-vis and Raman spectroscopy to study how different oxidation states of myoglobin are influenced by X-rays during crystallographic data collection. The spectroscopic changes have been monitored as a function of X-ray exposure (dose absorbed), and show that the different redox-states in myoglobin vary in how fast they are influenced by the X-rays (e.g. ferric Fe3+ myoglobin is faster reduced than ferryl FeIV=O myoglobin) [3]. The higher oxidation states are not reduced to normal ferrous Fe2+ or ferric Fe3+ states, but end up in some intermediate state. We have from these studies been able to estimate a lifedose (how much X-rays you can use before the crystal structure is too much influenced by the X-rays) for each of the states. By combining this with in-situ UV-vis light absorption and Raman spectroscopy we have been able to grasp the nature of the compound II state in myoglobin. We have also been able to use radiation damage to generate the otherwise unstable and unattainable peroxymyoglobin state by cryoradiolytic reduction of an oxymyoglobin equivalent [4]. References [1] H.-P. Hersleth, et al., J. Biol. Chem. 2007, 282, 23372-23386. [2] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Chem. Biodiv. 2008, 5, 2067-2089. [3] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011, In press. DOI: 10.1016/j.bbapap.2010.07.019 [4] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Biochem. J. 2008, 412, 257-264. Fig Crystal structure of the myoglobin compound II and the single-crystal UV-vis and Raman spectra showing the radiation-induced changes.

• Tomter, Ane Berg; Zoppellaro, Giorgio; Bell, Caleb B.; Andersen, Niels Højmark; Barra, Anne-Laure; Solomon, Edward I. & Andersson, K. Kristoffer (2011). Spectroscopic studies of the tyrosyl radical from the R2 subunit of ribonucleotide reductase from Bacillus cereus. Show summary

  Spectroscopic studies of the tyrosyl radical from the R2 subunit of ribonucleotide reductase from Bacillus cereus. A. B. Tomter1; G. Zoppellaro1; C. B. Bell2; N. H. Andersen1; A. Barra3; E. I. Solomon2; K. Andersson1 1. Department of Molecular Biosciences, University of Oslo, Oslo, Norway. 2. Department of Chemistry, Stanford University, Stanford, CA, United States. 3. Laboratoire National des Champs Magnétiques Intenses, LNCMI-G, CNRS, Grenoble, France. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to the corresponding deoxyribonucleotides in all living organisms, thereby providing and controlling the pool of building blocks necessary for DNA synthesis and repair. Class I RNR is oxygen dependent and consists of two non-identical homodimeric subunits, named R1 and R2 which can be assembled into enzymatically active tetramers or higher ordered oligomers. The R1 subunit contains the active site, and the R2 subunit a tyrosyl radical and a diiron-oxygen cofactor which are essential for initiation of the nucleotide reduction process in R1. The R2 subunit of the enzyme complex reacts with ferrous iron and dioxygen to generate a diferric iron-oxygen cluster and a tyrosyl radical that is essential for enzymatic activity [1]. We have studied the tyrosyl radical in the ferric active state, of the class Ib RNR R2F enzyme from Bacillus cereus, an opportunistic pathogen causing food poisoning. The tyrosyl radical was analysed by electron paramagnetic resonance (EPR/HF-EPR) and resonance Raman (rR) spectroscopy. In the B. cereus R2, the rR fingerprints and g-tensor values of tyrosyl radical resemble those featured by E. coli R2, although differences can be seen especially in the hyperfine A-tensor values, which indicate a different rotational conformation of the phenoxyl ring. We can also observe a highly active form with a radical in the manganese substituted B. cereus R2F with help of NrdI [2], similar to what has been observed for E. coli R2F [3]. In preliminary studies we can also observe formation of a radical with other metal-ions as well, which will be presented. [1] (a) M. Kolberg, K.R. Strand, P. Graff, K.K. Andersson, Biochim. Biophys. Acta-Proteins and Proteomics 2004, 1699, 1-34. (b) K. K. Andersson Ed. in Ribonucleotide reductase 2008, Nova Science, NY.[2] Å.K. Røhr, H.-P. Hersleth, K.K. Andersson, Angew. Chem. Int. Ed.. 2010, 49, 2324-2327[3] (a) J.A. Cortruvo, J. Stubbe, Biochemistry 2010, 49, 1297-1309. (b) N. Cox, H. Ogata, P. Stolle, E. Reijerse, G. Auling, W. Lubitz, J. Am. Chem. Soc. 2010, 132, 11197-11213 Fig. Resonance Raman spectra of the Fe-reconstituted B. cereus R2F, effects of 18O and D2O on tyrosyl radical phenyl-O stretch and oxy-Fe(III) vibrations

• Tomter, Ane Berg; Zoppellaro, Giorgio; Bell, Caleb B.; Andersen, Niels Højmark; Barra, Anne-Laure; Solomon, Edward I. & Andersson, K. Kristoffer (2011). Spectroscopic studies of the tyrosyl radical from the R2 subunit of ribonucleotide reductase from Bacillus cereus. Journal of Biological Inorganic Chemistry.  ISSN 0949-8257.  16 . doi: 10.1007/s00775-011-0862-z Show summary

  Spectroscopic studies of the tyrosyl radical from the R2 subunit of ribonucleotide reductase from Bacillus cereus. A. B. Tomter1; G. Zoppellaro1; C. B. Bell2; N. H. Andersen1; A. Barra3; E. I. Solomon2; K. Andersson1 1. Department of Molecular Biosciences, University of Oslo, Oslo, Norway. 2. Department of Chemistry, Stanford University, Stanford, CA, United States. 3. Laboratoire National des Champs Magnétiques Intenses, LNCMI-G, CNRS, Grenoble, France. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to the corresponding deoxyribonucleotides in all living organisms, thereby providing and controlling the pool of building blocks necessary for DNA synthesis and repair. Class I RNR is oxygen dependent and consists of two non-identical homodimeric subunits, named R1 and R2 which can be assembled into enzymatically active tetramers or higher ordered oligomers. The R1 subunit contains the active site, and the R2 subunit a tyrosyl radical and a diiron-oxygen cofactor which are essential for initiation of the nucleotide reduction process in R1. The R2 subunit of the enzyme complex reacts with ferrous iron and dioxygen to generate a diferric iron-oxygen cluster and a tyrosyl radical that is essential for enzymatic activity [1]. We have studied the tyrosyl radical in the ferric active state, of the class Ib RNR R2F enzyme from Bacillus cereus, an opportunistic pathogen causing food poisoning. The tyrosyl radical was analysed by electron paramagnetic resonance (EPR/HF-EPR) and resonance Raman (rR) spectroscopy. In the B. cereus R2, the rR fingerprints and g-tensor values of tyrosyl radical resemble those featured by E. coli R2, although differences can be seen especially in the hyperfine A-tensor values, which indicate a different rotational conformation of the phenoxyl ring. We can also observe a highly active form with a radical in the manganese substituted B. cereus R2F with help of NrdI [2], similar to what has been observed for E. coli R2F [3]. In preliminary studies we can also observe formation of a radical with other metal-ions as well, which will be presented. [1] (a) M. Kolberg, K.R. Strand, P. Graff, K.K. Andersson, Biochim. Biophys. Acta-Proteins and Proteomics 2004, 1699, 1-34. (b) K. K. Andersson Ed. in Ribonucleotide reductase 2008, Nova Science, NY.[2] Å.K. Røhr, H.-P. Hersleth, K.K. Andersson, Angew. Chem. Int. Ed.. 2010, 49, 2324-2327[3] (a) J.A. Cortruvo, J. Stubbe, Biochemistry 2010, 49, 1297-1309. (b) N. Cox, H. Ogata, P. Stolle, E. Reijerse, G. Auling, W. Lubitz, J. Am. Chem. Soc. 2010, 132, 11197-11213 Fig. Resonance Raman spectra of the Fe-reconstituted B. cereus R2F, effects of 18O and D2O on tyrosyl radical phenyl-O stretch and oxy-Fe(III) vibrations

• Tomter, Ane Berg; Zoppellaro, Giorgio; Schmitzberger, Florian; Andersen, Niels Højmark; Barra, Anne-Laure; Engman, H; Nordlund, Pär & Andersson, K. Kristoffer (2011). X-band EPR, HF-EPR, and Resonance Raman Spectroscopic Studies of the Ribonucleotide Reductase R2 Tyrosyl Radical from Epstein-Barr Virus. NBS-nytt.  ISSN 0801-3535.  s 57- 57 Show summary

  X-band EPR, HF-EPR, and Resonance Raman Spectroscopic Studies of the Ribonucleotide Reductase R2 Tyrosyl Radical from Epstein-Barr Virus A.B. Tomter1, G. Zoppellaro1, F. Schmitzberger2, N. H. Andersen1, A.-L. Barra3, H. Engman2, P. Nordlund2, and K. K. Andersson1 1Department of Molecular Biosciences, University of Oslo, Oslo 2 Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm 3 Laboratoire National des Champs Magnétiques Intenses, Grenoble Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. Class Ia RNRs consist of two homodimeric subunits R1, where the active site is found, and R2, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction. Here, the electronic and magnetic properties of the tyrosyl radical in the small subunit of RNR from EBV have been determined by X-band and high-field/high-frequency EPR spectroscopy recorded at cryogenic temperatures. We show that the radical has an unusually low g1-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results, we observe a relatively high stretching frequency associated with the tyrosyl radical (at 1508 cm-1) with resonance Raman spectroscopy. In contrast to mouse and HSV1 R2 we could not observe a deuterium shift in our Raman spectra. Thus we could not unequivocally identify a water molecule as a hydrogen bond donor moiety. Comparison with previous studies of RNR R2s from mouse, bacteria, and viruses, demonstrates that finely tuned electronic differences of the radical exist within the same RNR R2 Ia class.

• Andersen, Niels Højmark; Zoppellaro, Giorgio; Bubacco, L; Casella, L & Andersson, K. Kristoffer (2010). Raman Spectroscopic Evidence for a μ-η2:η2 Type Cu2O2 Core in Dodecameric Oxy-Hemocyanin from Carcinus Aestuarii. Show summary

  Hemocyanins (Hc) are oxygen carrier and storage proteins found in molluscs and arthropods.1 Arthropod Hc’s differ in being composed of subunits with a molecular mass in the range of 65-90 kDa, whereas molluscan Hc’s consist of much larger subunits. In case of Carcinus Aestuarii (Cae) (previously referred as Carcinus mediterraneus), Hc is present as dodecamers consisting of about 75kDa subunits containing 2 Copper atoms each.2 Calcium ions, L-Lactate3 and sugar moieties all play important structural roles for the stability of dodecamers over hexamers and other aggregation forms. The dodecamer Hc aggregate, that also exhibits Bohr effect, provides a complicated allosteric system for binding and release of molecular dioxygen. However, the Oxy-Hc structure is less sensitive to pH changes than the Cu(I)Cu(I) deoxy-Hc form.4,5 The two copper ions, each ligated by three histidines, are labeled CuA and CuB due to minor differences in structure and access to the solvent. The overall symmetry of the holohedrized His6Cu2O2 site, based on the only published crystal structure of oxy-Hc from the arthropod Limulus Polyphemus, is close to C2h if dihedral angles of the His-imidazole planes are not taken into account. This is different from the tyrosinases where the dicopper site symmetry is closer to D2d.1 In oxy form (oxy-Hc) the dioxygen is bound in the μ-η2:η2 mode as peroxide in a EPR-silent blue Cu(II)-O2 2–Cu(II) dicopper site. The resonance Raman spectrum shows a peroxide streching frequency at 746 cm–1, that downshifts to 706 cm–1 by oxygen-18 exchange. This compares well with oxy-Hc from L. Polyphemus that is known to have a μ-η2:η2 Cu2O2 core with peroxide characteristics, but is significantly lower than that of C. Magister.6 [1] E.Pidcock, H.V. Obias, M. Abe, H.-C. Liang, K.D. Karlin and E.I. Solomon J. Am. Chem. Soc. 1999, 121, 1299-1308. [2] P. Dolashka-Angelova, R. Hristova, S. Stoeva and W. Voelter Spectrochim. Acta A 1999, 55, 2927-2934. [3] S. Hirota, T. Kawahara, M. Beltramini, P.D. Muro, R.S. Magliozzo, J. Peisach, L.S. Powers, N. Tanaka, S. Nagao and L. Bubacco J. Biol. Chem 2010, accepted, Doi 10.1074/jbc.M803433200. [4] S. Hirota, N. Tanaka, I. Mičetić, P.D. Muro, S. Nagao, H. Kitagishi, K. Kano, R.S. Magliozzo, J. Peisach, M. Beltramini and L. Bubacco J. Biol. Chem 2010, accepted, Doi 10.1074/jbc.M109.076067. [5] S. Hirota, T. Kawahara, M. Beltramini, P.D. Muro, R.S. Magliozzo, J. Peisach, L.S. Powers, N. Tanaka, S. Nagao and L. Bubacco J. Biol. Chem 2008, 283, 31941-31948. [6] J. Ling. L.P. Nestor, R.S. Czernuszwicz, T.G. Spiro, R. Fraczkiewicz, K.D. Sharma, T.M. Loehr and J. Sanders-Loehr J. Am. Chem. Soc. 1994, 116, 7682-7691.

• Hersleth, Hans-Petter; Andersen, Niels Højmark & Andersson, K. Kristoffer (2010). Combining X-Ray Diffraction and in-situ Light Absorption and Raman Spectroscopy to Study Different Myoglobin Redox States. Show summary

  Combining X-Ray Diffraction and in-situ Light Absorption and Raman Spectroscopy to Study Different Myoglobin Redox States H.-P. Hersletha, N.H. Andersena, K.K. Anderssona aDepartment of Molecular Biosciences, Univeristy of Oslo, P.O.Box 1041 Blindern, 0315 Oslo, Norway, E-mail: h.p.hersleth@imbv.uio.no The influence of X-ray radiation damage to protein crystals is well known to occur even at cryogenic temperatures, and redox active sites like metal sites seems especially vulnerable for radiation-induced reduction. To correctly know the oxidation state of these metal sites in protein crystal structures is essential for interpreting the structure-function relation. We have used in-situ (online) UV-vis and Raman spectroscopy to study how different oxidation states of the haem protein myoglobin are influenced by X-rays during crystallographic data collection. The spectroscopic changes have been monitored as a function of X-ray exposure (dose absorbed), and show that the different redox-states in myoglobin vary considerably in how fast they are “reduced” by the X-rays (e.g. ferric Fe3+ myoglobin is faster reduced than ferryl FeIV=O myoglobin) [1]. The higher oxidation states are not reduced to normal ferrous Fe2+ or ferric Fe3+ states, but end up in some intermediate state. We have observed similar reduction effect of the flavoprotein NrdI [2]. The primary goal of the project has been to characterise and study the different intermediates in the reaction between myoglobin and peroxides [3,4]. The reaction intermediates generated appear biological relevant since myoglobin is proposed to function as scavenger of reactive oxygen species during oxidative stress [3]. We have also been able to use the radiation damage to generate an otherwise unstable and unattainable state by cryoradiolytic reduction of an oxymyoglobin equivalent (compound III) to generate and trap the so-called peroxymyoglobin intermediate. By annealing this compound the oxygen-oxygen bond is broken and the reaction propagates to the ferryl compound II intermediate [4,5]. Acknowledgments: The investigation has been supported by funding from the Norwegian Research Council: grant 177661/V30 (to K.K.A) and grant 138370/V30 (Synchrotron related research in the Oslo-region, SYGOR). The teams at the Swiss-Norwegian Beam Line (BM01) ESRF (Grenoble), at the MX-Beamlines ESRF (Grenoble), at X13 EMBL (Hamburg), at X10SA at SLS (Villigen) and Martin Weik are thanked for their valuable help. References [1] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2010, Submitted [2] Å.K. Røhr, H.-P. Hersleth, K.K. Andersson, Angew. Chem. Int. Ed.. 2010, 49, 2324-2327. [3] H.-P. Hersleth, T. Uchida, Å.K. Røhr, T. Teschner, V. Schünemann, T. Kitagawa, A.X. Trautwein, C.H. Görbitz, K.K. Andersson, J. Biol. Chem. 2007, 282, 23372-23386. [4] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Biochem. J. 2008, 412, 257-264. [5] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Chem. Biodiv. 2008, 5, 2067-2089.

• Sandvik, Guro Katrine; Tomter, Ane Berg; Røhr, Åsmund Kjendseth; Zoppellaro, Giorgio; Andersen, Niels Højmark; Bergan, Jonas; Ellefsen, Stian; Barra, Anne-Laure; Nilsson, Gøran Erik & Andersson, K. Kristoffer (2010). Class I Ribonucleotide reductase in an anoxia tolerant vertebrate, the crucian carp. Show summary

  Class I Ribonucleotide reductase in an anoxia tolerant vertebrate, the crucian carp Guro K. Sandvik1, Ane B. Tomter1, Åsmund K. Røhr, Giorgio Zoppellaro1, Niels H. Andersen1, Jonas Bergan1, Stian Ellefsen1, Anne-Laure Barra2, Göran E. Nilsson1 and K. Kristoffer Andersson1 1Department of Molecular Biosciences, University of Oslo, POBox 1041, NO-0316 Oslo, NORWAY 2 Grenoble High Magentic Field Laboratory, LCMI-CNRS, B.P. 166, FR- 38042 Grenoble cedex 9 FRANCE The fish crucian carp (Carrassius carrassius) can survive several months in anoxia, partly because of its extraordinary ability to convert lactate to ethanol, which allows it to avoid lactate accumulation. Consequently, the crucian carp are able to survive on anaerobic metabolism through the winter, when the pond they are living in gets covered with ice and snow, blocking oxygen exchange with the air. Remarkably, previous studies in our group have shown cell division in severe hypoxic crucian carp (in water with <0.1 mg O2/L). This led us to investigate the enzyme ribonucleotide reductase (RNR), because RNR is required for production of new nucleotides and is oxygen dependent in all vertebrates studied so far. RNR in crucian carp must therefore be exceptional in some way, possibly by forming an unusually stable radical, or by having an extremely high oxygen affinity. We have cloned and sequenced several variants of class I RNR in the crucian carp: two variants of the large RNR (R1) subunit, two variants of the small RNR (R2) subunit and two variants of the p53R2 subunit. Interestingly, quantitative real-time PCR measurements in normoxic and severe hypoxic crucian carp revealed that the expression of the small RNR subunit (R2), which is the subunit activated by oxygen, was maintained in severe hypoxia. In mammals, this subunit is normally only expressed in mitotically active cells, suggesting that the crucian carp may have a functional RNR in severe hypoxia. RNR proteins were also in vitro expressed in E. coli for structural and enzymatic studies. Amino acid analysis, EPR (electron paramagnetic resonance), HF-EPR and Raman spectroscopy show that the crucian carp RNR variants are structurally similar to mammalian RNR, but ongoing studies will reveal if their oxygen affinity or stability in hypoxia or anoxia are different. Supported by the Research Council of Norway.

• Sandvik, Guro Katrine; Tomter, Ane Berg; Røhr, Åsmund Kjendseth; Zoppellaro, Giorgio; Andersen, Niels Højmark; Bergan, Jonas; Ellefsen, Stian; Barra, Anne-Laure; Nilsson, Gøran Erik & Andersson, K. Kristoffer (2010). Class I Ribonucleotide reductase in an anoxia tolerant vertebrate, the crucian carp. Show summary

  Guro K. Sandvik1, Ane B. Tomter1, Åsmund K. Røhr, Giorgio Zoppellaro1, Niels H. Andersen1, Jonas Bergan1, Stian Ellefsen1, Anne-Laure Barra2, Göran E. Nilsson1 and K. Kristoffer Andersson1 1Department of Molecular Biosciences, University of Oslo, POBox 1041, NO-0316 Oslo, NORWAY 2 Grenoble High Magentic Field Laboratory, LCMI-CNRS, B.P. 166, FR- 38042 Grenoble cedex 9 FRANCE The fish crucian carp (Carrassius carrassius) can survive several months in anoxia, partly because of its extraordinary ability to convert lactate to ethanol, which allows it to avoid lactate accumulation. Consequently, the crucian carp are able to survive on anaerobic metabolism through the winter, when the pond they are living in gets covered with ice and snow, blocking oxygen exchange with the air. Remarkably, previous studies in our group have shown cell division in severe hypoxic crucian carp (in water with <0.1 mg O2/L). This led us to investigate the enzyme ribonucleotide reductase (RNR), because RNR is required for production of new nucleotides and is oxygen dependent in all vertebrates studied so far. RNR in crucian carp must therefore be exceptional in some way, possibly by forming an unusually stable radical, or by having an extremely high oxygen affinity. We have cloned and sequenced several variants of class I RNR in the crucian carp: two variants of the large RNR (R1) subunit, two variants of the small RNR (R2) subunit and two variants of the p53R2 subunit. Interestingly, quantitative real-time PCR measurements in normoxic and severe hypoxic crucian carp revealed that the expression of the small RNR subunit (R2), which is the subunit activated by oxygen, was maintained in severe hypoxia. In mammals, this subunit is normally only expressed in mitotically active cells, suggesting that the crucian carp may have a functional RNR in severe hypoxia. RNR proteins were also in vitro expressed in E. coli for structural and enzymatic studies. Amino acid analysis, EPR (electron paramagnetic resonance), HF-EPR and Raman spectroscopy show that the crucian carp RNR variants are structurally similar to mammalian RNR, but ongoing studies will reveal if their oxygen affinity or stability in hypoxia or anoxia are different. Supported by the Research Council of Norway.

• Tomter, Ane Berg; Barra, Anne-Laure; Sandvik, Guro Katrine; Zoppellaro, Giorgio; Andersen, Niels Højmark; Røhr, Åsmund Kjendseth; Bergan, Jonas; Ellefsen, Stian; Nilsson, Göran Erik; Bell III, Caleb; Schmitzberger, Florian; Nordlund, Pär; Solomon, Edward I. & Andersson, K. Kristoffer (2010). Spectroscopic and HF-EPR studies of the tyrosyl radical and the binuclear iron site of the R2F/R2 subunits of ribonucleotide reductase. Show summary

  Spectroscopic and HF-EPR Studies of the Tyrosyl Radical and the Binuclear Iron Site of the R2F/R2 subunits of Ribonucleotide Reductase Ane B. Tomter1, Anne-Laure Barra2, Guro K. Sandvik1, Giorgio Zoppellaro1, Niels H. Andersen1, Åsmund K. Røhr1, Jonas Bergan1, Stian Ellefsen1, Göran E. Nilsson1, Caleb B. Bell III3, Florian Schmitzberger4, Pär Nordlund4, Edward I. Solomon3, and K. Kristoffer Andersson1 1 Department of Molecular Biosciences, University of Oslo, Norway 2 Grenoble High Magnetic Field Laboratory, CNRS, Grenoble, France 3 Chemistry Department, Stanford University, Stanford, CA, USA 4 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to the corresponding deoxyribonucleotides in all living organisms, thereby providing and controlling the pool of building blocks necessary for DNA synthesis and repair. Class I RNR is oxygen dependent and consists of two non-identical homodimeric subunits, named R1 and R2 which can be assembled into enzymatically active tetramers (R12R22). As shown in Figure 1, the ribonucleotide binding and active site A is located in R1, while the small subunit R2 carries a stable tyrosyl radical which is necessary for enzymatic activity, close to a diiron metal site. The radical generated in the R2 subunit is shuttled to the active site of R1 subunit, about 35 Å from the radical iron cluster, through a conserved array of hydrogen bonded amino acids [1]. We have studied the reduced state of the class Ib RNR R2F enzyme from Bacillus cereus, an opportunistic pathogen causing food poisoning, using a combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature variable-field (VTVH) MCD spectroscopy [2]. Furthermore, the tyrosyl radical in the ferric active state was analysed by electron paramagnetic resonance (EPR/HF-EPR) [3] and resonance Raman (rR) spectroscopy of RNR from an anoxia tolerant vertebrate (crucian carp) and a virus. In the B. cereus R2, the rR fingerprints and g-tensor values of tyrosyl radical resemble those featured by E. coli R2, although differences can be seen especially in the hyperfine A-tensor values, which indicate a different rotational conformation of the phenoxyl ring. We can also observe a radical in the manganese substituted B. cereus R2F with help of NrdI [4], similar to what has been observed for E. coli R2F [5]. The results have been compared to the studies of E. coli R2, mouse R2, and p53R2 which all are of RNR class Ia. [1] (a) M. Kolberg, K.R. Strand, P. Graff, K.K. Andersson Biochim. Biophys. Acta 2004, 1699, 1-34. (b) Andersson K. K. Ed. in Ribonucleotide reductase 2008, Nova Science, New York. [2] A.B.Tomter, C.B. Bell, Å.K. Røhr, K.K. Andersson, E.I. Solomon Biochemistry 2008, 47, 11300-11309. [3] (a) K.K. Andersson, A.-L. Barra (2002) Spectrochim. Acta (A) Mol. Biomol. Spectrosc. 2002, 58, 1101-1112; (b) K.K. Andersson, P. P. Schmidt, B. Katterle, K.R. Stand, A. Palmer, S.-K. Lee, E. I. Solomon, A. Gräslund, A.-L. Barra (2003) J. Biol. Inorg. Chem. 2003, 8, 235-247(c) Barra A.-L., A. Gräslund and K. K. Andersson (2004) In Very High Frequency (VHR) ESR/EPR. Biol. Magn. Resonance 22, 145-164 [4] Å.K. Røhr, H.-P. Hersleth, K.K. Andersson, Angew. Chem. Int. Ed.. 2010, 49, 2324-2327 [5] J.A. Cortruvo, J. Stubbe, Biochemistry 2010, 49, 1297-1309

• Tomter, Ane Berg; Bell III, Caleb; Zoppellaro, Giorgio; Andersen, Niels Højmark; Barra, Anne-Laure; Solomon, Edward I. & Andersson, K. Kristoffer (2010). Spectroscopic Studies of the Tyrosyl Radical and the Binuclear Iron Site of the R2F subunit of Ribonucleotide Reductase from Bacillus cereus. Show summary

  Spectroscopic Studies of the Tyrosyl Radical and the Binuclear Iron Site of the R2F subunit of Ribonucleotide Reductase from Bacillus cereus A. B. Tomtera, C. B. Bell IIIb, G. Zoppellaroa, N. H. Anderssena, A.-L. Barrac, E. I. Solomonb, and K. K. Anderssona a.b.tomter@imbv.uio.no Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to the corresponding deoxyribonucleotides in all living organisms, thereby providing and controlling the pool of building blocks necessary for DNA synthesis and repair. Class I RNR is oxygen dependent and consists of two non-identical homodimeric subunits, named R1 and R2 which can be assembled into enzymatically active tetramers (R12R22). As shown in Figure 1, the ribonucleotide binding and active site A is located in R1, while the small subunit R2 carries a stable tyrosyl radical which is necessary for enzymatic activity, close to a diiron metal site. The radical generated in the R2 subunit is shuttled to the active site of R1 subunit, about 35 Å from the radical iron cluster, through a conserved array of hydrogen bonded amino acids [1]. We have studied the reduced state of the class Ib RNR R2F enzyme from Bacillus cereus, an opportunistic pathogen causing food poisoning, using a combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature variable-field (VTVH) MCD spectroscopy [2]. Furthermore, the tyrosyl radical in the ferric active state was analysed by electron paramagnetic resonance (EPR/HF-EPR) and resonance Raman (rR) spectroscopy. In the B. cereus R2, the rR fingerprints and g-tensor values of tyrosyl radical resemble those featured by E. coli R2, although differences can be seen especially in the hyperfine A-tensor values, which indicate a different rotational conformation of the phenoxyl ring. We can also observe a radical in the manganese substituted B. cereus R2F with help of NrdI [3], similar to what has been observed for E. coli R2F [4]. Spectral features of individual ferrous iron sites have been analyzed to obtain detailed geometric and electronic structural information. The results have been compared to the studies of E. coli R2, mouse R2, and p53R2 which all are of RNR class Ia.

• Tomter, Ane Berg; Zoppellaro, Giorgio; Bell III, Caleb; Røhr, Åsmund Kjendseth; Andersen, Niels Højmark; Barra, Anne-Laure; Solomon, Edward I. & Andersson, K. Kristoffer (2010). SPECTROSCOPIC STUDIES OF THE TYROSYL RADICAL AND THE BINUCLEAR IRON SITE OF THE R2 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE FROM BACILLUS CEREUS.
• Tomter, Ane Berg; Bell III, Caleb; Zoppellaro, Giorgio; Røhr, Åsmund Kjendseth; Andersen, Niels Højmark; Barra, Anne-Laure; Solomon, Edward I. & Andersson, K. Kristoffer (2009). Spectroscopic studies of the tyrosyl radical and the binuclear iron site of the R2 subunit of ribonucleotide reductase from Bacillus cereus.

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