2014-dd Principal Investigator and Senior Lecturer. Dept. of Biosciences, Section for Biochemistry and Molecular Biology, University of Oslo. From 2017 also at Dept. of Chemistry, Section for Chemical Life Sciences.

2007-2014 Post.doc./Researcher, Dept. of Biosciences, University of Oslo

2012-2012 High school teacher, Oslo Handelsgymnasium (60%)

2006-2007 Chemistry teacher on the PreCourse at Faculty of Engineering, Oslo University College (40%)

2003-2004 Teacher Education Program, (one-year), Department of Teacher Education and School Development, University of Oslo

2000-2007 PhD-Student (Dr. scient) in Protein Crystallography, Department of Chemistry, University of Oslo, with 40% teaching duties

1998-2000 Master (Cand.scient.) in Physical Chemistry (crystallography), Department of Chemistry, University of Oslo

1995-1998 Bachelor (Cand. mag.) in Mathematics, Physics and Chemistry, University of Oslo

Other

Organizing the Norwegian Chemistry Olympiad high-school competition (Norwegian Chemical Society) since 2004. Kjemiolympiaden

Tags: structural biology, biochemistry, protein crystallography, protein structure, diffraction, spectroscopy, Life sciences, Synchrotron, USA

Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). A Research-Inspired Biochemistry Laboratory Module - Combining Expression, Purification, Crystallization, Structure-Solving, and Characterization of a Flavodoxin-like Protein. Biochemistry and Molecular Biology Education. ISSN 1470-8175. 47(3), s 318- 332 . doi: 10.1002/bmb.21218 Full text in Research Archive.
Gudim, Ingvild; Hammerstad, Marta; Lofstad, Marie & Hersleth, Hans-Petter (2018). Characterization of different flavodoxin reductase-flavodoxin (FNR-Fld) interactions reveals an efficient FNR-Fld redox pair and identifies a novel FNR subclass. Biochemistry. ISSN 0006-2960. 57(37), s 5427- 5436 . doi: 10.1021/acs.biochem.8b00674 Full text in Research Archive.
Gudim, Ingvild; Lofstad, Marie; Van Beek, Wouter & Hersleth, Hans-Petter (2018). High-resolution crystal structures reveal a mixture of conformers of the Gly61-Asp62 peptide bond in an oxidized flavodoxin from Bacillus cereus. Protein Science. ISSN 0961-8368. 27(8), s 1439- 1449 . doi: 10.1002/pro.3436
Olsbu, Inger Kirstine; Zoppellaro, Giorgio; Andersson, Karl Kristoffer; Boucher, Jean-Luc & Hersleth, Hans-Petter (2018). Importance of Val567 on heme environment and substrate recognition of neuronal nitric oxide synthase. FEBS Open Bio. ISSN 2211-5463. 8(9), s 1553- 1566 . doi: 10.1002/2211-5463.12503 Full text in Research Archive.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST). Bio-protocol. ISSN 2331-8325. 7(8), s e2223 . doi: 10.21769/BioProtoc.2223 Full text in Research Archive.
Lofstad, Marie; Gudim, Ingvild; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2016). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus. Biochemistry. ISSN 0006-2960. 55(36), s 4998- 5001 . doi: 10.1021/acs.biochem.6b00699
Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). Crystal Structure of Bacillus cereus Class Ib Ribonucleotide Reductase Di-iron NrdF in Complex with NrdI. ACS Chemical Biology. ISSN 1554-8929. 9(2), s 526- 537 . doi: 10.1021/cb400757h Show summary
Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y•) cofactor in their NrdF (β2) subunit to initiate ribonucleotide reduction in the NrdE (α2) subunit. Contrary to the diferric tyrosyl radical (FeIII2-Y•) cofactor, which can self-assemble from FeII2-NrdF and O2, generation of the MnIII2-Y• cofactor requires the reduced form of a flavoprotein, NrdIhq, and O2 for its assembly. Here we report the 1.8 Å resolution crystal structure of Bacillus cereus Fe2-NrdF in complex with NrdI. Compared to the previously solved Escherichia coli NrdI-MnII2-NrdF structure, NrdI and NrdF binds similarly in Bacillus cereus through conserved core interactions. This protein–protein association seems to be unaffected by metal ion type bound in the NrdF subunit. The Bacillus cereus MnII2-NrdF and Fe2-NrdF structures, also presented here, show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of one of the metal-coordinating carboxylates is linked to the metal type present at the dimetal site and not associated with NrdI-NrdF binding. This carboxylate conformation seems to be vital for the water network connecting the NrdF dimetal site and the flavin in NrdI. From these observations, we suggest that metal-dependent variations in carboxylate coordination geometries are important for active Y• cofactor generation in class Ib RNRs. Additionally, we show that binding of NrdI to NrdF would structurally interfere with the suggested α2β2 (NrdE-NrdF) holoenzyme formation, suggesting the potential requirement for NrdI dissociation before NrdE-NrdF assembly after NrdI-activation. The mode of interactions between the proteins involved in the class Ib RNR system is, however, not fully resolved.
Rackwitz, Sergej; Faus, Isabelle; Schmitz, Markus; Kelm, Harald; Krüger, Hans-Jörg; Andersson, K. Kristoffer; Hersleth, Hans-Petter; Achterhold, Klaus; Schlage, Kai; Wille, Hans-Christian; Schünemann, Volker & Wolny, Juliusz A. (2014). A new sample environment for cryogenic nuclear resonance scattering experiments on single crystals and microsamples at P01, PETRA III. Hyperfine Interactions. ISSN 0304-3843. 226(1-3), s 673- 678 . doi: 10.1007/s10751-013-0981-8 Show summary
In order to carry out orientation dependent nuclear resonance scattering (NRS) experiments on small single crystals of e.g. iron proteins and/or chemical complexes but also on surfaces and other micrometer-sized samples a 2-circle goniometer including sample positioning optics has been installed at beamline P01, PETRA III, DESY, Hamburg. This sample environment is now available for all users of this beamline. Sample cooling is performed with a cryogenic gas stream which allows NRS measurements in the temperature range from 80 up to 400 K. In a first test this new sample environment has been used in order to investigate the orientation dependence of the nuclear inelastic scattering (NIS) signature of (i) a dinuclear iron(II) spin crossover (SCO) system and (ii) a hydrogen peroxide treated metmyoglobin single crystal.
Skråmo, Silje; Hersleth, Hans-Petter; Hammerstad, Marta; Andersson, K. Kristoffer & Røhr, Åsmund Kjendseth (2014). Cloning, expression, purification, crystallisation and preliminary X-Ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) from Bacillus cereus. Acta Crystallographica. Section F : Structural Biology and Crystallization Communications. ISSN 1744-3091. 70(No. 6. (May 2014)), s 777- 780 . doi: 10.1107/S2053230X14008334 Show summary
A ferredoxin/flavodoxin-NADP(H) oxidoreductase (FNR) from Bacillus cereus (Bc0385) belonging to the thioredoxin reductase-like class of FNRs, has been cloned, overexpressed, purified and crystallised. Diffraction data has been collected to 2.5 Å. Abstract [150 words] Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNR) are key enzymes involved in catalysing the electron transfer between ferredoxins/flavodoxins and NAD(P)H/NAD(P)+. In Bacillus cereus there are three genes that may encode FNRs, and the Bc0385 FNR has been cloned, overexpressed, purified and successfully crystallised in its NADPH/NADP+ free from. Diffraction data has been collected to 2.5 Å of crystals that belong to the orthorhombic space group P21212 with unit cell parameters a = 57.2, b = 164.3, c = 95.0 Å containing two FNR molecules in the asymmetric unit. The structure of the Bc0385 FNR has been solved by molecular replacement, and is a member of the homodimeric thioredoxin reductase-like class of FNRs.
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.
Zhao, Xiangbo; Hersleth, Hans-Petter; Zhu, Janan; Andersson, K. Kristoffer & Magliozzo, Richard S. (2013). Access channel residues Ser315 and Asp137 in Mycobacterium tuberculosis catalase-peroxidase (KatG) control peroxidatic activation of the pro-drug isoniazid. Chemical Communications. ISSN 1359-7345. 49(99), s 11650- 11652 . doi: 10.1039/c3cc47022a Show summary
Peroxidatic activation of the anti-tuberculosis pro-drug isoniazid by Mycobacterium tuberculosis catalase-peroxidase (KatG) is regulated by gating residues of a heme access channel. The steric restriction at the bottleneck of this channel is alleviated by replacement of residue Asp137 with Ser, according to crystallographic and kinetic studies.
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).
Hersleth, Hans-Petter & Andersson, K. Kristoffer (2011). How different oxidation states of crystalline myoglobin are influenced by X-rays. Biochimica et Biophysica Acta - Proteins and Proteomics. ISSN 1570-9639. 1814(6), s 785- 796 . doi: 10.1016/j.bbapap.2010.07.019
Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2011). ESRF Higlights 2010. Consequences of using X-rays when studying redox cofactors. ESRF Newsletter. ISSN 1011-9310. 2010, s 100- 101

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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.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2019). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; van Beek, Wouter & Hersleth, Hans-Petter (2019). New structural insight into the well known peptide flip observed in flavodoxins.
Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). Characterization of Proteins in the Ribonucleotide Reductase RedoxNetwork.
Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2019). Enzyme Activation by a Flavoprotein Redox Network.
Hammerstad, Marta; Zaltariov, Mirela F.; Arabshahi, Homayon John; Jovanovic, Katarina; Richter, Klaus W.; Cazacu, Maria; Shova, Sergiu; Balan, Mihaela; Radulovic, Sinisa; Reynisson, Jóhannes; Andersen, Niels Højmark; Hersleth, Hans-Petter; Andersson, K. Kristoffer & Arion, Vladimir B. (2019). New thiosemicarbazone derivatives and their copper(II) complexes as potential inhibitors against mammalian ribonucleotide reductase.
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.
Chaudhari, Sujata; Olsbu, Inger Kirstine; Alqarzaee, Abdulelah; Singh, Ryan; Schulze, Thomas; Zimmerman, Matthew; Hersleth, Hans-Petter & Thomas, Vinai Chittezham (2018). Endogenous nitric oxide synthase (NOS) activity reduces staphylococcal lifespan during stationary phase.
Gudim, Ingvild; Hersleth, Hans-Petter; Hammerstad, Marta & Sørlie, Morten (2018). Characterisation of flavodoxin and ferredoxin/flavodoxin reductases from Bacillus cereus and their interactions. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. 2000.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network. NBS-nytt. ISSN 0801-3535. s 40- 40
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2018). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus.
Olsbu, Inger Kirstine; Chaudhari, Sujata; Thomas, Vinai Chittezham & Hersleth, Hans-Petter (2018). Potential reductase partner for Staphylococcus aureus Nitric Oxide Synthase.
Olsbu, Inger Kirstine; Chaudhari, Sujata; Thomas, Vinai Chittezham & Hersleth, Hans-Petter (2018). Potential reductase partner for Staphylococcus aureus Nitric Oxide Synthase. NBS-nytt. ISSN 0801-3535. s 40- 40
Olsbu, Inger Kirstine; Hersleth, Hans-Petter & Sørlie, Morten (2018). Substrate recognition and redox partner identification in nitric oxide synthases. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. 2047.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Enzyme activation by a flavoprotein redox network.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class IB ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib RNR by a flavodoxin reductase in B. cereus. NSB-nytt. s 105- 105
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). A research-inspired biochemistry laboratory module – Combining, expression,purification, crystallisation, structure solving and characterisation of a flavdoxin-like protein.. NBS-nytt. ISSN 0801-3535. s 63- 63
Hammerstad, Marta; Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter; Zaltariov, Mirela-Fernanda; Arion, Vladimir B; Solomon, Edward I. & Andersson, K. Kristoffer (2017). INHIBITION OF CLASS 1A RIBONUCLEOTIDE REDUCTASE, AND A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE. Show summary
INHIBITION OF CLASS 1A RIBONUCLEOTIDE REDUCTASE, AND A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE Marta Hammerstad1*; Marie Lofstad1*; Lars H. Böttger2*; Åsmund Kjendseth Røhr3; Hans-Petter Hersleth1; Mirela F. Zaltariov4; Vladimir B. Arion4; Edward I. Solomon2 and K. Kristoffer Andersson1 1Department of Biosciences, University of Oslo, Pb.1066 Blindern, NO-0316 Oslo, Norway 2Department of Chemistry, Stanford University, Stanford, CA 94305, USA 3Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, NO-1432 Ås, Norway 4Institute of Inorganic Chemistry, Universität Wien, AT-1090 Vienna, Austria *These three authors contributed equally Presenting Author’s e-mail address: k.k.andersson@ibv.uio.no Ribonucleotide reductases (RNRs) are enzymes that convert RNA building blocks into DNA building blocks [1]. The reductive reaction by RNRs requires a cysteine thiyl radical, which, in the case of class Ia or Ib RNRs, is initiated by an FeIII2- or MnIII2-tyrosyl radical (Y•) cofactor in the R2 subunit of RNR. During enzymatic turnover, the cofactor is activated by oxygen, and generates a Y• that is transported from the smaller R2 subunit to the large catalytic subunit R1 of RNR, where DNA building blocks are formed. The small subunit of class Ia RNRs can be inhibited by several small compounds [2], through the inhibition of the active FeIII2- Y• cofactor. We have performed interaction studies and Kd measurements of a mammalian R2 protein with several newly synthesized compounds, and studied their potential inhibitory effect on the protein with EPR, showing promising results. Manganese catalase (MnCAT) enzymes [3] contain an active site that is similar in structure to the MnIII2 form of NrdF (the R2 subunit in class Ib RNR), characterized by a carboxylate-bridged MnIII-O-MnIII cofactor. However, it catalyzes a different reaction – the degradation of hydrogen peroxide to dioxygen and water. A still unresolved question is how these enzymes containing similar active sites can catalyze different reactions. A variety of spectroscopic methods have been used to try to resolve this question. Samples containing NrdF with active MnIII-O-MnIII cofactor have been prepared and studied by circular dichroism (CD) and magnetic CD (MCD) spectroscopy. The data show both similar and distinct features as compared to MnCAT. 1. A.B. Tomter; G. Zoppelaro; N.H. Andersen; H.-P. Hersleth; M. Hammerstad; Å.K. Røhr; G.K. Sandvik; G.E. Nilsson; C.B. Bell; A.L. Barra; E. Blasco; L. Le Pape; E.I. Solomon; and K.K. Andersson. Coord. Chem. Rev. (2013), 257, 3 2. A. Popovic-Bijelic A; C.R. Kowol; M.E.S. Lind; J. Luo; F. Himo; A.E. Enyedy; V.B. Arion; and A. Gräslund. (2011) J. Inorg. Biochem. 105, 1422-1431 3. T.C. Brunold; D.R. Gamelin; T.L. Stemmler; S.K. Mandal; W.H. Armstrong; J.E. Penner-Hahn; and E. I. Solomon. J. Am. Chem. Soc. (1998), 120, 8724
Hersleth, Hans-Petter (2017). A research-inspired biochemistry laboratory module – Combining, expression,purification, crystallisation, structure solving and characterisation of a flavdoxin-like protein..
Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus.
Hersleth, Hans-Petter (2017). Combining Protein X-ray Crystallography and in situ Single- Crystal UV-Vis and Raman Spectroscopy to Grasp the True Structure of Haem- and Flavoproteins.
Johannesen, Hedda; Cumar, Rohit; Hammerstad, Marta; Hersleth, Hans-Petter; Logan, Derek & Andersson, Karl Kristoffer (2017). Investigation of NrdD and NrdG from the two Firmicutes Bacillus cereus Lactococcus lactis.
Johannesen, Hedda; Hammerstad, Marta; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III.
Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III in Bacillus cereus.
Johannesen, Hedda; Kumar, Rohit; Hersleth, Hans-Petter; Hammerstad, Marta; Logan, Derek & Andersson, Karl Kristoffer (2017). A STRUCTURAL AND FUNCTIONAL INVESTIGATION OF RIBONUCLEOTIDE REDUCTASE CLASS III IN BACILLUS CEREUS.
Lofstad, Marie; Andersson, K. Kristoffer; Hersleth, Hans-Petter; Hammerstad, Marta & Kjendseth, Åsmund Røhr (2017). Activation Pathways of the Class Ib Ribonucleotide Reductase in Bacillus cereus. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. 1808.
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.
Olsbu, Inger Kirstine; Lombard, M; Hersleth, Hans-Petter; Boucher, Jean-Luc & Andersson, K. Kristoffer (2017). Key role of Val-567 on L-Arg analogues and heme ligands to neuronal nitric oxide syntase.
Shoor, Marita; Gudim, Ingvild; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Structural and functional characterization of redox proteins in an enzyme activating network involving the thioredoxin reductase in Bacillus cereus.
Shoor, Marita; Hersleth, Hans-Petter; Gudim, Ingvild & Hammerstad, Marta (2017). Structural and functional characterization of the redox protein Thioredoxin reductase from Bacillus cereus.
Gudim, Ingvild; Lofstad, Marie; Andersson, K. Kristoffer; Hammerstad, Marta & Hersleth, Hans-Petter (2016). Probing enzyme activation networks ‐ structural and functional studies of flavoproteins in Bacillus cereus. NBS-nytt. ISSN 0801-3535. s 45- 45
Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2016). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus.
Hammerstad, Marta; Hersleth, Hans-Petter; Lofstad, Marie; Johannesen, Hedda; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2016). Structural Insight into the Function of Ribonucleotide Reductase.
Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2016). Structural Insight into the Function of Ribonucleotide Reductase. NBS-nytt. ISSN 0801-3535. s 64- 64
Hersleth, Hans-Petter (2016). Probing enzyme activation networks - structural and functional studies of flavoproteins in Bacillus cereus.
Hersleth, Hans-Petter (2016). Probing enzyme activation networks - structural and functional studies of flavoproteins in Bacillus cereus.
Hersleth, Hans-Petter (2016). Probing enzyme activation networks ‐ structural and functional studies of flavoproteins in Bacillus cereus.
Hersleth, Hans-Petter; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr; Van beek, Wouter; Giullaume, Pompidor; Mehmet, Can; Xiangbo, Zhao; Magliozzo, Richard s.; Bren, Kara L & Andersson, K. Kristoffer (2016). Grasping the True Structure of Haem- and Flavoproteins – Combining X-RAY Crystallography and In Situ Single-Crystal UV-VIS and Raman Spectroscopy.
Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A Structural and Functional Investigation of Ribonucleotide Reductase Class III In Bacillus Cereus.
Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A structural and functional investigation of Ribonucleotide reductase Class III in Bacillus cereus. NBS-nytt. ISSN 0801-3535. s 65- 65
Johannesen, Hedda; Hersleth, Hans-Petter; Logan, Derek; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A structural and functional investigation of Ribonucleotide reductase Class III in Bacillus cereus- Investigating the interaction and mechanism of the NrdD and NrdG complex.
Johannesen, Hedda; Hersleth, Hans-Petter; Logan, Derek; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A structural and functional investigation of Ribonucleotide reductase Class III in Bacillus cereus- Investigating the interaction and mechanism of the NrdD and NrdG complex.
Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter; Hammerstad, Marta; Solomon, Edward I. & Andersson, K. Kristoffer (2016). A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE BY CD AND MCD SPECTROSCOPY.
Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Hammerstad, Marta; Solomon, Edward I. & Andersson, K. Kristoffer (2016). A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy. NBS-nytt. ISSN 0801-3535. s 45- 45
Lofstad, Marie; Gudim, Ingvild; Kjendseth, Åsmund Røhr; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2016). Activation of Class Ib Ribonucleotide Reductase by NRDI and Its Reductase Partner in Bacillus cereus.
Lofstad, Marie; Gudim, Ingvild; Van beek, Wouter; Pompidor, Guillaume; Kjendseth, Åsmund Røhr; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2016). Radiation damage of haem- and flavoproteins – combining X-ray crystallography and single-crystal UV-Vis and Raman spectroscopy.
Lofstad, Marie; Gudim, Ingvild; Van beek, Wouter; Pompidor, Guillaume; Kjendseth, Åsmund Røhr; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2016). Radiation damage of haem- and flavoproteins –combining X-ray crystallography and single-crystal UV-Vis and Raman spectroscopy. 9th International Workshop on X-ray Radiation Damage to Biological Crystalline Samples.
Olsbu, Inger Kirstine; Murielle, Lombard; Hersleth, Hans-Petter; Jean-Luc, Boucher & Andersson, K. Kristoffer (2016). Key Role of VAL567 on L-Argenine Analogues and HEME Ligands Binding to Neuronal Nitric Oxide Synthase.
Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structure of a ferredoxin/flavodoxin-NADP(H) oxidoreductase in Bacillus cereus.. NBS-nytt. ISSN 0801-3535. s 96- 96
Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structure of a ferredoxin/flavodoxin-NADP(H) oxidoreductase in Bacillus cereus..
Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structures of ferredoxin/flavodoxin-NADP(H) oxidoreductases in Bacillus cereus.
Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structures of ferredoxin/flavodoxin-NADP(H) oxidoreductases in Bacillus cereus.. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. A71, s s205- s205 . doi: 10.1107/S2053273315096916
Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2015). Structural Insight into the Function of Ribonucleotide Reductase. NBS-nytt. ISSN 0801-3535. s 47- 47 Show summary
Structural Insight into the Function of Ribonucleotide Reductase Marta Hammerstad*, Hans-Petter Hersleth*, Ane B. Tomter*, Åsmund K. Røhr*, and K. Kristoffer Andersson* *Department of Biosciences, University of Oslo, Oslo, Norway Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y•) cofactor in their NrdF (2) subunit for ribonucleotide reduction in the NrdE (2) subunit. Contrary to the diferric tyrosyl radical (FeIII2-Y•) cofactor, which can self-assemble from FeII2-NrdF and O2, generation of the MnIII2-Y• cofactor requires the reduced form of a flavoprotein, NrdIhq, and O2 for its assembly. Here we report the 1.8 Å resolution crystal structure of Bacillus cereus Fe2-NrdF in complex with NrdI. Compared to the Escherichia coli NrdI-MnII2-NrdF structure, NrdI and NrdF binds similarly in B. cereus through conserved interactions. In addition to the B. cereus NrdI-Fe2-NrdF structure, the MnII2-NrdF and Fe2-NrdF structures show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of a metal-coordinating carboxylate seems to be is linked to the metal type. This carboxylate conformation is likely vital for the NrdF di-metal site and the flavin. We also have new results regarding the Mycobacterium tuberculosis class Ib RNR. References Hammerstad M, et al., ACS Chem. Biol. 9 (2), 526–537 (2014) Tomter AB, et al., Coord. Chem. Rev. 257, 3-26 (2013) Hammerstad M., et al., J. Biol. Inorg. Chem. 19, 893-902 (2014) Andersson KK (Ed.) Ribonucleotide reductase, Nova Science Publishers, ISBN: 978-1-60456-199-9 (2008) Røhr AK, et al., Angew. Chem. Int. Ed. 49, 2324-2327 (2010) Boal AK, et al., Science. 329, 1526 (2010)
Hersleth, Hans-Petter (2015). Combining X-ray crystallography and in situ single-crystal UV-Vis and Raman spectroscopy to study redox proteins.
Hersleth, Hans-Petter & Røhr, Åsmund Kjendseth (2015). Teaching a general molecular bioscience master course as a project ”From gene to structure”..
Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Van beek, Wouter; Pompidor, Guillaume & Andersson, K. Kristoffer (2015). Combining X-ray crystallography and in situ single-crystal UV-Vis and Raman spectroscopy to study haem- and flavoproteins.. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. A71, s s489- s489
Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Van beek, Wouter; Pompidor, Guillaume & Andersson, K. Kristoffer (2015). Combining X-ray crystallography and in situ single-crystal UV-Vis and Raman spectroscopy to study redox proteins. http://www.esrf.eu/files/live/sites/www/files/UsersAndScience/Experiments/CRG/BM01/SNSeminar/Program.pdf. 1, s 2- 2
Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hammerstad, Marta; Hersleth, Hans-Petter; Solomon, Edward I. & Andersson, K. Kristoffer (2015). A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy.
Monka, Susanne; Andersson, K. Kristoffer; Hersleth, Hans-Petter & Hammerstad, Marta (2015). Structural and functional characterisation of ferredoxins in Bacillus cereus.
Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2015). On the way to determine the structure of a ferredoxin in B. cereus. Show summary
Ferredoxins are proteins responsible for the transfer of electrons from NADPH dependent ferredoxin reductases to different enzymes in bacteria that need electrons for activation. Up to now two genes for ferredoxins have been identified in B. cereus: the bcBC 2795 gene is assumed to code for a ferredoxin with a 2Fe-2S cluster as co-factor and BCbc 1483 for a 4Fe-4S cluster carrying ferredoxin. The 2Fe-2S ferredoxin gene was cloned into a pET-22b vector and overexpressed in E. coli. The purification procedure involved ammonium sulphate precipitation, ion exchange chromatography using Q Sepharose or DEAE columns, and size-exclusion chromatography with a Superdex 75 column. The protein detection with UV-Vis spectroscopy (A280 nm) presented a challenge due to the absence of tryptophan and tyrosine residues. Therefore, the protein concentration was determined by Bradford assays. The faint yellowish colour of the purified protein (106 amino acids, 11.4 kDa) indicated the presence of an apoprotein. The crystallization screening with JCSG+ Suite resulted in several hits, of which X-ray diffraction data was collected to 2.3 Å. Solving the structure by molecular replacement has so far been unsuccessful, possibly due to a large unit cell of 220 x 220 x 220 Å3. Further studies are going to involve the reconstitution of iron-sulfur clusters, further characterization and screening for new crystallization conditions.
Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Van Beek, Wouter; Diadkin, Vadim; Wiker, Geir; Chernyshov, Dmitry & Andersson, K. Kristoffer (2015). Combining Protein X-Ray Crystallography and Single-Crystal Spectroscopy - A New in situ Setup at BM01A at ESRF. Show summary
1Department of Biosciences, Section for Biochemistry and Molecular Biology, University of Oslo, Norway 2Swiss-Norwegian Beam Lines, European Synchrotron Radiation Facility, Grenoble, France E-mail: h.p.hersleth@ibv.uio.no Redox proteins are essential for all organisms, with functions ranging from substrate oxidation to respiration and photosynthesis. Central to many of these proteins are redox active cofactor as haems, iron-sulfur clusters, flavins, disulfides, quionens or NADPH. To be able to understand the structure-function relation of these proteins, structural studies are performed with X-ray diffraction. However, for redox proteins the crystal structures are missing key information as oxidation state, protonation or spin state. These informations can be essential for understanding the reaction mechanisms of these proteins. Therefore, a combination of X-ray diffraction and spectroscopic methods like UV-vis and Raman spectroscopy is vital to obtain a deeper understanding of these redox proteins. Additionally, the redox sites are very labile for X-ray induced radiation damage and reduction during crystallographic data collection at synchrotrons. To determine the redox state of the crystals as well as monitoring potential radiation damage, we have combined the X-ray diffraction setup at the Swiss-Norwegian Beam Lines BM01A (ESRF) with an in situ setup for measuring UV-vis and Raman spectroscopy. The centering and optimisation are performed manually, but the running of the combined setup performing alternating X-ray diffraction experiments and UV-vis and Raman spectroscopy can be programmed and performed in an automatic way.
Wu, Bernt; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2015). Structural and functional characterization of flavohemoglobin from Bacillus Cereus.
Wu, Bernt; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2015). Purification and characterization of Flavohemoglobin A flavoheme enzyme.
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
Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). Crystal Structure of B. cereus Class Ib Ribonucleotide Reductase NrdI-Fe2-NrdF Complex.
Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). Structural insight Into the Function of Ribonucleotide Reductase.
Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). Structure of B. cereus Class Ib Ribonucleotide Reductase NrdI-Fe2-NrdF Complex. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. 70, s C434- C434 Show summary
Structure of B. cereus Class Ib Ribonucleotide Reductase NrdI-Fe2-NrdF Complex M. Hammerstad1, H. Hersleth1, A. Tomter1, Å. Røhr1, K. Andersson1 1University of Oslo, Department of Biosciences, Oslo, Norway Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y•) cofactor in their NrdF (beta2) subunit to initiate ribonucleotide reduction in the NrdE (beta2) subunit. Contrary to the diferric tyrosyl radical (FeIII2-Y•) cofactor, which can selfassemble from FeII2-NrdF and O2, generation of the MnIII2-Y• cofactor requires the reduced form of a flavoprotein, NrdIhq, and O2 for its assembly. Here we report the 1.8 Å resolution crystal structure of Bacillus cereus Fe2-NrdF in complex with NrdI. Compared to the previously solved Escherichia coli NrdI-MnII2-NrdF structure, NrdI and NrdF binds similarly in Bacillus cereus through conserved core interactions. This protein-protein association seems to be unaffected by metal ion type bound in the NrdF subunit. The Bacillus cereus MnII2-NrdF and Fe2-NrdF structures, also presented here, show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of one of the metal-coordinating carboxylates is linked to the metal type present at the di-metal site, and not associated with NrdI-NrdF binding. This carboxylate conformation seems to be vital for the water network connecting the NrdF di-metal site and the flavin in NrdI. From these observations, we suggest that metal-dependent variations in carboxylate coordination geometries are important for active Y• cofactor generation in class Ib RNRs. Additionally, we show that binding of NrdI to NrdF would structurally interfere with the suggested alfa2beta2 (NrdE-NrdF) holoenzyme formation, suggesting the potential requirement for NrdI dissociation before NrdE-NrdF assembly after NrdI-activation. The mode of interactions between the proteins involved in the class Ib RNR system is, however, not fully resolved. [1] [1] M. Hammerstad, H.-P. Hersleth, A.B.Tomter et al, ACS Chem. Biol., 2014, DOI: 10.1021/cb400757h
Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). The Bacillus cereus class Ib ribonucleotide reductase NrdIFe2- NrdF protein complex structure favors a metaldependent conformational basis for cofactor activation.
Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). The Bacillus cereus class lb ribonucleotide reductase Nrdl-Fe-2-NrdF protein complex structure favors a metal-dependent conformational basis for cofactor activation. Journal of Biological Inorganic Chemistry. ISSN 0949-8257. 19, s S268- S268
Hersleth, Hans-Petter; Xhao, Xiahong; Magliozzo, Richard S. & Andersson, K. Kristoffer (2014). Structural insight into the function and anti-TB pro-drug activation by KatG. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. 70, s C707- C707
Lofstad, Marie; Gudim, Ingvild; Skråmo, Silje; Hammerstad, Marta; Røhr, Åsmund Kjendseth; Tomter, Ane Berg; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). Crystallisation of ferredoxin/flavodoxin-NADP(H) oxidoreductases, flavodoxins, ferredoxins and redox partners in Bacillus cereus. Show summary
Crystallisation of ferredoxin/flavodoxin-NADP(H) oxidoreductases, flavodoxins, ferredoxins and redox partners in Bacillus cereus Lofstad M1, Gudim I1, Skråmo S1, Hammerstad, M1, Røhr ÅK1, Tomter AB1, Andersson KK1, Hersleth H-P1 1Depatment of Biosciences, Section for Biochemistry and Molecular Biology, University of Oslo, Oslo, Norway Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNRs) catalyses the reversible redox reaction between ferredoxins (Fds) or flavodoxins (Flds) and NAD(P)+/NAD(P)H. In oxygenic photosynthetic organisms FNRs catalyse the reduction of NADP+ by photosynthetically reduced Fd, while in many heterotrophs, FNRs catalyse the NADPH-dependent reduction of Fd/Fld to provide subsequently reducing power to different Fd/Fld-dependent enzyme systems. In Bacillus cereus there are three annotated FNRs that can be redox partners for three Flds and two Fds. We have without tags purified and crystallised several of these redox proteins in Bacillus cereus, and some of their redox partners. A goal of the project is to understand the recognition, selectivity and flexibility of these FNRs, Flds and Fds with respect to each other and their redox partners. References Skråmo S, et al., Acta Cryst. F70, 777-780 (2014) Hammerstad M, et al., ACS Chem. Biol. 9, 526-537 (2014) Røhr ÅK, et al., Angew. Chem. Int. Ed. 49, 2324-2327 (2010)
Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2014). Nitric oxide synthase and possible redox partners in Bacillus cereus. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. 70, s C1657- C1657 Show summary
Nitric oxide synthase and possible redox partners in Bacillus cereus M. Lofstad1, H. Hersleth1, Å. Røhr1, M. Hammerstad1, K. Andersson1 1University of Oslo, Department of Biosciences, Oslo, Norway Nitric oxide synthase (NOS), a BH4-dependent heme-enzyme, is the only enzyme that specifically produces NO in mammals. NO is produced by the NOS homodimer in two multistep reaction cycles involving electron transfer from a reducing domain to the heme active site. The importance of NO in mammals is due to its function in signalling, vasodilation and immune response. Some bacterial species also contain NOS-encoding genes, but these bacterial NOSs are differently organized – they contain no reducing domain – and their functions and mechanism are not fully resolved [1]. Bacterial NOSs are potential drug targets, because of their role in protection against antibiotics and oxidative stress in some pathogenic bacterial species (e.g. Bacillus anthracis) [2]. Flavodoxins (Flds) have been shown to be relevant redox partners for bacterial NOSs [3], but the specificity of the interaction between NOS and Flds remains poorly understood. We have investigated the NOS protein system in Bacillus cereus, whose genome encodes NOS and two Flds, by combining crystallographic and spectroscopic methods. So far the structures of the two Flds have been solved to 0.98 Å and 2.75 Å resolution, while NOS has been solved to 2.9 Å resolution. An important part of the study has been to investigate the effect of synchrotron X-ray radiation on the oxidation state and structure of the Flds, due to their radiation sensitive cofactor flavin mononucleotide (FMN). The high-resolution (0.98 Å), oxidized structure of one Fld indicates that X-rays induce structural changes around the FMN cofactor. Another important part of the study has been to gain further insight into the specificity and flexibility of the interactions between ferredoxin/flavodoxin-NADP+ reductases, Flds and NOS in Bacillus cereus, as well as the possible mechanism of bacterial NOSs. [1] J. Sudhamsu, B. Crane, Trends in microbiology, 2009, 17, 212-218, [2] I. Gusarov, K. Shatalin, M. Starodubtseva et al, Science, 2009, 325, 1380- 1384, [3] Z. Wang, R. Lawson, M. Buddha et al, Journal of Biological Chemistry, 2007, 282, 196- 202 http://www.eiseverywhere.com/image.php?acc=4087&id=283682 Keywords: Nitric oxide synthase, Flavodoxin, Radiation damage
Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2014). Nitric oxide synthase and possible redox partners in Bacillus cereus. Journal of Biological Inorganic Chemistry. ISSN 0949-8257. 19, s S265- S265
Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). On the way to determine the structure of a ferredoxin in B. cereus.
Rackwitz, Sergej; Faus, Isabelle; Schmitz, Markus; Krüger, Hans-Jörg; Andersson, K. Kristoffer; Hersleth, Hans-Petter; Achterhold, Klaus; Schlage, Kai; Wille, Hans-Christian; Wolny, Juliusz A. & Schünemann, Volker (2014). Nuclear resonance vibrational spectroscopy on iron protein single crystals.
Skråmo, Silje; Lofstad, Marie; Monka, Susanne; Hammerstad, Marta; Røhr, Åsmund Kjendseth; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). Structural studies of ferredoxin/ﬂavodoxin-NADPH reductases, ﬂavodoxins, ferredoxins and redox partners in B. cereus.
Wu, Bernt; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). Structural and functional characterization of flavohemoglobin from Bacillus Cereus.
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).
Hammerstad, Marta; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2013). Crystal Structure of the B. cereus Class Ib Ribonucleotide Reductase NrdF Subunit in Complex with the Flavoprotein NrdI. NBS-nytt. ISSN 0801-3535. 1, s 49- 49
Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2013). The Bacillus cereus class Ib ribonucleotide reductase NrdI-Fe2-NrdF protein complex structure favors a metal-dependent conformational basis for cofactor activation.
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 (2013). Grasping the Nature of Compound II in Myoglobin by Combining X-Ray Diffraction and in-situ Light Absorption and Raman Spectroscopy.
Hersleth, Hans-Petter (2013). Protein Crystal Spectroscopy.
Hersleth, Hans-Petter (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.
Hersleth, Hans-Petter (2013). What to be aware of when solving the structure of redoxproteins?.
Hersleth, Hans-Petter; Lofstad, Marie; Røhr, Åsmund Kjendseth; 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 structure of haem and flavoproteins.
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.
Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Lofstad, Marie; Skråmo, Silje; Van beek, Wouter; Pompidor, Guillaume & Andersson, K. Kristoffer (2013). What to be aware of when solving the structure of redoxproteins?. NBS-nytt. ISSN 0801-3535. 1, s 49- 49
Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Nitric oxide synthase and possible redox partners in Bacillus cereus.
Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Nitric oxide synthase and possible redox partners in Bacillus cereus.
Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Structural and functional studies of nitric oxide synthase and flavodoxins in Bacillus cereus. NBS-nytt. ISSN 0801-3535. 1, s 84- 84 Show summary
Abstract P6, poster Structural and functional studies of nitric oxide synthase and flavodoxins in Bacillus cereus Marie Lofstad1, Hans-Petter Hersleth1, Åsmund Kjendseth Røhr1, Marta Hammerstad1 and K. Kristoffer Andersson1 1Department of Molecular Biosciences, University of Oslo, PO Box 1041, Blindern, Oslo, Norway Nitric oxide synthase (NOS) is the only protein that specifically produces NO in mammals. The NO is produced in two multistep reaction cycles involving electron deliveries to the heme active site, and its importance is due to its function in signaling, vasodilation and immune response. Some bacterial species also contain NOS-encoding genes; however, these bacterial NOSs are differently organized and their functions are not fully understood. In this project we have investigated the NOS protein system in Bacillus cereus by combining crystallographic and spectroscopic methods. The genes encoding NOS and two flavodoxins – thought to transfer reduction equivalents to the catalytic site in NOS – from Bacillus cereus were cloned and the proteins were expressed and purified. Protein crystals were obtained from the three proteins and X-ray data was collected at synchrotrons, the resulting data being used to solve the structures of one flavodoxin (resolution 0.98 Å) and NOS (resolution 2.9 Å). The high-resolution structure of the flavodoxin, in addition to data from single-crystal spectroscopy experiments, suggested that the flavin cofactor of the flavodoxin was one-electron reduced to its semiquinone state during data collection, inducing a peptide flip of a glycine residue in the backbone of the protein. This effect of radiation has not been observed in the structure of another flavodoxin protein of Bacillus cereus. Last update: 2012-11-23 10:51:36
Schünemann, Volker; Rackwitz, Sergej; Faus, Isabelle; Wolny, Juliusz A.; Schmitz, Markus; Kelm, Harald; Krüger, Hans-Jörg; Andersson, K. Kristoffer; Hersleth, Hans-Petter; Achterhold, Klaus; Schlage, Kai & Wille, Hans-Christian (2013). A New Sample Environment for Cryogenic Nuclear Resonance Scattering Experiments on Single Crystals and Microsamples at P01, PETRA III.
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.
Hammerstad, Marta; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Högbom, Martin; Graslund, Astrid & Andersson, K. Kristoffer (2012). Studies of the class Ib ribonucleotide reductase system in Bacillus cereus and Mycobacterium tuberculosis. Show summary
Studies of the class Ib ribonucleotide reductase system in Bacillus cereus and Mycobacterium tuberculosis Hammerstad, M. a; Hersleth, H.-P. a; Røhr, Å. K. a; Högbom, M. b; Gräslund, A. b and Andersson, K. K a a Department of Molecular Biosciences, University of Oslo, Postboks 1041, Blindern, 0316 Oslo, Norway, marta.hammerstad@imbv.uio.no b Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius Väg 16C, SE-106 91, Stockholm, Sweden Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class I RNRs (further subdivided into class Ia, Ib and Ic) require a dinuclear metal cluster for employing a radical chemistry catalytic mechanism [1]. M. tuberculosis and B. cereus (or B. anthracis) both contain the class Ib RNR, and might require either a di-iron or di-manganese metallocofactor in their radical-generating subunits, R2F2 and R2F, respectively [2,3]. The RNR systems in both organisms include a variety of interacting proteins, such as a thioredoxin-like protein and a flavoprotein [4,5]. Biochemical, spectroscopic and structural studies of some of these proteins will be presented. Also, EPR studies indicate that a second, alternate subunit, R2F1, in M. tuberculosis might serve as an additional radical-generating subunit in its class Ib RNR. References [1] K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science Publishers, Inc. Hauppauge, N.Y. USA , ISBN: 978-1-60456-199-9 [2] A.B. Tomter, et al., Plos One. 2012, 7, e33436. [3] M. Crona, et al., J. Biol. Chem. 2011, 286, 33053 [4] A.B. Tomter, et al., Coordin. Chem. Rev. 2012, Submitted. [5] Å.K. Røhr, et al., Angew. Chem. Int. Ed. 2010, 49, 2324
Hammerstad, Marta; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Högbom, Martin; Graslund, Astrid & Andersson, K. Kristoffer (2012). Studies of the class Ib ribonucleotide reductase system in Bacillus cereus and Mycobacterium tuberculosis. Show summary
Studies of the class Ib ribonucleotide reductase system in Bacillus cereus and Mycobacterium tuberculosis M. Hammerstad1, H.-P. Hersleth1, Å.K. Røhr1, M. Högbom2, A. Gräslund2, K.K. Andersson1 1Department of Molecular Biosciences, University of Oslo, Oslo, Norway 2Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class I RNRs (further subdivided into class Ia, Ib and Ic) require a dinuclear metal cluster for employing a radical chemistry catalytic mechanism [1]. M. tuberculosis and B. cereus (or B. anthracis) both contain the class Ib RNR, and might requires either a di-iron or di-manganese metallocofactor in their radical-generating subunits, R2F2 and R2F, respectively [2,3]. The RNR systems in both organisms include a variety of interacting proteins, such as a thioredoxin-like protein and a flavoprotein [4]. Biochemical, spectroscopic and structural studies of some of these proteins will be presented here. Also, EPR studies indicate that a second, alternate subunit, R2F1, in M. tuberculosis might serve as an additional radical-generating subunit in its class Ib RNR. References [1] K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science, New York. [2] A.B. Tomter, et al. Plos One. 2012, 7,3,e33436. [3] M. Crona, et al. J. Biol. Chem. 2011, 286,38. [4] A.B. Tomter, et al. CCR. 2012, Submitted.
Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2012). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987.
Hersleth, Hans-Petter (2012). Radiation damage in haem and flavoproteins studied by in-situ single-crystal pectroscopy. Show summary
Radiation damage in haem and flavoproteins studied by in-situ single-crystal spectroscopy. Hans-Petter Hersleth,a Åsmund K. Røhr,a Wouter van Beek,b Guillaume Pompidor,c K. Kristoffer Andersson,a aDepartament of Molecular Biosciences, University of Oslo, Norway, bSwiss–Norwegian Beamlines at ESRF, France, cSwiss Light Source, Paul Scherrer Institute, Switzerland E-mail: h.p.hersleth@imbv.uio.no To be able to correctly interpret the crystal structure of redox- and metalloproteins caution must be employed. 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 seem especially vulnerable for radiation-induced reduction [1,2,3]. We have used in-situ (online) UV-vis and Raman spectroscopy to study how different haem and flavoproteins are influenced by X-rays during crystallographic data collection [1,2]. The spectroscopic changes have been monitored as a function of X-ray exposure (dose absorbed), Our studies show that these redox states are very fast reduced by X-rays resulting in very short lifedoses. Structurally we have observed for haem proteins a lengthening of the Fe-O bond, and for flavoproteins a bending of the flavin ring during X-ray induced radiation damage, in agreement with DFT [1,2,3]. We have recently started to investigate if varying the doserates and wavelengths can increase the lifedoses. In general our studies show the need for combining protein crystallography with in-situ single-crystal spectroscopy when redox and metalloproteins are studied. [1] Hersleth, H.-P. et al. (2011). Biochim. Biophys. Acta 1814, 785-796. [2] Røhr, Å. K. et al. (2010). Angew. Chem. Int. Ed. 49, 2324-2327. [3] Hersleth, H.-P. et al. (2008). Chem. Biodiv. 5, 2067-2089. Acknowledgements. Financial support by the Norwegian Research Council through projects 177661/V30 / 214239/F20 / 218412/F50 / 138370/V30 / 216625/F50 /. Keywords: protein crystallography; radiation damage; spectroscopy
Hersleth, Hans-Petter & Andersson, K. Kristoffer (2012). Combining X-Ray Diffraction and in-situ Single-Crystal UV-Vis and Raman Spectroscopy to Study Haem Proteins. Show summary
Combining X-Ray Diffraction and in-situ Single-Crystal UV-Vis and Raman Spectroscopy to Study Haem Proteins. Hans-Petter Hersleth & K. Kristoffer Andersson Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, 0316 Oslo 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 seem especially vulnerable for radiation-induced reduction. It is essential to correctly know the oxidation state of these metal sites in protein crystal structures 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 [1]. 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. These lifedoses are about 1000 times lower than the Henderson limit used for general radiation damage. Additionally, the higher oxidation states of myoglobin are not reduced to normal ferrous Fe2+ or ferric Fe3+ states, but end up in some intermediate state. One of the primary goal of the project has been to characterise and study the different intermediates in the reaction between myoglobin and peroxides [2,3]. The reaction intermediates generated appear biological relevant since myoglobin is proposed to function as scavenger of reactive oxygen species during oxidative stress [2]. 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 [3]. [1] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011, 1814, 785-796. [2] 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.. [3] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Biochem. J. 2008, 412, 257-264.
Hersleth, Hans-Petter & Andersson, K. Kristoffer (2012). How fast are the redox and metallo sites in protein crystal reduced by radiation damage during crystallographic data collection?.
Hersleth, Hans-Petter; Lofstad, Marie; Røhr, Åsmund Kjendseth; Van Beek, Wouter; Can, Mehmet; Zhao, X; Pompidor, Guillaume; Magliozzo, Richard S.; Bren, Kara L & Andersson, K. Kristoffer (2012). 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. Show summary
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. Hersleth, H.-P.a.; Lofstad, M.a.; Røhr, Å.K.a.; van Beek, W.b.; Can, M.c; Zhao, X.d.; Pompidor, G.e.; Magliozzo, R.S.d.; Bren, K.L.c.; Andersson, K.K.a. a Department of Molecular Biosciences, University of Oslo, P.O.Box 1041 Blindern, NO-0316 Oslo, Norway, h.p.hersleth@imbv.uio.no b Swiss–Norwegian Beamlines at ESRF, BP 220, 38043 Grenoble Cedex, France c Department of Chemistry, University of Rochester, New York 14627-0216, USA d Department of Chemistry, Brooklyn College, Brooklyn, New York 11210, USA e Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland To be able to correctly interpret the crystal structure of redox- and metalloproteins caution must be employed. 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 seem especially vulnerable for radiation-induced reduction [1,2,3]. We have used in situ (online) UV-vis and Raman spectroscopy to study how different haem and flavoproteins are influenced by X-rays during crystallographic data collection [1,2]. The spectroscopic changes have been monitored as a function of X-ray exposure (dose absorbed), Our studies show that these redox states are very fast reduced by X-rays resulting in very short lifedoses. Structurally we have observed for haem proteins a lengthening of the Fe-O bond, and for flavoproteins a bending of the flavin ring during X-ray induced radiation damage, in agreement with DFT [1,2,3]. We have recently started to investigate if varying the doserates and wavelengths can increase the lifedoses. In general our studies show the need of combining protein crystallography with in situ single-crystal spectroscopy when redox and metalloproteins are studied. Acknowledgements. Financial support by the Norwegian Research Council through projects 177661/V30 / 214239/F20 / 218412/F50 / 138370/V30 / 216625/F50 /. References [1] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011: 1814, 785 [2] Å.K. Røhr, H.-P. Hersleth, K.K. Andersson, Angew. Chem. Int. Ed. 2010, 49, 2324 [3] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Chem. Biodiv. 2008, 5, 2067
Hersleth, Hans-Petter; Lofstad, Marie; Van Beek, Wouter; Pompidor, Guillaume & Andersson, K. Kristoffer (2012). 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 proteins. Show summary
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 proteins. Hersleth, H.-P.a.; Lofstad, M.a.; van Beek, W.b.; Pompidor, G.e.; Andersson, K.K.a. a Department of Molecular Biosciences, University of Oslo, P.O.Box 1041 Blindern, NO-0316 Oslo, Norway, h.p.hersleth@imbv.uio.no b Swiss–Norwegian Beamlines at ESRF, BP 220, 38043 Grenoble Cedex, France e Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland To be able to correctly interpret the crystal structure of redox- and metalloproteins caution must be employed. 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 seem especially vulnerable for radiation-induced reduction [1,2,3]. We have used in situ (online) UV-vis and Raman spectroscopy to study how different haem and flavoproteins are influenced by X-rays during crystallographic data collection [1,2]. The spectroscopic changes have been monitored as a function of X-ray exposure (dose absorbed), Our studies show that these redox states are very fast reduced by X-rays resulting in very short lifedoses. Structurally we have observed for haem proteins a lengthening of the Fe-O bond, and for flavoproteins a bending of the flavin ring during X-ray induced radiation damage, in agreement with DFT [1,2,3]. We have recently started to investigate if varying the doserates and wavelengths can increase the lifedoses. In general our studies show the need of combining protein crystallography with in situ single-crystal spectroscopy when redox and metalloproteins are studied. Acknowledgements. Financial support by the Norwegian Research Council through projects 177661/V30 / 214239/F20 / 218412/F50 / 138370/V30 / 216625/F50 /. References [1] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011: 1814, 785 [2] Å.K. Røhr, H.-P. Hersleth, K.K. Andersson, Angew. Chem. Int. Ed. 2010, 49, 2324 [3] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Chem. Biodiv. 2008, 5, 2067
Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Van Beek, Wouter; Pompidor, Guillaume & Andersson, K. Kristoffer (2012). Radiation damage in haem and flavoproteins studied by in-situ single-crystal pectroscopy. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. 68A Show summary
Radiation damage in haem and flavoproteins studied by in-situ single-crystal spectroscopy. Hans-Petter Hersleth,a Åsmund K. Røhr,a Wouter van Beek,b Guillaume Pompidor,c K. Kristoffer Andersson,a aDepartament of Molecular Biosciences, University of Oslo, Norway, bSwiss–Norwegian Beamlines at ESRF, France, cSwiss Light Source, Paul Scherrer Institute, Switzerland E-mail: h.p.hersleth@imbv.uio.no To be able to correctly interpret the crystal structure of redox- and metalloproteins caution must be employed. 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 seem especially vulnerable for radiation-induced reduction [1,2,3]. We have used in-situ (online) UV-vis and Raman spectroscopy to study how different haem and flavoproteins are influenced by X-rays during crystallographic data collection [1,2]. The spectroscopic changes have been monitored as a function of X-ray exposure (dose absorbed), Our studies show that these redox states are very fast reduced by X-rays resulting in very short lifedoses. Structurally we have observed for haem proteins a lengthening of the Fe-O bond, and for flavoproteins a bending of the flavin ring during X-ray induced radiation damage, in agreement with DFT [1,2,3]. We have recently started to investigate if varying the doserates and wavelengths can increase the lifedoses. In general our studies show the need for combining protein crystallography with in-situ single-crystal spectroscopy when redox and metalloproteins are studied. [1] Hersleth, H.-P. et al. (2011). Biochim. Biophys. Acta 1814, 785-796. [2] Røhr, Å. K. et al. (2010). Angew. Chem. Int. Ed. 49, 2324-2327. [3] Hersleth, H.-P. et al. (2008). Chem. Biodiv. 5, 2067-2089. Acknowledgements. Financial support by the Norwegian Research Council through projects 177661/V30 / 214239/F20 / 218412/F50 / 138370/V30 / 216625/F50 /. Keywords: protein crystallography; radiation damage; spectroscopy
Hersleth, Hans-Petter; Van Beek, Wouter; Pompidor, Guillaume & Andersson, K. Kristoffer (2012). Using in-situ Spectroscopy to Study X-ray Induced Radiation Damage to Haem Protein Crystals. Show summary
Seventh International Workshop on X-ray Radiation Damage to Biological Crystalline Samples Diamond Light Source, UK, March 14th-16th 2012 Using in-situ Spectroscopy to Study X-ray Induced Radiation Damage to Haem Protein Crystals. P7, page 67 Hans-Petter Hersleth, Wouter van Beek, Guillaume Pompidor, K. Kristoffer Andersson The influence of X-ray radiation damage to protein crystals is well known, and redox active sites like metal sites seem especially vulnerable for radiation-induced reduction. It is essential to correctly know the oxidation state of these metal sites in protein crystal structures, to be able to interpret 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 in how fast they are "reduced" by the X-rays [1]. The higher oxidation states of myoglobin are not reduced to normal ferrous Fe2+ or ferric Fe3+ states, but end up in some intermediate states. One of the goals has been to characterise and study different intermediates in the reaction between myoglobin and peroxides [2,3]. For the so-called compound II intermediate (ferryl Fe4+O), we have shown by combining X-ray diffraction with in-situ UV-vis and Raman spectroscopy that the Fe-O bond increases during X-ray exposure and crystallographic data collection from a double-bond to a single-bond. The lifedose of this state have been estimated to be only ~0.02 MGy [1], and the effect of different dose-rates has been investigated. We have previously 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 compound II intermediate [3,4]. References [1] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011,1814, 785-796. [2] H.-P. Hersleth, et al., J. Biol. Chem. 2007, 282, 23372-23386. [3] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Biochem. J. 2008, 412, 257-264. [4] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Chem. Biodiv. 2008, 5, 2067-2089.
Lofstad, Marie; Hammerstad, Marta; Olsbu, Inger Kirstine; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2012). Structural and Functional Studies of Nitric Oxide Synthase and Flavodoxines in Bacillus cereus. Show summary
Structural and Functional Studies of Nitric Oxide Synthase and Flavodoxines in Bacillus cereus Marie Lofstad, Marta. Hammerstad, Inger .K. Olsbu, Åsmund .K. Røhr, Hand-Petter.-P. Hersleth and K. Kristoffer.K. Andersson Department of Molecular Biosciences, University of Oslo, PO Box 1041, Blindern, Oslo, Norway Marie Lofstad <marie.lofstad@imbv.uio.no> Nitric Oxide Synthase (NOS) is the enzyme that produces nitric oxide in the body from arginine and oxygen. In mammals three different NOS isoforms with different functions exist: endothelial eNOS, neuronal nNOS and inducible iNOS. The mammalian NOS consists of one oxygenase domain with heme/biopterin (NOSoxy), andone reductase domain (with FMN, FAD and NADPH) and can have a calmodulin domain . The reductase domain latter transports electrons to the heme. NOSoxy. Bacterial NOSs, on the other hand, consist only of an oxygenase domain, and separate proteins deliver the electrons to this domain. The possible functions of bacterial NOSs are not fully understood. We have studied a NOS-like protein from Bacillus cereus, BC5444, which is of interest since it is a close relative to the pathogen Bacillus anthracis. Like other bacterial NOSs, it is lacking a reductase domain; however, several possible reductase partners exist in the genome. Flavodoxines are most likely candidates, due to their capacity to deliver electrons through their co-factor flavin mononucleotide (FMN). Two such flavodoxines, BC1376 and BC3541, have been further investigated. How electrons are transferred by these two flavodoxines from a flavodoxin reductase to NOS, is currently not known. Another intriguing question, concerns the specificity of the flavodoxines – is one specific flavodoxin a more efficient electron donor for NOS than the other, or are they equally good? Different biochemical and structural techniques is are going to be applied in order to try to answer these questions. So far BC5444, BC3541 and BC1376 have been cloned, expressed, purified and crystallized, and the structure of the flavodoxin BC1365 has been solved at high resolution of 1Å using X-ray crystallography. Crystals of the flavodoxin BC3541 and the NOS BC5444 have diffracted to approximately 3Å. Supported by the national Ph.D. school, Biostruct.
Lofstad, Marie; Hammerstad, Marta; Olsbu, Inger Kirstine; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2012). Structural and spectroscopic characterization of the Nitric Oxide Synthase protein system in Bacillus cereus.
Lofstad, Marie; Hammerstad, Marta; Olsbu, Inger Kirstine; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2012). Structural studies of Nitric Oxide Synthase and Flavodoxines in Bacillus cereus.
Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Pompidor, Guillaume & Andersson, K. Kristoffer (2012). Monitoring flavin X-ray radiation damage using single crystal spectroscopy. Show summary
RøHR, Åsmund Kjendseth1; HERSLETH, Hans-Petter1; POMPIDOR, Guillaume2; ANDERSSON, K. Kristoffer1 1University of Oslo, Norway 2SLS, Paul Scherrer Institut, Switzerland Considering that more than one thousand structures in the Protein Data Bank contain flavin cofactors [1], it is of interest to verify the flavin geometry and electronic state when using these structures for deducing reaction mechanisms and when analyzing the conformational interplay between the cofactor and its protein scaffold. Inspecting flavin structure with QM/MM methods and monitoring of flavin vibrational modes with single-crystal spectroscopic methods during X-ray data collection provide important information regarding the actual flavin state. Here we present data collected from crystals of the flavoprotein NrdI, comparing high resolution crystal structures, geometry optimized models, and single crystal Raman spectra, showing that flavin geometry and state indeed are changed when exposed to Xray radiation [2]. References: [1] Senda, T., Senda, M., Kimura, S., Ishida, T. (2009) Antioxid.Redox Signal. 11, 1741-1766. [2] RøHR, A.K., Hersleth, H.-P., Andersson, K.K. (2010), Angew. Chem. Int. Ed. Engl. 49, 2324-2327
Skråmo, Silje; Hersleth, Hans-Petter; Hammerstad, Marta; Røhr, Åsmund Kjendseth; Tomter, Ane Berg & Andersson, K. Kristoffer (2012). Structural and spectroscopic studies of electron transport from FldR to the Ribonucleotide reductase system in B. cereus.
Skråmo, Silje; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2012). Studies of Electron Transfer from Flavodoxin Reductase to NrdI in Bacillus cereus.
Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2011). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987. Show summary
Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 M. Hammerstad1; Å. K. Røhr1; H. Hersleth1; A. B. Tomter1; K. K. Andersson1 1. University of Oslo, Oslo, Norway. Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication [1]. RNR was the first enzyme discovered to use thioredoxins (Trxs), small ubiquitous proteins containing a redox-active cysteine dithiol/cysteine disulfide. The oxidoreductases perform the fast and reversible thiol-disulfide exchange between their active site cysteines and cysteines in the substrate [2]. All Trx-like proteins share an overall α/β/α sandwich fold, in addition to the conserved C-X-X-C motif [3]. E. coli NrdH-redoxins, also described as the reductants of NrdE of bacterial class Ib RNR [4], have a different active site environment compared to the typical Trx [5]. A protein homologous to the NrdH-redoxins, BC3987, has been located in the B. cereus genome, showing higher activity as an electron donor for the Mn-cofactor form than the Fe-cofactor form of the class Ib RNR [6]. Sequence similarity between these two Trx-like proteins suggests a common catalytic mechanism, involving conserved Thr residues adjacent to the active site. These threonines are believed to influence the protonation state of the C-terminal Cys of the C-X-X-C motif [7]. The function and structure of BC3987 has been characterized. The crystal structures of mutant proteins have been solved using X-ray crystallography. Also, determinations of active site cysteine pKa values were performed, enhancing our understanding of the unusual catalytic mechanism regarding these Trx-like enzymes. References [1] K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science, New York. [2] D. Ritz, J. Beckwith, Annu. Rev. Microbiol . 2001, 55,21. [3] J.L. Martin, Nature. 1995. 3,245. [4] A. Jordan, F. Aslund, E. Pontis, P. Reichard, A. Holmgren, J. Biol. Chem. 1997, 272,18044. [5] G.B. Kallis, A. Holmgren, J. Biol. Chem. 1980, 255, 261. [6] A.B. Tomter, (2010) Ph.D. Thesis, University of Oslo. [7] Å.K. Røhr, (2010) Ph.D. Thesis, University of Oslo. Fig. Electron density map surrounding the BC3987 T53A mutant active site.
Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2011). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987. NBS-nytt. ISSN 0801-3535. s 111- 111 Show summary
Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 M. Hammerstad1, Å.K. Røhr1, H.-P. Hersleth1, A.B. Tomter1 and K.K. Andersson1 1Department of Molecular Biosciences, University of Oslo, Norway Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms [1,2]. RNR was the first enzyme discovered to use thioredoxins (Trxs), small ubiquitous proteins containing a redox-active cysteine dithiol/cysteine disulfide, for the reduction of its active site cysteines. These thiol-disulfide oxidoreductases perform the fast and reversible thiol-disulfide exchange between their active site cysteines and cysteines in the substrate protein [3]. All thioredoxin-like proteins share an overall // sandwich fold, in addition to the conserved C-X-X-C motif [4]. The fundamental reaction mechanism for electron transfer from Trx to its substrate was proposed by Kallis and Holmgren in 1980 [5]. E. coli NrdH-redoxins, also described as the reductants of NrdE of bacterial class Ib RNR [6], have a different active site environment compared to the typical Trx, although possessing Trx functionality. A protein homologous to the NrdH-redoxins, BC3987, has been located in the B. cereus genome [7], also possibly functioning as an electron donor for class Ib RNR [8]. Sequence similarity between these two Trx-like proteins suggests a common catalytic mechanism, involving a conserved threonine residue adjacent to the active site. This threonine is believed to influence the protonation state of the C-terminal Cys of the C-X-X-C motif [7]. The function and structure of BC3987 has been characterized using various biochemical techniques. The crystal structures of two mutant proteins have been solved using X-ray crystallography. Also, determinations of active site cysteine pKa values and redox potential determination of active site cysteine thiol/disulfide measurements were performed, enhancing our understanding regarding the unusual catalytic mechanism regarding these Trx-like enzymes. References [1] M. Kolberg, K.R. Strand, P.Graff, K.K. Andersson, Biochim. Biophys. Acta. 2004, 1699, 1. [2] K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science, New York. [3] D. Ritz, J. Beckwith, Annu. Rev. Microbiol . 2001, 55,21. [4] J.L. Martin, Nature. 1995. 3,245. [5] G.B. Kallis, A. Holmgren, J. Biol. Chem. 1980, 255, 261. [6] A. Jordan, F. Aslund, E. Pontis, P. Reichard, A. Holmgren, J. Biol. Chem. 1997, 272,18044. [7] Å.K. Røhr, K.K. Andersson, J. Biol. Chem (Submitted). 2010. [8] A.B. Tomter, G. Zoppelaro, C.B. Bell III, A.-L. Barra, N.H. Andersen, E.I. Solomon, K.K. Andersson, J. Biol. Chem (to be Submitted). 2011
Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2011). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987. Journal of Biological Inorganic Chemistry. ISSN 0949-8257. 16 . doi: 10.1007/s00775-011-0862-z Show summary
Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 M. Hammerstad1; Å. K. Røhr1; H. Hersleth1; A. B. Tomter1; K. K. Andersson1 1. University of Oslo, Oslo, Norway. Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication [1]. RNR was the first enzyme discovered to use thioredoxins (Trxs), small ubiquitous proteins containing a redox-active cysteine dithiol/cysteine disulfide. The oxidoreductases perform the fast and reversible thiol-disulfide exchange between their active site cysteines and cysteines in the substrate [2]. All Trx-like proteins share an overall α/β/α sandwich fold, in addition to the conserved C-X-X-C motif [3]. E. coli NrdH-redoxins, also described as the reductants of NrdE of bacterial class Ib RNR [4], have a different active site environment compared to the typical Trx [5]. A protein homologous to the NrdH-redoxins, BC3987, has been located in the B. cereus genome, showing higher activity as an electron donor for the Mn-cofactor form than the Fe-cofactor form of the class Ib RNR [6]. Sequence similarity between these two Trx-like proteins suggests a common catalytic mechanism, involving conserved Thr residues adjacent to the active site. These threonines are believed to influence the protonation state of the C-terminal Cys of the C-X-X-C motif [7]. The function and structure of BC3987 has been characterized. The crystal structures of mutant proteins have been solved using X-ray crystallography. Also, determinations of active site cysteine pKa values were performed, enhancing our understanding of the unusual catalytic mechanism regarding these Trx-like enzymes. References [1] K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science, New York. [2] D. Ritz, J. Beckwith, Annu. Rev. Microbiol . 2001, 55,21. [3] J.L. Martin, Nature. 1995. 3,245. [4] A. Jordan, F. Aslund, E. Pontis, P. Reichard, A. Holmgren, J. Biol. Chem. 1997, 272,18044. [5] G.B. Kallis, A. Holmgren, J. Biol. Chem. 1980, 255, 261. [6] A.B. Tomter, (2010) Ph.D. Thesis, University of Oslo. [7] Å.K. Røhr, (2010) Ph.D. Thesis, University of Oslo. Fig. Electron density map surrounding the BC3987 T53A mutant active site.
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.
Hersleth, Hans-Petter & Andersson, K. Kristoffer (2011). Combining X-Ray Diffraction and in-situ Single-Crystal Light Absorption and Raman Spectroscopy to Study Different Redox States of Myoglobin. Show summary
Combining X-Ray Diffraction and in-situ Single-Crystal Light Absorption and Raman Spectroscopy to Study Different Redox States of Myoglobin H.-P. Hersletha, K.K. Anderssona aDepartment of Molecular Biosciences, Univeristy of Oslo, P.O.Box 1041 Blindern, 0316 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. It is essential to correctly know the oxidation state of these metal sites in protein crystal structures 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 in how fast they are “reduced” by the X-rays (e.g. ferric Fe3+ myoglobin is faster reduced than ferryl FeIV=O myoglobin) [1]. 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. These lifedoses are about 1000 times lower than the Henderson limit used for general radiation damage. Additionally, the higher oxidation states of myoglobin are not reduced to normal ferrous Fe2+ or ferric Fe3+ states, but end up in some intermediate state. One of the primary goal of the project has been to characterise and study the different intermediates in the reaction between myoglobin and peroxides [2,3]. The reaction intermediates generated appear biological relevant since myoglobin is proposed to function as scavenger of reactive oxygen species during oxidative stress [2]. 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 [3]. [1] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011, In press. DOI: 10.1016/j.bbapap.2010.07.019. [2] 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.. [3] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Biochem. J. 2008, 412, 257-264.
Hersleth, Hans-Petter & Andersson, K. Kristoffer (2011). Grasping the Nature of the Compound II state in Myoglobin by combining X-Ray Diffraction and in-situ Single-Crystal Light Absorption and Raman Spectroscopy. Show summary
Grasping the Nature of the Compound II state in Myoglobin by combining X-Ray Diffraction and in-situ Single-Crystal Light Absorption and Raman Spectroscopy Hans-Petter Hersleth and K. Kristoffer Andersson Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway h.p.hersleth@imbv.uio.no 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]. 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. 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 [2]. 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 [3]. References [1] H.-P. Hersleth et al., J. Biol. Chem. 2007, 282, 23372-23386. [2] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011, In press. DOI: 10.1016/j.bbapap.2010.07.019 [3] H.-P. Hersleth et al.,, Biochem. J. 2008, 412, 257-264.
Hersleth, Hans-Petter; Xiangbo, Zhao; Richard S., Magliozzo & Andersson, K. Kristoffer (2011). Combining X-Ray Diffraction and in-situ Spectroscopy to Study Haem Proteins. Show summary
Combining X-Ray Diffraction and in-situ Spectroscopy to Study Haem Proteins. Hans-Petter Hersleth,a Xiangbo Zhao,b Richard S. Magliozzo,b K. Kristoffer Andersson,a aDepartament of Molecular Biosciences, University of Oslo (Norway). bDepartment of Chemistry, Brooklyn College, New York (USA). 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. It is essential to correctly know the oxidation state of these metal sites in protein crystal structures 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 proteins myoglobin and catalase-peroxidase 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 “reduced” by the X-rays (e.g. ferric Fe3+ myoglobin is faster reduced than ferryl FeIV=O myoglobin) [1], and there is also considerable differences between myoglobin and catalase-peroxidase. The higher oxidation states of myoglobin are not reduced to normal ferrous Fe2+ or ferric Fe3+ states, but end up in some intermediate state. One of the primary goal of the project has been to characterise and study the different intermediates in the reaction between myoglobin and peroxides [2,3]. The reaction intermediates generated appear biological relevant since myoglobin is proposed to function as scavenger of reactive oxygen species during oxidative stress. 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 [3,4]. [1] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011, In press. DOI: 10.1016/j.bbapap.2010.07.019. [2] H.-P. Hersleth, et al., J. Biol. Chem. 2007, 282, 23372-23386. [3] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Biochem. J. 2008, 412, 257-264. [4] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Chem. Biodiv. 2008, 5, 2067-2089. Keywords: haem protein, radiation damage, spectroscopy
Hersleth, Hans-Petter; Zhao, Xiangbo; Magliozzo, Richard S. & Andersson, K. Kristoffer (2011). Combining X-Ray Diffraction and in-situ Spectroscopy to Study Haem Proteins. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. A67, s C159- C160 Show summary
Combining X-Ray Diffraction and in-situ Spectroscopy to Study Haem Proteins. Hans-Petter Hersleth,a Xiangbo Zhao,b Richard S. Magliozzo,b K. Kristoffer Andersson,a aDepartament of Molecular Biosciences, University of Oslo (Norway). bDepartment of Chemistry, Brooklyn College, New York (USA). 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 seem especially vulnerable for radiation-induced reduction. It is essential to correctly know the oxidation state of these metal sites in protein crystal structures, to be able to interpret the structure-function relation. We have used in-situ (online) UV-vis and Raman spectroscopy to study how different oxidation states of the haem proteins myoglobin and catalase-peroxidase 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 “reduced” by the X-rays (e.g. ferric Fe3+ myoglobin is faster reduced than ferryl FeIV=O myoglobin) [1], and there is also differences between the ferric myoglobin and catalase-peroxidase. The higher oxidation states of myoglobin are not reduced to normal ferrous Fe2+ or ferric Fe3+ states, but end up in some intermediate state. One of the primary goal of the project has been to characterise and study the different intermediates in the reaction between myoglobin and peroxides [2,3]. The reaction intermediates generated in this reaction appear biological relevant since myoglobin is proposed to function as scavenger of reactive oxygen species during oxidative stress. 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 [3,4]. [1] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011, In press. DOI: 10.1016/j.bbapap.2010.07.019. [2] H.-P. Hersleth, et al., J. Biol. Chem. 2007, 282, 23372-23386. [3] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Biochem. J. 2008, 412, 257-264. [4] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Chem. Biodiv. 2008, 5, 2067-2089. Keywords: haem proteins, radiation damage, spectroscopy
Hersleth, Hans-Petter; Zhao, Xiangbo; Magliozzo, Richard S. & Andersson, K. Kristoffer (2011). Combining X-Ray Diffraction and in-situ Spectroscopy to Study Haem Proteins. Show summary
Combining X-Ray Diffraction and in-situ Spectroscopy to Study Haem Proteins. Hans-Petter Hersleth,a Xiangbo Zhao,b Richard S. Magliozzo,b K. Kristoffer Andersson,a aDepartament of Molecular Biosciences, University of Oslo (Norway). bDepartment of Chemistry, Brooklyn College, New York (USA). 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 seem especially vulnerable for radiation-induced reduction. It is essential to correctly know the oxidation state of these metal sites in protein crystal structures, to be able to interpret the structure-function relation. We have used in-situ (online) UV-vis and Raman spectroscopy to study how different oxidation states of the haem proteins myoglobin and catalase-peroxidase 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 “reduced” by the X-rays (e.g. ferric Fe3+ myoglobin is faster reduced than ferryl FeIV=O myoglobin) [1], and there is also differences between the ferric myoglobin and catalase-peroxidase. The higher oxidation states of myoglobin are not reduced to normal ferrous Fe2+ or ferric Fe3+ states, but end up in some intermediate state. One of the primary goal of the project has been to characterise and study the different intermediates in the reaction between myoglobin and peroxides [2,3]. The reaction intermediates generated in this reaction appear biological relevant since myoglobin is proposed to function as scavenger of reactive oxygen species during oxidative stress. 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 [3,4]. [1] H.-P. Hersleth, K.K. Andersson, Biochim. Biophys. Acta 2011, In press. DOI: 10.1016/j.bbapap.2010.07.019. [2] H.-P. Hersleth, et al., J. Biol. Chem. 2007, 282, 23372-23386. [3] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Biochem. J. 2008, 412, 257-264. [4] H.-P. Hersleth, Y.-W. Hsiao, U. Ryde, C.H. Görbitz, K.K. Andersson, Chem. Biodiv. 2008, 5, 2067-2089. Keywords: haem proteins, radiation damage, spectroscopy
Lofstad, Marie; Hammerstad, Marta; Olsbu, Inger Kirstine; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2011). Structural studies of Nitric Oxide Synthase and Flavodoxines in Bacillus cereus. Show summary
Structural studies of Nitric Oxide Synthase and Flavodoxines in Bacillus cereus Author list: Marie Lofstad, Marta Hammerstad, Inger K. Olsbu, Åsmund K. Røhr, Hans-Petter Hersleth, K. Kristoffer Andersson Nitric Oxide Synthase (NOS) produces NO in the body from arginine and oxygen. In mammals three different NOS isoforms exist: endothelial eNOS, neuronal nNOS and inducible iNOS. The mammalian NOS consists of one oxygenase domain with heme (NOSoxy) and one reductase domain (with FMN, FAD and NADPH) which transports electrons to NOSoxy. Bacterial NOS, on the other hand, consists only of an oxygenase domain, and have separate proteins that deliver the electrons to this domain. The redox partner of NOS in Bacillus cereus is not known, but the three flavodoxins in Bacillus cereus are the most probable candiates. In this project we will try to structurally characterize the Bacillus cereus NOS (bcNOS) system: try to determine the natural electron donors of bcNOS, find out how specific these donors are, and understand the electron transfer between bcNOS and its electron donors. The results from these studies are going to be compared with the knowledge about the mammalian NOS reaction. I have so far in my master project cloned bcNOS and two flavodoxins. The main purpose of the project is to solve the structure of these proteins, as well as performimg activity assays and spectroscopic studies. For the flavodoxin BC1376 some nice crystals that diffracted to better than 1.0 Å, have already been obtained, and I am currently working on solving the structure.
Olsbu, Inger Kirstine; Lombard, M; Hersleth, Hans-Petter; Andersson, K. Kristoffer & Boucher, Jean-Luc (2011). Key role of Val567 on L-Arginine analogues and heme ligands binding to nNOS.
Olsbu, Inger Kirstine; Lombard, M; Hersleth, Hans-Petter; Andersson, K. Kristoffer & Boucher, Jean-Luc (2011). Key role of Val567 on L-Arginine analogues and heme ligands binding to nNOS. NBS-nytt. ISSN 0801-3535. s 94- 94
Pompidor, G; Dworkowski, F.S.N.; Thominet, V; Hough, M A; Hersleth, Hans-Petter & Fuchs, Martin R (2011). Combination of in-situ optical and macromolecular crystallography. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. s C55- C556
Pompidor, G; Dworkowski, F.S.N.; Thominet, V; Hough, M A; Hersleth, Hans-Petter; Schulze-Briese, C & Fuchs, Martin R (2011). Combining X-ray diffraction and vibrational spectroscopy in structural biology. Show summary
Combining X-ray diffraction and vibrational spectroscopy in structural biology G. Pompidor,1* F.S.N Dworkowski,1 V. Thominet,1 M.A. Hough,2 H.-P. Hersleth,3 C. Schulze-Briese,1M.R. Fuchs1 1Swiss Light Source, Paul Scherrer Institute, Villigen, Swizterland 2Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, United Kingdom 3Department cDepartment of Biological Sciences, University Of Oslo, Norway *e-mail contact (guillaume.pompidor@psi.ch) Over the past few years spectroscopy has become a complementary method of choice to X-ray diffraction in structural biology. Spectroscopy on single crystals allows to identify and to characterize chemical species which still remain ambiguous on the basis of macromolecular crystallography alone. By the use of the in-situ micro-spectrophotometer available at beamline X10SA of the Swiss Light Source, one can perform diffraction and Raman spectroscopy measurements concurrently on the same protein crystal. The on-axis geometry of the instrument ensures that both diffraction and spectroscopic techniques probe the same volume of the sample. This is of great importance for studying radiation damage processes and/or X-ray induced reactions. Resonant Raman spectroscopy measurements using either a Kr+ laser (413 nm) or a solid state laser (405 nm) have been carried out on 2 different hemoproteins, myoglobin form horse heart and cytochrome c' from Alcaligenes xylosoxidans. The spectroscopic results complement nicely the picture of the active site provided by crystallography and permit the clear identification of the ligand bound to the heme. Non-resonant Raman spectroscopy in the NEAR-INFRARED domain (785 nm), potentially applicable to every protein sample as the presence of a colored co-factor is not required, has been successfully performed on two test proteins, horse insulin and hen egg-white lysozyme. The disulfide bond breakage due to X-ray exposure during diffraction data collection has been monitored by the decrease in the intensity of the S-S stretch band in the Raman spectra.
Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2011). Monitoring protein bound flavin X-ray radiation damage.
Skråmo, Silje; Hersleth, Hans-Petter; Hammerstad, Marta; Røhr, Åsmund Kjendseth; Tomter, Ane Berg & Andersson, K. Kristoffer (2011). Structural and spectroscopic studies of electron transport from FldR to the RNR system in B. cereus. Show summary
Structural and spectroscopic studies of electron transport from FldR to the RNR system in B. cereus Silje Skråmo1*, Hans-Petter Hersleth1, Marta Hammerstad1, Åsmund K. Røhr1, Ane B. Tomter and K. Kristoffer Andersson1 1 Department of Molecular Biosciences, University of Oslo, Norway * silje.skramo@imbv.uio.no The Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides. The aim of the project is to study how the flavodoxin reductases (FldR) BC4926 and BC0385 transport electrons via NrdI to the RNR system in Bacillus cereus and how each of the transports affects the activity of RNR. The project is accomplished by cloning (BC0385), proteine isolation (BC0385, BC4926), activity assay measurement (FldR and NrdI, FldR, NrdI and RNR), crystallisation (BC4926 and BC0385, as well as co-crystallisation with NrdI for both FldR) and crystal structure solving. The function of NrdI in the class Ib RNR system still remains unclear. To chart the mechanism of the NrdI it is important to have an assay where the function can be studied in detail. One of the two different FldR in B. cereus is transferring electrones from NADPH to NrdI. How the reduced NrdI in turn is working on the RNR system via NrdF is strongly dependent on the FldR. Therefore, by examining both FldR, it may be possible to suggest which one is working best in vitro and maybe also in vivo. This charting may lead to possibilities of studying the interaction between NrdI and NrdF and the synthethis of the metal cofactor in the system. Structure studies of the NrdI-FldR-complex are of great interest because there exists no similar structure at this point of time.
Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2010). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 Mutants. Show summary
Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 Mutants Marta Hammerstad,a Åsmund K. Røhr, a Hans-Petter Hersleth, a Ane B. Tomter a and K. Kristoffer Andersson a a Department of Molecular Biosciences, University of Oslo, Norway E-mail; martaham@imbv.uio.no Thioredoxin (Trx) is a small ubiquitous protein containing a redox-active cysteine dithiol/cysteine disulfide. These thiol-disulfide oxidoreductases all share an overall // sandwich fold, in addition to the conserved C-X-X-C motif [1]. The E. coli NrdH-redoxins, described as the reductants of NrdE of bacterial class Ib RNR [2], and C. pasteurianum Cp9-redoxins, involved in the reduction of various hydroperoxide substrates [3] both possess Trx functionality. However, an active site environment differing from traditional Trxs has been proposed for these enzymes. A protein homologous to the NrdH-redoxins and Cp9-redoxins has recently been located in the B. cereus genome, BC3987, with unknown function [4]. The crystal structures of two BC3987 mutants, T53A and D11W, have been solved using X-ray crystallography. Also, for better understanding of the active site chemistry performed by the above mentioned enzymes, determination of the active site cysteine pKa values and redox potential determinations of the mutants were performed. The resulting data revealed slight alternations compared to the native protein, contributing to more knowledge about the reaction mechanism performed by these Trx-like protein. References [1] J.L. Martin, Nature. 1995. 3,245. [2] M. Kolberg et al., Biochim. Biophys. Acta. 2004, 1699, 1. [3] C.M. Reynolds et al., Biochemistry. 2002. 41,1990. [4] Å.K. Røhr and K.K. Andersson, J. Biol. Chem (Submitted). 2010.

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Published Nov. 4, 2010 2:40 PM - Last modified Mar. 9, 2020 3:39 PM

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