Hans-Petter Hersleth

• Hammerstad, Marta; Steinvik, Erlend & Hersleth, Hans-Petter (2025). The methyltransferase NmbA methylates the low-molecular weight thiol bacillithiol, and displays a specific structural architecture. Redox Biology. 88. doi: 10.1016/j.redox.2025.103937. Full text in Research Archive Show summary

  Low-molecular-weight (LMW) thiols maintain the cellular redox balance and protect cells against reactive species, heavy metals, toxins, and antibiotics. Despite having similar metabolic functions, structurally distinct LMW thiols are widespread in nature, with bacillithiol (BSH) being the predominant LMW thiol in bacteria. The LMW thiol N-methyl-BSH (N-Me-BSH) has been identified in the green sulfur bacterium Chlorobaculum tepidum, revealing the presence of a putative S-adenosyl-l-methionine (SAM)-dependent methyltransferase (MT), NmbA, which could catalyze the final biosynthetic step of N-Me-BSH. In this study, we report biochemical evidence for NmbA's specific function as an MT of the N-atom of the BSH cysteine moiety. We also present the crystal structure of NmbA, confirming that NmbA is a Class I SAM-dependent MT, however, displaying a unique three-dimensional architecture that differs from those of other natural product MTs (NPMTs). The NmbA active site has a narrow molecular basket structure resulting from an unusual organization of the variable Cap domain, and our docking calculations suggests that it can specifically accommodate the BSH substrate. Our research provides a valuable overview of the phylogenetic distribution of N-Me-BSH in bacteria, alongside essential functional and structural insight into a new class of N-directed NPMTs. These findings contribute to the field of SAM-dependent MTs and may allow for targeting distinct bacterial defense mechanisms involving LMW thiols with potential environmental, biotechnological, and medical implications.

• Schroer, Lisa; Weirich, Sara; Hammerstad, Marta; Hersleth, Hans-Petter; Grønsberg, Ida Andrietta & Hagen, Lars [Show all 11 contributors for this article] (2025). Orthologues of the human protein histidine methyltransferase METTL9 display distinct substrate specificities. Journal of Biological Chemistry. ISSN 0021-9258. 301(7). doi: 10.1016/j.jbc.2025.110318. Full text in Research Archive Show summary

  The human (Homo sapiens; Hs) methyltransferase (MTase) METTL9 is the first enzyme shown to generate 1-methylhistidine (π-methylhistidine) in proteins. METTL9 preferentially methylates an alternating histidine (HxH) motif, where “x” is a small, uncharged amino acid, and multiple substrates have been identified. Putative METTL9 orthologues are found in most eukaryotes, and we have here investigated the activity of such enzymes from several species, representing all five eukaryotic supergroups. The majority of the tested enzymes demonstrated in vitro MTase activity on the prototype HsMETTL9 substrates ARMC6 and DNAJB12. We also detected protein methylation activity of the Caenorhabditis elegans METTL9 which had previously been suggested to be a DNA MTase. However, METTL9 from the fruit fly (Drosophila melanogaster; Dm) and the picoplankton Ostreococcus tauri (Ot) displayed distinct substrate specificities, differing from each other and from that of HsMETTL9. These differences were observed when recombinant proteins and short peptides were used as METTL9 substrates. To further analyze substrate specificity, we used peptide arrays to systematically replace the “x” residue and the residues flanking the HxH motif in a substrate peptide. This revealed varying degrees of tolerance among the METTL9 orthologues (Hs > Dm > Ot) for substitutions at these positions. Our results show that the METTL9 orthologues, although requiring an HxH target site, have evolved different substrate specificities, likely due to differing biologically relevant substrates in the respective organisms. Furthermore, we solved the X-ray structure of OtMETTL9, revealing several differences from the previously published HsMETTL9 structures that may explain its distinct substrate specificity.

• Fernandez, Javier Gutierrez; Hersleth, Hans-Petter & Hammerstad, Marta (2024). The crystal structure of myco­thiol di­sulfide reductase (Mtr) provides mechanistic insight into the specific low-molecular-weight thiol reductase activity of Actinobacteria. Acta Crystallographica Section D: Structural Biology. 80(3), p. 181–193. doi: 10.1107/S205979832400113X. Full text in Research Archive Show summary

  Low-molecular-weight (LMW) thiols are involved in many processes in all organisms, playing a protective role against reactive species, heavy metals, toxins and antibiotics. Actinobacteria, such as Mycobacterium tuberculosis, use the LMW thiol mycothiol (MSH) to buffer the intracellular redox environment. The NADPH-dependent FAD-containing oxidoreductase mycothiol disulfide reductase (Mtr) is known to reduce oxidized mycothiol disulfide (MSSM) to MSH, which is crucial to maintain the cellular redox balance. In this work, the first crystal structures of Mtr are presented, expanding the structural knowledge and understanding of LMW thiol reductases. The structural analyses and docking calculations provide insight into the nature of Mtrs, with regard to the binding and reduction of the MSSM substrate, in the context of related oxidoreductases. The putative binding site for MSSM suggests a similar binding to that described for the homologous glutathione reductase and its respective substrate glutathione disulfide, but with distinct structural differences shaped to fit the bulkier MSSM substrate, assigning Mtrs as uniquely functioning reduc- tases. As MSH has been acknowledged as an attractive antitubercular target, the structural findings presented in this work may contribute towards future anti- tuberculosis drug development.

• Hammerstad, Marta; Rugtveit, Anne Kristine; Dahlen, Sondov Åsmundson Braathen; Andersen, Hilde Kristin & Hersleth, Hans-Petter (2023). Functional Diversity of Homologous Oxidoreductases—Tuning of Substrate Specificity by a FAD-Stacking Residue for Iron Acquisition and Flavodoxin Reduction. Antioxidants. 12(6). doi: 10.3390/antiox12061224. Full text in Research Archive Show summary

  Although bacterial thioredoxin reductase-like ferredoxin/flavodoxin NAD(P)+ oxidoreduc- tases (FNRs) are similar in terms of primary sequences and structures, they participate in diverse biological processes by catalyzing a range of different redox reactions. Many of the reactions are critical for the growth, survival of, and infection by pathogens, and insight into the structural basis for substrate preference, specificity, and reaction kinetics is crucial for the detailed understanding of these redox pathways. Bacillus cereus (Bc) encodes three FNR paralogs, two of which have assigned distinct biological functions in bacillithiol disulfide reduction and flavodoxin (Fld) reduction. Bc FNR2, the endogenous reductase of the Fld-like protein NrdI, belongs to a distinct phylogenetic cluster of homologous oxidoreductases containing a conserved His residue stacking the FAD cofactor. In this study, we have assigned a function to FNR1, in which the His residue is replaced by a conserved Val, in the reduction of the heme-degrading monooxygenase IsdG, ultimately facilitating the release of iron in an important iron acquisition pathway. The Bc IsdG structure was solved, and IsdG-FNR1 interactions were proposed through protein–protein docking. Mutational studies and bioinformatics analyses confirmed the importance of the conserved FAD-stacking residues on the respective reaction rates, proposing a division of FNRs into four functionally unique sequence similarity clusters likely related to the nature of this residue.

• Shoor, Marita; Gudim, Ingvild; Hersleth, Hans-Petter & Hammerstad, Marta (2021). Thioredoxin reductase from Bacillus cereus exhibits distinct reduction and NADPH-binding properties. FEBS Open Bio. 11(11), p. 3019–3031. doi: 10.1002/2211-5463.13289. Full text in Research Archive Show summary

  Low molecular weight (low Mr) thioredoxin reductases (TrxRs) are homodimeric NADPH-dependent dithiol flavoenzymes that reduce thioredoxins (Trxs) or Trx-like proteins involved in the activation networks of enzymes, such as the bacterial class Ib ribonucleotide reductase (RNR). During the last few decades, TrxR-like ferredoxin/flavodoxin NADP+ oxidoreductases (FNRs) have been discovered and characterized in several types of bacteria, including those not encoding the canonical plant-type FNR. In Bacillus cereus, a TrxR-like FNR has been shown to reduce the flavodoxin-like protein NrdI in the activation of class Ib RNR. However, some species only encode TrxR, and lack the homologous TrxR-like FNR. Due to the structural similarity between TrxRs and TrxR-like FNRs, as well as variations in their occurrence in different microorganisms, we hypothesized that low Mr TrxR may be able to replace TrxR-like FNR in, for example, the reduction of NrdI. In this study, characterization of TrxR from B. cereus has revealed a weak FNR activity towards NrdI reduction. Additionally, the crystal structure shows that only one out of two binding sites of the B. cereus TrxR homodimer is occupied with NADPH, indicating a possible asymmetric co-substrate binding in TrxR.

• Hammerstad, Marta & Hersleth, Hans-Petter (2021). Overview of structurally homologous flavoprotein oxidoreductases containing the low Mr thioredoxin reductase-like fold – A functionally diverse group. Archives of Biochemistry and Biophysics. ISSN 0003-9861. 702, p. 1–19. doi: 10.1016/j.abb.2021.108826. Full text in Research Archive Show summary

  Structural studies show that enzymes have a limited number of unique folds, although structurally related enzymes have evolved to perform a large variety of functions. In this review, we have focused on enzymes containing the low molecular weight thioredoxin reductase (low Mr TrxR) fold. This fold consists of two domains, both containing a three-layer ββα sandwich Rossmann-like fold, serving as flavin adenine dinucleotide (FAD) and, in most cases, pyridine nucleotide (NAD(P)H) binding-domains. Based on a search of the Protein Data Bank for all published structures containing the low Mr TrxR-like fold, we here present a comprehensive overview of enzymes with this structural architecture. These range from TrxR-like ferredoxin/flavodoxin NAD(P)+ oxidoreductases, through glutathione reductase, to NADH peroxidase. Some enzymes are solely composed of the low Mr TrxR-like fold, while others contain one or two additional domains. In this review, we give a detailed description of selected enzymes containing only the low Mr TrxR-like fold, however, catalyzing a diversity of chemical reactions. Our overview of this structurally similar, yet functionally distinct group of flavoprotein oxidoreductases highlights the fascinating and increasing number of studies describing the diversity among these enzymes, especially during the last decade(s).

• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2020). The Crystal Structures of Bacillithiol Disulfide Reductase Bdr (YpdA) Provide Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductase. Biochemistry. ISSN 0006-2960. 59(51), p. 4793–4798. doi: 10.1021/acs.biochem.0c00745. Full text in Research Archive Show summary

  Low G+C Gram-positive Firmicutes, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the low-molecular weight thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein YpdA has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial for maintaining the reduced pool of BSH and cellular redox balance. In this work, we present the first crystallographic structures of YpdAs, namely, those from S. aureus and B. cereus. Our analyses reveal a uniquely organized biological tetramer; however, the structure of the monomeric subunit is highly similar to those of other flavoprotein disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in the proximity of the FAD cofactor. We also report enzymatic activities for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.

• 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), p. 318–332. doi: 10.1002/bmb.21218. Full text in Research Archive Show summary

  Many laboratory courses consist of short and seemingly unconnected individual laboratory exercises. To increase the course consistency, relevance, and student engagement, we havedevelopedaresearch-inspiredandproject-basedmodule, “From Gene to Structure and Function”. This 2.5-week full-day biochemistry and structural biology module covers protein expression, purification, structure solving, and characterization.Themoduleiscenteredaroundthe flavodoxin-likeprotein NrdI, involved in the activation of the bacterial ribonucleotide reductase enzyme system. Through an in-depth focus on one specific protein, the students will learn the basic laboratory skills needed in order to generate a broader knowledge and breadth within the field. With respect to generic skills, the studentsreporttheir findingsasascientificarticle,withtheaim to learn to present concise research results and write scientific papers. The current research-inspired project has the potential ofbeingfurtherdevelopedintoamorediscovery-drivenproject and extended to include other molecular biological techniques orbiochemical/biophysicalcharacterizations.Instudentevaluations, this research-inspired laboratory course has received very high ratings and been highly appreciated, where the students have gained research experience for more independent future work in the laboratory. © 2019 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry andMolecularBiology,47(3):318–332,2019.

• 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. 8(9), p. 1553–1566. doi: 10.1002/2211-5463.12503. Full text in Research Archive Show summary

  Nitric oxide (NO) produced by mammalian nitric oxide synthases (mNOSs) is an important mediator in a variety of physiological functions. Crystal structures of mNOSs have shown strong conservation of the active‐site residue Val567 (numbering for rat neuronal NOS, nNOS). NOS‐like proteins have been identified in several bacterial pathogens, and these display striking sequence identity to the oxygenase domain of mNOS (NOSoxy), with the exception of a Val to Ile mutation at the active site. Preliminary studies have highlighted the importance of this Val residue in NO‐binding, substrate recognition, and oxidation in mNOSs. To further elucidate the role of this valine in substrate and substrate analogue recognition, we generated five Val567 mutants of the oxygenase domain of the neuronal NOS (nNOSoxy) and used UV‐visible and EPR spectroscopy to investigate the effects of these mutations on the heme distal environment, the stability of the heme‐FeII‐CO complexes, and the binding of a series of substrate analogues. Our results are consistent with Val567 playing an important role in preserving the integrity of the active site for substrate binding, stability of heme‐bound gaseous ligands, and potential NO production.

• 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), p. 5427–5436. doi: 10.1021/acs.biochem.8b00674. Full text in Research Archive Show summary

  Flavodoxins (Flds) are small, bacterial proteins that transfer electrons to various redox enzymes. Flavodoxins are reduced by ferredoxin/flavodoxin NADP+ oxidoreductases (FNRs), but little is known of the FNR-Fld interaction. Here, we compare the interactions of two flavodoxins (Fld1–2), one flavodoxin-like protein (NrdI), and three different thioredoxin reductase (TrxR)-like FNRs (FNR1–3), all from Bacillus cereus. Steady-state kinetics shows that the FNR2-Fld2 electron transfer pair is particularly efficient, and redox potential measurements also indicate that this is the most favorable electron donor/acceptor pair. Furthermore, crystal structures of FNR1 and FNR2 show that the proteins have crystallized in different conformations, a closed and an open conformation, respectively. We suggest that a large-scale conformational rearrangement takes place during the FNR catalytic cycle to allow for the binding and reduction of the Fld and, subsequently, the re-reduction of the FNR by NADPH. Finally, inspection of the residues surrounding the FAD cofactor in the FNR active site shows that a key isoalloxazine ring-stacking residue is different in FNR1 and FNR2, which could explain the large difference in catalytic efficiency between the two FNRs. To date, all of the characterized TrxR-like FNRs have a residue with aromatic character stacking against the FAD isoalloxazine ring, and this has been thought to be a conserved feature of this class of FNRs. FNR1, however, has a valine in this position. Bioinformatic analysis shows that the TrxR-like FNRs can actually be divided into two groups, one group where the FAD-stacking residue has aromatic character and another group where it is valine.

• Gudim, Ingvild; Lofstad, Marie; Beek, Wouter Van & 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), p. 1439–1449. doi: 10.1002/pro.3436. 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. 7(8), p. e2223–e2223. doi: 10.21769/BioProtoc.2223. Full text in Research Archive Show summary

  This protocol describes how to measure protein-protein interactions by microscale thermophoresis (MST) using the MonolithTM NT.115 instrument (NanoTemper). We have used the protocol to determine the binding affinities between three different flavodoxin reductases (FNRs) and a flavodoxin-like protein, NrdI, from Bacillus cereus (Lofstad et al., 2016). NrdI is essential in the activation of the manganese-bound form of the class Ib ribonucleotide reductase (RNR) system. RNRs, in turn, are the only source of the de novo synthesis of deoxyribonucleotides required for DNA replication and repair in all living organisms.

• 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), p. 4998–5001. doi: 10.1021/acs.biochem.6b00699. Full text in Research Archive
• Rackwitz, Sergej; Faus, Isabelle; Schmitz, Markus; Kelm, Harald; Krüger, Hans-Jörg & Andersson, K. Kristoffer [Show all 12 contributors for this article] (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), p. 673–678. doi: 10.1007/s10751-013-0981-8. Full text in Research Archive 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.

• 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), p. 526–537. doi: 10.1021/cb400757h. Full text in Research Archive 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.

• 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. 70(No. 6. (May 2014) ), p. 777–780. doi: 10.1107/S2053230X14008334. Full text in Research Archive 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.

• Tomter, Ane Berg; Zoppellaro, Giorgio; Andersen, Niels Højmark; Hersleth, Hans-Petter; Hammerstad, Marta & Røhr, Åsmund Kjendseth [Show all 15 contributors for this article] (2013). Ribonucleotide reductase class I with different radical generating clusters. Coordination chemistry reviews. ISSN 0010-8545. 257(1), p. 3–26. doi: 10.1016/j.ccr.2012.05.021. Full text in Research Archive 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), p. 11650–11652. doi: 10.1039/c3cc47022a. Full text in Research Archive 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.

• Can, Mehmet; Krucinska, Jolanta; Zoppellaro, Giorgio; Andersen, Niels Højmark; Wedekind, Joseph E. & Hersleth, Hans-Petter [Show all 8 contributors for this article] (2013). Structural Characterization of Nitrosomonas europaea Cytochrome c-552 Variants with Marked Differences in Electronic Structure. ChemBioChem. ISSN 1439-4227. 14(14), p. 1828–1838. doi: 10.1002/cbic.201300118. Full text in Research Archive 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.

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• Hammerstad, Marta; Gudim, Ingvild; Andersen, Hilde Kristin; Aracena-Solem, Matias & Hersleth, Hans-Petter (2026). Metals and thiols in bacterial defense and survival. Full text in Research Archive
• Hammerstad, Marta & Hersleth, Hans-Petter (2025). Structural studies of flavodoxins from Bacillus cereus in different redox states. Full text in Research Archive
• Hammerstad, Marta; Karlsen, Marthe Lindahl; Gudim, Ingvild; Gutiérrez-Fernández, Javier & Hersleth, Hans-Petter (2025). Structural and functional studies of enzymes involved in thiol-based redox pathways. Full text in Research Archive
• Karlsen, Marthe Lindahl; Hersleth, Hans-Petter & Hammerstad, Marta (2025). Structural and functional insights into bacillithiol disulfide reductase – A potential antimicrobial target. Full text in Research Archive
• Hammerstad, Marta; Karlsen, Marthe Lindahl; Gutiérrez-Fernández, Javier; Gudim, Ingvild & Hersleth, Hans-Petter (2024). Structures of bacilli­thiol and myco­thiol di­sulfide reductases of bacilli and mycobacteria. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. 80. doi: 10.1107/S2053273324099200. Full text in Research Archive
• Hersleth, Hans-Petter (2024). Structures of bacillithiol and mycothiol disulfide reductases of bacilli and mycobacteria. Full text in Research Archive
• Hammerstad, Marta; Karlsen, Marthe Lindahl; Fernandez, Javier Gutierrez; Gudim, Ingvild & Hersleth, Hans-Petter (2024). Structures of bacillithiol and mycothiol disulfide reductases of Firmicutes and Actinobacteria. Full text in Research Archive
• Karlsen, Marthe Lindahl; Hersleth, Hans-Petter & Hammerstad, Marta (2024). Structural and functional investigation of bacillithiol disulfide reductases. Full text in Research Archive
• Hammerstad, Marta; Andersen, Hilde Kristin; Rugtveit, Anne Kristine; Dahlen, Sondov Åsmundson Braathen & Hersleth, Hans-Petter (2023). Functional diversity among flavin-containing oxidoreductases involved in redox protection, iron homeostasis, and ribonucleotide reduction. Full text in Research Archive
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases. Full text in Research Archive
• Karlsen, Marthe Lindahl; Hersleth, Hans-Petter & Hammerstad, Marta (2023). Diversity among bacillithiol disulfide reductases – a structural and functional investigation. Full text in Research Archive
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases. Full text in Research Archive
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases. Full text in Research Archive
• Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2023). A Research-inspired Laboratory Module on a Redox Protein Combining Biochemistry and Structural Biology. Full text in Research Archive
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Structural determination of Mycobacterium tuberculosis and Rhodococcus erythropolis mycothiol disulphide reductases. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2022). Redox protection of pathogens by low molecular weight thiols. Full text in Research Archive
• Rese, Morten; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Diversity in myoglobin function. Full text in Research Archive
• Andersen, Hilde Kristin; Hersleth, Hans-Petter & Hammerstad, Marta (2022). Structure-function studies of ferredoxin-flavodoxin NADP+ oxidoreductase 1 from Bacillus cereus as an iron-uptake oxidoreductase. Full text in Research Archive
• Rese, Morten; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Diversity in myoglobin function. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2022). Redox protection of pathogens by low molecular weight thiols. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2020). The Crystal Structures of Bacillithiol Disulfide Reductase YpdA Reveal Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductases. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2019). Enzyme Activation by a Flavoprotein Redox Network. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Beek, Wouter van & Hersleth, Hans-Petter (2019). New structural insight into the well known peptide flip observed in flavodoxins. Acta Crystallographica Section A: Foundations and Advances. A75, p. e123–e123. doi: 10.1107/S2053273319094336. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). Enzyme activation by a flavoprotein redox network. Acta Crystallographica Section A: Foundations and Advances. A75, p. e128–e128. doi: 10.1107/S2053273319094282. Full text in Research Archive
• Kendall-Price, Sophie; Evans, Rhiannon M; Rowbotham, Jack S; Reeve, Holly A; Carr, Stephen B & Frogley, Mark D [Show all 11 contributors for this article] (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, p. 425–425. Full text in Research Archive
• 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. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Beek, Wouter van & Hersleth, Hans-Petter (2019). New structural insight into the well known peptide flip observed in flavodoxins. Full text in Research Archive
• Hammerstad, Marta; Zaltariov, Mirela F.; Arabshahi, Homayon John; Jovanovic, Katarina; Richter, Klaus W. & Cazacu, Maria [Show all 14 contributors for this article] (2019). New thiosemicarbazone derivatives and their copper(II) complexes as potential inhibitors against mammalian ribonucleotide reductase. Full text in Research Archive
• 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. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). Characterization of Proteins in the Ribonucleotide Reductase RedoxNetwork. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Chaudhari, Sujata; Olsbu, Inger Kirstine; Alqarzaee, Abdulelah; Singh, Ryan; Schulze, Thomas & Zimmerman, Matthew [Show all 8 contributors for this article] (2018). Endogenous nitric oxide synthase (NOS) activity reduces staphylococcal lifespan during stationary phase. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network. NBS-nytt. ISSN 0801-3535. p. 40–40. Full text in Research Archive
• Olsbu, Inger Kirstine; Chaudhari, Sujata; Thomas, Vinai Chittezham & Hersleth, Hans-Petter (2018). Potential reductase partner for Staphylococcus aureus Nitric Oxide Synthase. Full text in Research Archive
• 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. p. 40–40. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Enzyme activation by a flavoprotein redox network. Full text in Research Archive
• 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. Full text in Research Archive
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III in Bacillus cereus. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. p. 105–105. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus. Full text in Research Archive
• 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. Full text in Research Archive
• Johannesen, Hedda; Hammerstad, Marta; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III. Full text in Research Archive
• 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. p. 63–63. Full text in Research Archive
• Hersleth, Hans-Petter (2017). A research-inspired biochemistry laboratory module – Combining, expression,purification, crystallisation, structure solving and characterisation of a flavdoxin-like protein. Full text in Research Archive
• Lofstad, Marie; Gudim, Ingvild; Kjendseth, Åsmund Røhr; Andersen, Niels Højmark; Beek, Wouter van & Andersson, K. Kristoffer [Show all 7 contributors for this article] (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. Full text in Research Archive
• 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. Full text in Research Archive
• Hammerstad, Marta; Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter & Zaltariov, Mirela-Fernanda [Show all 9 contributors for this article] (2017). INHIBITION OF CLASS 1A RIBONUCLEOTIDE REDUCTASE, AND A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE. Full text in Research Archive 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

• Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Hammerstad, Marta & Solomon, Edward I. [Show all 7 contributors for this article] (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. p. 45–45. Full text in Research Archive
• Hammerstad, Marta; Hersleth, Hans-Petter; Lofstad, Marie; Johannesen, Hedda; Tomter, Ane Berg & Røhr, Åsmund Kjendseth [Show all 7 contributors for this article] (2016). Structural Insight into the Function of Ribonucleotide Reductase. Full text in Research Archive
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A Structural and Functional Investigation of Ribonucleotide Reductase Class III In Bacillus Cereus. Full text in Research Archive
• 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. Full text in Research Archive
• Hersleth, Hans-Petter (2016). Probing enzyme activation networks - structural and functional studies of flavoproteins in Bacillus cereus. Full text in Research Archive
• 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. Full text in Research Archive
• Hersleth, Hans-Petter; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr; beek, Wouter Van & Giullaume, Pompidor [Show all 11 contributors for this article] (2016). Grasping the True Structure of Haem- and Flavoproteins – Combining X-RAY Crystallography and In Situ Single-Crystal UV-VIS and Raman Spectroscopy. Full text in Research Archive
• Hersleth, Hans-Petter (2016). Probing enzyme activation networks - structural and functional studies of flavoproteins in Bacillus cereus. Full text in Research Archive
• Hersleth, Hans-Petter (2016). Probing enzyme activation networks ‐ structural and functional studies of flavoproteins in Bacillus cereus. Full text in Research Archive
• Lofstad, Marie; Gudim, Ingvild; beek, Wouter Van; Pompidor, Guillaume; Kjendseth, Åsmund Røhr & Andersson, K. Kristoffer [Show all 7 contributors for this article] (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. Full text in Research Archive
• Lofstad, Marie; Gudim, Ingvild; beek, Wouter Van; Pompidor, Guillaume; Kjendseth, Åsmund Røhr & Andersson, K. Kristoffer [Show all 7 contributors for this article] (2016). Radiation damage of haem- and flavoproteins – combining X-ray crystallography and single-crystal UV-Vis and Raman spectroscopy. Full text in Research Archive
• Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter; Hammerstad, Marta & Solomon, Edward I. [Show all 7 contributors for this article] (2016). A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE BY CD AND MCD SPECTROSCOPY. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. p. 65–65. Full text in Research Archive
• 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. p. 64–64. Full text in Research Archive
• 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. p. 45–45. Full text in Research Archive
• Hersleth, Hans-Petter & Røhr, Åsmund Kjendseth (2015). Teaching a general molecular bioscience master course as a project ”From gene to structure”. Full text in Research Archive
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structure of a ferredoxin/flavodoxin-NADP(H) oxidoreductase in Bacillus cereus. Full text in Research Archive
• Hersleth, Hans-Petter (2015). Combining X-ray crystallography and in situ single-crystal UV-Vis and Raman spectroscopy to study redox proteins. Full text in Research Archive
• 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, p. s205–s205. doi: 10.1107/S2053273315096916. Full text in Research Archive
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structure of a ferredoxin/flavodoxin-NADP(H) oxidoreductase in Bacillus cereus. NBS-nytt. ISSN 0801-3535. p. 96–96. Full text in Research Archive
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structures of ferredoxin/flavodoxin-NADP(H) oxidoreductases in Bacillus cereus. Full text in Research Archive
• Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; beek, Wouter Van; 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, p. s489–s489. Full text in Research Archive
• Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; beek, Wouter Van; 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, p. 2–2. Full text in Research Archive
• Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hammerstad, Marta; Hersleth, Hans-Petter & Solomon, Edward I. [Show all 7 contributors for this article] (2015). A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy. Full text in Research Archive
• Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2015). On the way to determine the structure of a ferredoxin in B. cereus. Full text in Research Archive 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.

• Wu, Bernt; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2015). Structural and functional characterization of flavohemoglobin from Bacillus Cereus. Full text in Research Archive
• Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Beek, Wouter Van; Diadkin, Vadim; Wiker, Geir & Chernyshov, Dmitry [Show all 7 contributors for this article] (2015). Combining Protein X-Ray Crystallography and Single-Crystal Spectroscopy - A New in situ Setup at BM01A at ESRF. Full text in Research Archive 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.

• 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. p. 47–47. Full text in Research Archive 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)

• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Rackwitz, Sergej; Faus, Isabelle; Schmitz, Markus; Krüger, Hans-Jörg; Andersson, K. Kristoffer & Hersleth, Hans-Petter [Show all 11 contributors for this article] (2014). Nuclear resonance vibrational spectroscopy on iron protein single crystals. Full text in Research Archive
• 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, p. C707–C707. Full text in Research Archive
• 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, p. C434–C434. Full text in Research Archive 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

• 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, p. C1657–C1657. Full text in Research Archive 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

• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). Structural insight Into the Function of Ribonucleotide Reductase. Full text in Research Archive
• Skråmo, Silje; Lofstad, Marie; Monka, Susanne; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer [Show all 7 contributors for this article] (2014). Structural studies of ferredoxin/flavodoxin-NADPH reductases, flavodoxins, ferredoxins and redox partners in B. cereus. Full text in Research Archive
• Lofstad, Marie; Gudim, Ingvild; Skråmo, Silje; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Tomter, Ane Berg [Show all 8 contributors for this article] (2014). Crystallisation of ferredoxin/flavodoxin-NADP(H) oxidoreductases, flavodoxins, ferredoxins and redox partners in Bacillus cereus. Full text in Research Archive 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. JBIC Journal of Biological Inorganic Chemistry. ISSN 0949-8257. 19, p. S265–S265. Full text in Research Archive
• 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. JBIC Journal of Biological Inorganic Chemistry. ISSN 0949-8257. 19, p. S268–S268. Full text in Research Archive
• 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. JBIC Journal of Biological Inorganic Chemistry. ISSN 0949-8257. 19, p. S339–S339. Full text in Research Archive
• Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). On the way to determine the structure of a ferredoxin in B. cereus. Full text in Research Archive
• Wu, Bernt; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). Structural and functional characterization of flavohemoglobin from Bacillus Cereus. Full text in Research Archive
• Hersleth, Hans-Petter (2013). Protein Crystal Spectroscopy. Full text in Research Archive
• Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Lofstad, Marie; Hammerstad, Marta; Skråmo, Silje & Andersen, Niels Højmark [Show all 13 contributors for this article] (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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Nitric oxide synthase and possible redox partners in Bacillus cereus. Full text in Research Archive
• Hammerstad, Marta; Tomter, Ane Berg; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Zoppellaro, Giorgio & Andersen, Niels Højmark [Show all 12 contributors for this article] (2013). Studies of the tyrosyl radicals and metal clusters in R2 of class Ia and Ib ribonucleotide reductase. Full text in Research Archive
• 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). Full text in Research Archive
• Schünemann, Volker; Rackwitz, Sergej; Faus, Isabelle; Wolny, Juliusz A.; Schmitz, Markus & Kelm, Harald [Show all 12 contributors for this article] (2013). A New Sample Environment for Cryogenic Nuclear Resonance Scattering Experiments on Single Crystals and Microsamples at P01, PETRA III. Full text in Research Archive
• Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Nitric oxide synthase and possible redox partners in Bacillus cereus. Full text in Research Archive
• 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. Full text in Research Archive
• Hersleth, Hans-Petter; Lofstad, Marie; Røhr, Åsmund Kjendseth; Can, Mehmet; Zhao, Xiangbo & Pompidor, Guillaume [Show all 9 contributors for this article] (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. Full text in Research Archive
• Tomter, Ane Berg; Zoppellaro, Giorgio; Hersleth, Hans-Petter; Andersen, Niels Højmark; Barra, Anne-Laure & Sandvik, Guro Katrine [Show all 13 contributors for this article] (2013). Spectroscopic, structural and DFT studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase. NBS-nytt. ISSN 0801-3535. 1, p. 74–74. Full text in Research Archive 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.

• 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, p. 84–84. Full text in Research Archive 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

• Hersleth, Hans-Petter (2013). What to be aware of when solving the structure of redoxproteins? Full text in Research Archive
• Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Lofstad, Marie; Skråmo, Silje; beek, Wouter Van & Pompidor, Guillaume [Show all 7 contributors for this article] (2013). What to be aware of when solving the structure of redoxproteins? NBS-nytt. ISSN 0801-3535. 1, p. 49–49. Full text in Research Archive
• 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, p. 49–49. Full text in Research Archive
• Hersleth, Hans-Petter; Beek, Wouter Van; Pompidor, Guillaume & Andersson, K. Kristoffer (2012). Using in-situ Spectroscopy to Study X-ray Induced Radiation Damage to Haem Protein Crystals. Full text in Research Archive 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.

• Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Beek, Wouter Van; 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. Full text in Research Archive 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. Full text in Research Archive 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.

• Andersen, Hilde Kristin; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Struktur-funksjonsstudier av ferredoksin-flavodoksin NADP+ oksidoreduktase 1 fra Bacillus cereus som en jern-opptak oksidoreduktase. Universitetet i Oslo. Full text in Research Archive Show summary

  Bacillus cereus har tre enzymer som er annotert som ferredoksin-flavodoksin NADP+ oksidoreduktaser (FNRer). FNR3 er vist å redusere disulfidformen av bacillitiol, FNR2 er vist å effektivt redusere flavodoksiner, men det er enda ikke funnet noen god redokspartner til FNR1. FNR1 er vist å skille seg fra FNR2 ved å være del av en fylogenetisk undergruppe av tioredoksinreduktase-aktige FNRer hvor en konservert aromatisk residu i C-terminalen, som stabiliserer FAD-kofaktoren er erstattet med valin sammenlignet med FNR2-gruppen. Staphylococcus aureus har en nær homolog til FNR1 som deler den konserverte valinresiduen, som har blitt vist å kunne fungere som en jern-opptak-oksidoreduktase (IruO). IruO kan tilgjengeliggjøre jern for bakterier ved å redusere hemoksygenasene iron-regulated surface determinant (Isd)G og IsdI som degraderer hem, eller sideroforer. B. cereus har kun en homolog til IsdG, men ikke IsdI. I dette prosjektet har det blitt utført aktivitetsstudier for å undersøke om B. cereus FNR1 kan fungere som en IruO, og redusere IsdG for å degradere hem, ved bruk av UV-vis-spektroskopi. FNR1 og IsdG har blitt uttrykt og renset. Strukturen til IsdG er blitt løst ved bruk av proteinkrystallografi. IsdG viste å ha svært lik struktur i B. cereus og S. aureus, med ferredoksinfolding og de konserverte residuene Asn7, Trp67 og His77 i bindingssetet til hem. Aktivitetsstudiene viste at FNR1 kunne redusere IsdG for å degradere hem og tilgjengeliggjøre jern. FNR1 er anslått å ha evner i samme størrelsesorden til å redusere IsdG som til å redusere flavodoksiner. Aktivitetsstudiene tyder på at hem blir degradert ved først å redusere Fe(III) til Fe(II) og at det går via Fe(II)-O2 som i kanoniske hemoksygenaser.

• Rese, Morten; Hersleth, Hans-Petter; Hammerstad, Marta & Andersen, Niels Højmark (2022). Functional diversity of myoglobin isozymes from Carassius auratus - Biophysical study of myoglobin as a peroxidase. Universitetet i Oslo. Full text in Research Archive Show summary

  Myoglobin (Mb) is a heme protein expressed in oxidative muscle tissue and functions in oxygen storage and transport. Mb can also display complex redox chemistry involved in several important physiological processes including scavenging of reactive oxygen species and nitric oxide metabolism. Goldfish and the common carp are the only vertebrates known today that express two paralogs of Mb (Mb1 and Mb2) as a result of a recent whole genome duplication. Mb1 is expressed in gills, liver, and skeletal muscle tissue, whereas Mb2 is exclusively expressed in the brain. The tissue-specific expression led to work showing that Mb2 in carp fish is a more effective peroxidase. In this study, work has been done to gain detailed structural and functional insight into the differences of the goldfish Mb1 and Mb2 isozymes by biophysical characterization and 3D structure analysis. The intensively studied Mb from horse heart (hMb) has been studied together with Mb1 and Mb2 as a reference. The reaction of Mb with hydrogen peroxide (H2O2) has been of particular interest. In addition, studies on how radiation damage during diffraction data collection affects redox states of Mb have been performed. This study shows that goldfish Mb2 reacts with H2O2 significantly faster than Mb1. Structural modeling of Mb1 and Mb2 indicates that no differences around the active site are responsible for the difference in reactivity. The structural basis of this functional difference is therefore complex in nature and might be explained by altered dynamics as a consequence of differing amino acid composition towards the surface of the proteins. The ferric and ferryl redox states of Mb are damaged by X-ray radiation during diffraction data collection. The ferric state is significantly more susceptible to radiation damage compared to the ferryl state. This study shows the importance of performing a time/dose calculation on the amount of radiation a specific redox state can handle when collecting diffraction data.

• Hersleth, Hans-Petter; Hammerstad, Marta & Dahlen, Sondov Åsmundson Braathen (2021). Structure and function of FNR1 from Bacillus cereus undergoing a mutation. Universitetet i Oslo. Full text in Research Archive
• Rugtveit, Anne Kristine; Hammerstad, Marta & Hersleth, Hans-Petter (2021). Structural and functional studies of a ferredoxin/flavodoxin NADP+-oxidoreductase mutant from Bacillus cereus. Universitetet i Oslo. Full text in Research Archive
• Gudim, Ingvild; Hersleth, Hans-Petter; Hammerstad, Marta & Sørlie, Morten (2018). Characterisation of flavodoxin and ferredoxin/flavodoxin reductases from Bacillus cereus and their interactions. Reprosentralen, University of Oslo. Full text in Research Archive
• Olsbu, Inger Kirstine; Hersleth, Hans-Petter & Sørlie, Morten (2018). Substrate recognition and redox partner identification in nitric oxide synthases. Reprosentralen, University of Oslo. Full text in Research Archive
• Shoor, Marita; Hersleth, Hans-Petter; Gudim, Ingvild & Hammerstad, Marta (2017). Structural and functional characterization of the redox protein Thioredoxin reductase from Bacillus cereus. Universitetet i Oslo. Full text in Research Archive
• 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. Universitet i Oslo. Full text in Research Archive
• Wu, Bernt; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2015). Purification and characterization of Flavohemoglobin - A flavoheme enzyme. Universitetet i Oslo. Full text in Research Archive Show summary

  Flavohemoglobin is a flavoheme enzyme that catalyzes dioxygenation of nitric oxide (NO) to nitrate NO3–. NO is diatomic radical gas produced by enzymatic and non-enzymatic oxidation of reduced nitrogenous compounds. NO acts as a membrane-permeable signal molecule in mammals, in addition to being a toxin at higher concentrations and being utilized by the immune system against invading pathogens. FHb is involved in protection against the cytotoxic effects of NO (termed nitrosative stress) and is found in a range of bacterial species and some fungi. FHbs are not found in higher eukaryotes, but is employed by a range of pathogens, and is a potential therapeutic target. In this thesis we sought to investigate the FHb from Bacillus cereus by determination of the X-ray crystal structure as only a few structures of FHb from different organisms have been obtained. The protein was successfully cloned and expressed and a purification protocol has been developed, with ammonium sulfate precipitation, anion exchange chromatography and gel filtration. A high yield with high purity was obtained, but with heme and FAD at substoichiometric levels. Crystallization screening resulted in some small needles grown from precipitate, although they were non-reproducible and no crystal structure was obtained. A heme assay was performed in order to identify the heme/protein ratio and protein assays was performed to calculate protein concentration. Testing of reconstitution with heme and FAD was performed with promising results.

• Monka, Susanne; Andersson, K. Kristoffer; Hersleth, Hans-Petter & Hammerstad, Marta (2015). Structural and functional characterisation of ferredoxins in Bacillus cereus. Universitetet i Oslo. Full text in Research Archive Show summary

  Ferredoxins are iron-sulfur proteins that transfer electrons in different cellular processes. In Bacillus cereus there are two genes for ferredoxins that have been identified, one present with a [2Fe-2S] cluster (BC2795) and one with a [4Fe- 4S] cluster (BC1483). These proteins can potentially receive electrons from three possible ferredoxin-NADP+-reductases, and further activate different enzymes. In this project, the starting point was to purify and structurally characterize the two ferredoxins in B. cereus and potentially their interactions with redox partners. For the [4Fe-4S] ferredoxin several attempts to overexpress this protein was unsuccessful. However, the [2Fe-2S] ferredoxin (106 amino acids, 11.4 kDa) was successfully overexpressed in Escherichia coli. This [2Fe-2S] ferredoxin was purified in a series of purification steps involving ammonium sulphate, desalting column, Q Sepharose or DEAE columns, and Superdex 75 column. The purification procedure showed to be troublesome because of possible low iron-sulfur cluster content, and because of low UV-vis detection due to the absence of tryptophanes and tyrosines in the protein. Due to the high content of apoprotein, it was also tried to establish a reconstitution procedure for BC2795. The apoform of the protein was crystallized. Nevertheless, it was not possible to solve the structure.

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