Hans-Petter Hersleth

• 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. ISSN 2059-7983. 80(3), s. 181–193. doi: 10.1107/S205979832400113X. Fulltekst i vitenarkiv Vis sammendrag

  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. ISSN 2076-3921. 12(6). doi: 10.3390/antiox12061224. Fulltekst i vitenarkiv Vis sammendrag

  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. ISSN 2211-5463. 11(11), s. 3019–3031. doi: 10.1002/2211-5463.13289. Fulltekst i vitenarkiv Vis sammendrag

  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, s. 1–19. doi: 10.1016/j.abb.2021.108826. Fulltekst i vitenarkiv Vis sammendrag

  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), s. 4793–4798. doi: 10.1021/acs.biochem.0c00745. Fulltekst i vitenarkiv Vis sammendrag

  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), s. 318–332. doi: 10.1002/bmb.21218. Fulltekst i vitenarkiv
• Gudim, Ingvild; Lofstad, Marie; Van Beek, Wouter & Hersleth, Hans-Petter (2018). High-resolution crystal structures reveal a mixture of conformers of the Gly61-Asp62 peptide bond in an oxidized flavodoxin from Bacillus cereus. Protein Science. ISSN 0961-8368. 27(8), s. 1439–1449. doi: 10.1002/pro.3436.
• Gudim, Ingvild; Hammerstad, Marta; Lofstad, Marie & Hersleth, Hans-Petter (2018). Characterization of different flavodoxin reductase-flavodoxin (FNR-Fld) interactions reveals an efficient FNR-Fld redox pair and identifies a novel FNR subclass. Biochemistry. ISSN 0006-2960. 57(37), s. 5427–5436. doi: 10.1021/acs.biochem.8b00674. Fulltekst i vitenarkiv
• Olsbu, Inger Kirstine; Zoppellaro, Giorgio; Andersson, Karl Kristoffer; Boucher, Jean-Luc & Hersleth, Hans-Petter (2018). Importance of Val567 on heme environment and substrate recognition of neuronal nitric oxide synthase. FEBS Open Bio. ISSN 2211-5463. 8(9), s. 1553–1566. doi: 10.1002/2211-5463.12503. Fulltekst i vitenarkiv
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST). Bio-protocol. ISSN 2331-8325. 7(8). doi: 10.21769/BioProtoc.2223. Fulltekst i vitenarkiv
• Lofstad, Marie; Gudim, Ingvild; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2016). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus. Biochemistry. ISSN 0006-2960. 55(36), s. 4998–5001. doi: 10.1021/acs.biochem.6b00699.

Se alle arbeider i Cristin

• Gutierrez Fernandez, Javier; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases.
• Gutierrez Fernandez, Javier; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases.
• Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2023). A Research-inspired Laboratory Module on a Redox Protein Combining Biochemistry and Structural Biology.
• Gutierrez Fernandez, Javier; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases.
• Karlsen, Marthe Lindahl; Hersleth, Hans-Petter & Hammerstad, Marta (2023). Diversity among bacillithiol disulfide reductases – a structural and functional investigation.
• 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.
• Gutierrez Fernandez, Javier; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Structural determination of Mycobacterium tuberculosis and Rhodococcus erythropolis mycothiol disulphide reductases.
• Rese, Morten; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Diversity in myoglobin function.
• 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.
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2022). Redox protection of pathogens by low molecular weight thiols.
• Rese, Morten; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Diversity in myoglobin function.
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2022). Redox protection of pathogens by low molecular weight thiols.
• 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.
• 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. ISSN 2053-2733. A75, s. e128–e128. doi: 10.1107/S2053273319094282.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; van Beek, Wouter & Hersleth, Hans-Petter (2019). New structural insight into the well known peptide flip observed in flavodoxins. Acta Crystallographica Section A: Foundations and Advances. ISSN 2053-2733. A75, s. e123–e123. doi: 10.1107/S2053273319094336.
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2019). Enzyme Activation by a Flavoprotein Redox Network.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; van Beek, Wouter & Hersleth, Hans-Petter (2019). New structural insight into the well known peptide flip observed in flavodoxins.
• Hammerstad, Marta; Zaltariov, Mirela F.; Arabshahi, Homayon John; Jovanovic, Katarina; Richter, Klaus W. & Cazacu, Maria [Vis alle 14 forfattere av denne artikkelen] (2019). New thiosemicarbazone derivatives and their copper(II) complexes as potential inhibitors against mammalian ribonucleotide reductase.
• 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.
• Kendall-Price, Sophie; Evans, Rhiannon M; Rowbotham, Jack S; Reeve, Holly A; Carr, Stephen B & Frogley, Mark D [Vis alle 11 forfattere av denne artikkelen] (2019). Generating Single Metalloprotein Crystals in Well-Defined Redox States – Electrochemical Microspectroscopy As a Tool for Mechanistic Studies. Meeting Abstracts - The Electrochemical Society [ECS]. ISSN 1091-8213. 9, s. 425–425.
• Andersen, Niels Højmark; Hammerstad, Marta; Zaltariov, Mirela F.; Rapta, Peter; Arion, Vladimir B. & Hersleth, Hans-Petter (2019). Interaction of Thiosemicarbazones with the Ribonucleotide Reductase R2 subunit Studied by Resonance Raman Spectroscopy.
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). Characterization of Proteins in the Ribonucleotide Reductase RedoxNetwork .
• Chaudhari, Sujata; Olsbu, Inger Kirstine; Alqarzaee, Abdulelah; Singh, Ryan; Schulze, Thomas & Zimmerman, Matthew [Vis alle 8 forfattere av denne artikkelen] (2018). Endogenous nitric oxide synthase (NOS) activity reduces staphylococcal lifespan during stationary phase.
• 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.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2018). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus.
• Olsbu, Inger Kirstine; Chaudhari, Sujata; Thomas, Vinai Chittezham & Hersleth, Hans-Petter (2018). Potential reductase partner for Staphylococcus aureus Nitric Oxide Synthase.
• Olsbu, Inger Kirstine; Chaudhari, Sujata; Thomas, Vinai Chittezham & Hersleth, Hans-Petter (2018). Potential reductase partner for Staphylococcus aureus Nitric Oxide Synthase. NBS-nytt. ISSN 0801-3535. s. 40–40.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network. NBS-nytt. ISSN 0801-3535. s. 40–40.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network.
• Johannesen, Hedda; Hammerstad, Marta; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III.
• 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.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Enzyme activation by a flavoprotein redox network .
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
• Hersleth, Hans-Petter (2017). Combining Protein X-ray Crystallography and in situ Single- Crystal UV-Vis and Raman Spectroscopy to Grasp the True Structure of Haem- and Flavoproteins.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class IB ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
• Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus.
• 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.
• 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.
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III in Bacillus cereus.
• 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.
• Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). A research-inspired biochemistry laboratory module – Combining, expression,purification, crystallisation, structure solving and characterisation of a flavdoxin-like protein. NBS-nytt. ISSN 0801-3535. s. 63–63.
• Hersleth, Hans-Petter (2017). A research-inspired biochemistry laboratory module – Combining, expression,purification, crystallisation, structure solving and characterisation of a flavdoxin-like protein.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib RNR by a flavodoxin reductase in B. cereus. NSB-nytt. s. 105–105.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
• Hammerstad, Marta; Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter & Zaltariov, Mirela-Fernanda [Vis alle 9 forfattere av denne artikkelen] (2017). INHIBITION OF CLASS 1A RIBONUCLEOTIDE REDUCTASE, AND A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE. Vis sammendrag

  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

• 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.
• Lofstad, Marie; Gudim, Ingvild; Kjendseth, Åsmund Røhr; Andersen, Niels Højmark; van Beek, Wouter & Andersson, K. Kristoffer [Vis alle 7 forfattere av denne artikkelen] (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.
• Johannesen, Hedda; Hersleth, Hans-Petter; Logan, Derek; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A structural and functional investigation of Ribonucleotide reductase Class III in Bacillus cereus- Investigating the interaction and mechanism of the NrdD and NrdG complex.
• Johannesen, Hedda; Hersleth, Hans-Petter; Logan, Derek; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A structural and functional investigation of Ribonucleotide reductase Class III in Bacillus cereus- Investigating the interaction and mechanism of the NrdD and NrdG complex .
• Hersleth, Hans-Petter (2016). Probing enzyme activation networks - structural and functional studies of flavoproteins in Bacillus cereus.
• Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter; Hammerstad, Marta & Solomon, Edward I. [Vis alle 7 forfattere av denne artikkelen] (2016). A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE BY CD AND MCD SPECTROSCOPY.
• 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.
• 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.
• 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.
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A Structural and Functional Investigation of Ribonucleotide Reductase Class III In Bacillus Cereus.
• Hersleth, Hans-Petter; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr; Van beek, Wouter & Giullaume, Pompidor [Vis alle 11 forfattere av denne artikkelen] (2016). Grasping the True Structure of Haem- and Flavoproteins – Combining X-RAY Crystallography and In Situ Single-Crystal UV-VIS and Raman Spectroscopy.
• Hersleth, Hans-Petter (2016). Probing enzyme activation networks - structural and functional studies of flavoproteins in Bacillus cereus.
• Lofstad, Marie; Gudim, Ingvild; Van beek, Wouter; Pompidor, Guillaume; Kjendseth, Åsmund Røhr & Andersson, K. Kristoffer [Vis alle 7 forfattere av denne artikkelen] (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.
• Lofstad, Marie; Gudim, Ingvild; Van beek, Wouter; Pompidor, Guillaume; Kjendseth, Åsmund Røhr & Andersson, K. Kristoffer [Vis alle 7 forfattere av denne artikkelen] (2016). Radiation damage of haem- and flavoproteins – combining X-ray crystallography and single-crystal UV-Vis and Raman spectroscopy.
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A structural and functional investigation of Ribonucleotide reductase Class III in Bacillus cereus. NBS-nytt. ISSN 0801-3535. s. 65–65.
• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2016). Structural Insight into the Function of Ribonucleotide Reductase. NBS-nytt. ISSN 0801-3535. s. 64–64.
• Gudim, Ingvild; Lofstad, Marie; Andersson, K. Kristoffer; Hammerstad, Marta & Hersleth, Hans-Petter (2016). Probing enzyme activation networks ‐ structural and functional studies of flavoproteins in Bacillus cereus. NBS-nytt. ISSN 0801-3535. s. 45–45.
• Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Hammerstad, Marta & Solomon, Edward I. [Vis alle 7 forfattere av denne artikkelen] (2016). A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy. NBS-nytt. ISSN 0801-3535. s. 45–45.
• Hammerstad, Marta; Hersleth, Hans-Petter; Lofstad, Marie; Johannesen, Hedda; Tomter, Ane Berg & Røhr, Åsmund Kjendseth [Vis alle 7 forfattere av denne artikkelen] (2016). Structural Insight into the Function of Ribonucleotide Reductase.
• Hersleth, Hans-Petter (2016). Probing enzyme activation networks ‐ structural and functional studies of flavoproteins in Bacillus cereus.
• Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Van beek, Wouter; Pompidor, Guillaume & Andersson, K. Kristoffer (2015). Combining X-ray crystallography and in situ single-crystal UV-Vis and Raman spectroscopy to study redox proteins. http://www.esrf.eu/files/live/sites/www/files/UsersAndScience/Experiments/CRG/BM01/SNSeminar/Program.pdf. 1, s. 2–2.
• Hersleth, Hans-Petter (2015). Combining X-ray crystallography and in situ single-crystal UV-Vis and Raman spectroscopy to study redox proteins.
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structures of ferredoxin/flavodoxin-NADP(H) oxidoreductases in Bacillus cereus.
• Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hammerstad, Marta; Hersleth, Hans-Petter & Solomon, Edward I. [Vis alle 7 forfattere av denne artikkelen] (2015). A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy.
• Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Van beek, Wouter; Pompidor, Guillaume & Andersson, K. Kristoffer (2015). Combining X-ray crystallography and in situ single-crystal UV-Vis and Raman spectroscopy to study haem- and flavoproteins. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. A71, s. s489–s489.
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structures of ferredoxin/flavodoxin-NADP(H) oxidoreductases in Bacillus cereus. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. A71, s. s205–s205. doi: 10.1107/S2053273315096916.
• Hersleth, Hans-Petter & Røhr, Åsmund Kjendseth (2015). Teaching a general molecular bioscience master course as a project ”From gene to structure”. .
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structure of a ferredoxin/flavodoxin-NADP(H) oxidoreductase in Bacillus cereus. NBS-nytt. ISSN 0801-3535. s. 96–96.
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structure of a ferredoxin/flavodoxin-NADP(H) oxidoreductase in Bacillus cereus.
• Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2015). On the way to determine the structure of a ferredoxin in B. cereus. Vis sammendrag

  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.
• Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Van Beek, Wouter; Diadkin, Vadim; Wiker, Geir & Chernyshov, Dmitry [Vis alle 7 forfattere av denne artikkelen] (2015). Combining Protein X-Ray Crystallography and Single-Crystal Spectroscopy - A New in situ Setup at BM01A at ESRF. Vis sammendrag

  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.

• 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.
• 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.
• Hersleth, Hans-Petter; Hammerstad, Marta & Dahlen, Sondov Åsmundson Braathen (2021). Structure and function of FNR1 from Bacillus cereus undergoing a mutation. Universitetet i Oslo.
• 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.
• Olsbu, Inger Kirstine; Hersleth, Hans-Petter & Sørlie, Morten (2018). Substrate recognition and redox partner identification in nitric oxide synthases. Reprosentralen, University of Oslo. ISSN 1501-7710.
• 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. ISSN 1501-7710.
• 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.
• 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. ISSN 1501-7710.
• Monka, Susanne; Andersson, K. Kristoffer; Hersleth, Hans-Petter & Hammerstad, Marta (2015). Structural and functional characterisation of ferredoxins in Bacillus cereus. Universitetet i Oslo.
• Wu, Bernt; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2015). Purification and characterization of Flavohemoglobin A flavoheme enzyme . Universitetet i Oslo.

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