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  • 1. Ahlstrand, Tuuli
    et al.
    Torittu, Annamari
    Elovaara, Heli
    Välimaa, Hannamari
    Pöllänen, Marja T.
    Kasvandik, Sergo
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ihalin, Riikka
    Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake2018In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 9, no 1, p. 1205-1223Article in journal (Refereed)
    Abstract [en]

    Naturally competent bacteria acquire DNA from their surroundings to survive in nutrient-poor environments and incorporate DNA into their genomes as new genes for improved survival. The secretin HofQ from the oral pathogen Aggregatibacter actinomycetemcomitans has been associated with DNA uptake. Cytokine sequestering is a potential virulence mechanism in various bacteria and may modulate both host defense and bacterial physiology. The objective of this study was to elucidate a possible connection between natural competence and cytokine uptake in A. actinomycetemcomitans. The extramembranous domain of HofQ (emHofQ) was shown to interact with various cytokines, of which IL-8 exhibited the strongest interaction. The dissociation constant between emHofQ and IL-8 was 43nM in static settings and 2.4M in dynamic settings. The moderate binding affinity is consistent with the hypothesis that emHofQ recognizes cytokines before transporting them into the cells. The interaction site was identified via crosslinking and mutational analysis. By structural comparison, relateda type I KH domain with a similar interaction site was detected in the Neisseria meningitidis secretin PilQ, which has been shown to participate in IL-8 uptake. Deletion of hofQ from the A. actinomycetemcomitans genome decreased the overall biofilm formation of this organism, abolished the response to cytokines, i.e., decreased eDNA levels in the presence of cytokines, and increased the susceptibility of the biofilm to tested -lactams. Moreover, we showed that recombinant IL-8 interacted with DNA. These results can be used in further studies on the specific role of cytokine uptake in bacterial virulence without interfering with natural-competence-related DNA uptake.

  • 2.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berthold, Catrine
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A Dynamic C-terminal Segment in the Mycobacterium tuberculosis Mn/Fe R2lox Protein can Assume a Helical Structure with Possible Functional ConsequencesManuscript (preprint) (Other academic)
  • 3.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berthold, Catrine L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A dynamic c terminal segment in the mycobacterium tuberculosis mn/fe r2lox protein can adopt a helical structure with possible functional consequences2012In: Chemistry and Biodiversity, ISSN 1612-1872, E-ISSN 1612-1880, Vol. 9, no 9, p. 1981-1988Article in journal (Refereed)
    Abstract [en]

    Mycobacterium tuberculosis R2-like ligand-binding oxidase (MtR2lox) belongs to a recently discovered group of proteins that are homologous to the ribonucleotide reductase R2 proteins. MtR2lox carries a heterodinuclear Mn/Fe cofactor and, unlike R2 proteins, a large ligand-binding cavity. A unique tyrosine-valine cross link is also found in the vicinity of the active site. To date, all known structures of R2 and R2lox proteins show a disordered C-terminal segment. Here, we present two new crystal forms of MtR2lox, revealing an ordered helical C-terminal. The ability of alternating between an ordered and disordered state agrees well with bioinformatic analysis of the protein sequence. Interestingly, ordering of the C-terminal helix shields a large positively charged patch on the protein surface, potentially used for interaction with other cellular components. We hypothesize that the dynamic C-terminal segment may be involved in control of protein function in vivo.

  • 4.
    Andersson, Charlotta S
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A Mycobacterium tuberculosis ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 1091-6490, Vol. 106, no 14, p. 5633-8Article in journal (Refereed)
    Abstract [en]

    Chlamydia trachomatis R2c is the prototype for a recently discovered group of ribonucleotide reductase R2 proteins that use a heterodinuclear Mn/Fe redox cofactor for radical generation and storage. Here, we show that the Mycobacterium tuberculosis protein Rv0233, an R2 homologue and a potential virulence factor, contains the heterodinuclear manganese/iron-carboxylate cofactor but displays a drastic remodeling of the R2 protein scaffold into a ligand-binding oxidase. The first structural characterization of the heterodinuclear cofactor shows that the site is highly specific for manganese and iron in their respective positions despite a symmetric arrangement of coordinating residues. In this protein scaffold, the Mn/Fe cofactor supports potent 2-electron oxidations as revealed by an unprecedented tyrosine-valine crosslink in the active site. This wolf in sheep's clothing defines a distinct functional group among R2 homologues and may represent a structural and functional counterpart of the evolutionary ancestor of R2s and bacterial multicomponent monooxygenases.

  • 5.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundgren, Camilla A. K.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Magnúsdóttir, Auður
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Martinez Molina, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The Mycobacterium tuberculosis Very-Long-Chain Fatty Acyl-CoA Synthetase: Structural Basis for Housing Lipid Substrates Longer than the Enzyme2012In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 20, no 6, p. 1062-1070Article in journal (Refereed)
    Abstract [en]

    The Mycobacterium tuberculosis acid-induced operon MymA encodes the fatty acyl-CoA synthetase FadD13 and is essential for virulence and intracellular growth of the pathogen. Fatty acyl-CoA synthetases activate lipids before entering into the metabolic pathways and are also involved in transmembrane lipid transport. Unlike soluble fatty acyl-CoA synthetases, but like the mammalian integral-membrane very-long-chain acyl-CoA synthetases, FadD13 accepts lipid substrates up to the maximum length tested (C-26). Here, we show that FadD13 is a peripheral membrane protein. The structure and mutational studies reveal an arginine- and aromatic-rich surface patch as the site for membrane interaction. The protein accommodates a hydrophobic tunnel that extends from the active site toward the positive patch and is sealed by an arginine-rich lid-loop at the protein surface. Based on this and previous data, we propose a structural basis for accommodation of lipid substrates longer than the enzyme and transmembrane lipid transport by vectorial CoA-esterification.

  • 6.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhrström, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Popović-Bijelić, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The manganese ion of the heterodinuclear Mn/Fe cofactor in Chlamydia trachomatis ribonucleotide reductase R2c is located at metal position 1.2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 1, p. 123-125Article in journal (Refereed)
    Abstract [en]

    The essential catalytic radical of Class-I ribonucleotide reductase is generated and delivered by protein R2, carrying a dinuclear metal cofactor. A new R2 subclass, R2c, prototyped by the Chlamydia trachomatis protein was recently discovered. This protein carries an oxygen-activating heterodinuclear Mn(II)/Fe(II) metal cofactor and generates a radical-equivalent Mn(IV)/Fe(III) oxidation state of the metal site, as opposed to the tyrosyl radical generated by other R2 subclasses. The metal arrangement of the heterodinuclear cofactor remains unknown. Is the metal positioning specific, and if so, where is which ion located? Here we use X-ray crystallography with anomalous scattering to show that the metal arrangement of this cofactor is specific with the manganese ion occupying metal position 1. This is the position proximal to the tyrosyl radical site in other R2 proteins and consistent with the assumption that the high-valent Mn(IV) species functions as a direct substitute for the tyrosyl radical.

  • 7.
    Andersson, Martin E
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rinaldo-Matthis, Agnes
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Blodig, Wolfgang
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liang, Yuhe
    Persson, Bert Ove
    Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Su, Xiao-Dong
    Nordlund, Pär
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural and mutational studies of the carboxylate cluster in iron-free ribonucleotide reductase R2.2004In: Biochemistry, ISSN 0006-2960, Vol. 43, no 24, p. 7966-72Article in journal (Refereed)
    Abstract [en]

    The R2 protein of ribonucleotide reductase features a di-iron site deeply buried in the protein interior. The apo form of the R2 protein has an unusual clustering of carboxylate side chains at the empty metal-binding site. In a previous study, it was found that the loss of the four positive charge equivalents of the diferrous site in the apo protein appeared to be compensated for by the protonation of two histidine and two carboxylate side chains. We have studied the consequences of removing and introducing charged residues on the local hydrogen-bonding pattern in the region of the carboxylate cluster of Corynebacterium ammoniagenes and Escherichia coli protein R2 using site-directed mutagenesis and X-ray crystallography. The structures of the metal-free forms of wild-type C. ammoniagenes R2 and the mutant E. coli proteins D84N, S114D, E115A, H118A, and E238A have been determined and their hydrogen bonding and protonation states have been structurally assigned as far as possible. Significant alterations to the hydrogen-bonding patterns, protonation states, and hydration is observed for all mutant E. coli apo proteins as compared to wild-type apo R2. Further structural variations are revealed by the wild-type apo C. ammoniagenes R2 structure. The protonation and hydration effects seen in the carboxylate cluster appear to be due to two major factors: conservation of the overall charge of the site and the requirement of electrostatic shielding of clustered carboxylate residues. Very short hydrogen-bonding distances between some protonated carboxylate pairs are indicative of low-barrier hydrogen bonding.

  • 8. Andersson, ME
    et al.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rinaldo-Matthis, Agnes
    Andersson, KK
    Sjöberg, BM
    Nordlund, Pär
    The crystal structure of an azide complex of the diferrous R2 subunit of ribonucleotide reductase displays a novel carboxylate shift with important mechanistic implications for diiron-catalyzed oxygen activation1999In: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, ISSN 0002-7863, Vol. 121, no 11, p. 2346-2352Article in journal (Refereed)
  • 9.
    Assarsson, Maria
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersson, M E
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Persson, B O
    Sahlin, M
    Barra, A L
    Sjöberg, B M
    Nordlund, P
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli.2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 29, p. 26852-26859Article in journal (Refereed)
    Abstract [en]

    The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.

  • 10. Atanassova, Anelia
    et al.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zamble, Deborah B
    High throughput methods for analyzing transition metals in proteins on a microgram scale.2008In: Structural Proteomics: High-Throughput Methods, 2008Chapter in book (Refereed)
    Abstract [en]

    Transition metals are among the most common ligands that contribute to the biochemical and physiological properties of proteins. In the course of structural proteomic projects, the detection of transition metal cofactors prior to the determination of a high-resolution structure is extremely beneficial. This information can be used to select tractable targets from the proteomic pipeline because the presence of a metal often improves protein stability and can be used to help solve the phasing problem in x-ray crystallography. Recombinant proteins are often purified with substoichiometric amounts of metal loaded, so additional metal may be needed to obtain the homogeneous protein solution crucial for structural analysis. Furthermore, identifying a metal cofactor provides a clue about the nature of the biological role of an unclassified protein and can be applied with structural data in the assignation of a putative function. Many of the existing methods for transition metal analysis of purified proteins have limitations, which include a requirement for a large quantity of protein or a reliance on equipment with a prohibitive cost.The authors have developed two simple high throughput methods for identifying metalloproteins on a microgram scale. Each of the techniques has distinct advantages and can be applied to address divergent experimental goals. The first method, based on simple luminescence and colorimetric reactions, is fast, cheap, and semiquantitative. The second method, which employs HPLC separation, is accurate and affords unambiguous metal identification.

  • 11.
    Bennett, Matthew
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal structure of the essential biotin-dependent carboxylase AccA3 from Mycobacterium tuberculosis2017In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 7, no 5, p. 620-626Article in journal (Refereed)
    Abstract [en]

    Biotin-dependent acetyl-CoA carboxylases catalyze the committed step in type II fatty acid biosynthesis, the main route for production of membrane phospholipids in bacteria, and are considered a key target for antibacterial drug discovery. Here we describe the first structure of AccA3, an essential component of the acetyl-CoA carboxylase system in Mycobacterium tuberculosis (MTb). The structure, sequence comparisons, and modeling of ligand-bound states reveal that the ATP cosubstrate-binding site shows distinct differences compared to other bacterial and eukaryotic biotin carboxylases, including all human homologs. This suggests the possibility to design MTb AccA3 subtype-specific inhibitors.

  • 12.
    Berntsson, Ronnie P. -A.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Odegrip, Richard
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sehlén, Wilhelmina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Skaar, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Svensson, Linda M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Massad, Tariq
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haggard-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P22014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, p. 2725-2735Article in journal (Refereed)
    Abstract [en]

    The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 angstrom resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

  • 13.
    Berthold, Catrine L.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wang, He
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 34, p. 14247-14252Article in journal (Refereed)
    Abstract [en]

    ADP-ribosylation is a ubiquitous regulatory posttranslational modification involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dinitrogenase reductase-activating glycohydrolase (DraG), a dimanganese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity. DraG has long served as a model protein for ADP-ribosylhydrolases. Here, we present the crystal structure of DraG in the holo and ADP-ribose bound forms. We also present the structure of a reaction intermediate analogue and propose a detailed catalytic mechanism for protein de-ADP-ribosylation involving ring opening of the substrate ribose. In addition, the particular manganese coordination in DraG suggests a rationale for the enzyme's preference for manganese over magnesium, although not requiring a redox active metal for the reaction.

  • 14. Collins, Ruairi
    et al.
    Johansson, Ann-Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karlberg, Tobias
    Markova, Natalia
    van den Berg, Susanne
    Olesen, Kenneth
    Hammarstrom, Martin
    Flores, Alex
    Schuler, Herwig
    Schiavone, Lovisa Holmberg
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Arner, Elias S. J.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biochemical Discrimination between Selenium and Sulfur 1: A Single Residue Provides Selenium Specificity to Human Selenocysteine Lyase2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 1, p. e30581-Article in journal (Refereed)
    Abstract [en]

    Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.

  • 15. Covarrubias, Adrian Suarez
    et al.
    Bergfors, Terese
    Jones, T Alwyn
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural mechanics of the pH-dependent activity of beta-carbonic anhydrase from Mycobacterium tuberculosis.2006In: J Biol Chem, ISSN 0021-9258, Vol. 281, no 8, p. 4993-9Article in journal (Refereed)
  • 16.
    Covarrubias, Adrian Suarez
    et al.
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Högbom, Martin
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Bergfors, Terese
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Carroll, Paul
    Institute for Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London.
    Mannerstedt, Karin
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Oscarson, Stefan
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Parish, Tanya
    Institute for Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London.
    Jones, T Alwyn
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Mowbray, Sherry L
    Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala, Sweden.
    Structural, biochemical, and in vivo investigations of the threonine synthase from Mycobacterium tuberculosis.2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 381, no 3, p. 622-33Article in journal (Refereed)
    Abstract [en]

    Threonine biosynthesis is a general feature of prokaryotes, eukaryotic microorganisms, and higher plants. Since mammals lack the appropriate synthetic machinery, instead obtaining the amino acid through their diet, the pathway is a potential focus for the development of novel antibiotics, antifungal agents, and herbicides. Threonine synthase (TS), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the final step in the pathway, in which L-homoserine phosphate and water are converted into threonine and inorganic phosphate. In the present publication, we report structural and functional studies of Mycobacterium tuberculosis TS, the product of the rv1295 (thrC) gene. The structure gives new insights into the catalytic mechanism of TSs in general, specifically by suggesting the direct involvement of the phosphate moiety of the cofactor, rather than the inorganic phosphate product, in transferring a proton from C4' to C(gamma) in the formation of the alphabeta-unsaturated aldimine. It further provides a basis for understanding why this enzyme has a higher pH optimum than has been reported elsewhere for TSs and gives rise to the prediction that the equivalent enzyme from Thermus thermophilus will exhibit similar behavior. A deletion of the relevant gene generated a strain of M. tuberculosis that requires threonine for growth; such auxotrophic strains are frequently attenuated in vivo, indicating that TS is a potential drug target in this organism.

  • 17.
    Griese, Julia J.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Uppsala University, Sweden.
    Branca, Rui M. M.
    Srinivas, Vivek
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ether cross-link formation in the R2-like ligand-binding oxidase2018In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 23, no 6, p. 879-886Article in journal (Refereed)
    Abstract [en]

    R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of Mn-II and Fe-II in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine-valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.

  • 18.
    Griese, Julia J.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Uppsala University, Sweden.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion: Q-XAD2019In: acta crystallographica section d structural biology, ISSN 2059-7983, Vol. 75, p. 764-771Article in journal (Refereed)
    Abstract [en]

    Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a heterodinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of high-quality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided.

  • 19.
    Griese, Julia J.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    X ray reduction correlates with soaking accessibility as judged from four non crystallographically related diiron sites2012In: METALLOMICS, ISSN 1756-5901, Vol. 4, no 9, p. 894-898Article in journal (Refereed)
    Abstract [en]

    X-ray crystallography is extensively used to determine the atomic structure of proteins and their cofactors. Though a commonly overlooked problem, it has been shown that structural damage to a redox active metal site may precede loss of diffractivity by more than an order of magnitude in X-ray dose. Therefore the risk of misassigning redox states is great. Adequate treatment and consideration of this issue is of paramount importance in metalloprotein science, from experimental design to interpretation of the data and results. Some metal sites appear to be much more amenable to reduction than others, but the underlying processes are poorly understood. Here, we have analyzed the four non-crystallographically related diiron sites in a crystal of the ribonucleotide reductase R2F protein from Corynebacterium ammoniagenes. We conclude that the amount of X-ray reduction a metal site suffers correlates with its soaking accessibility. This direct observation supports the hypothesis that a diffusion component is involved in the X-ray reduction process.

  • 20.
    Griese, Julia J.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Uppsala University, Sweden.
    Kositzki, Ramona
    Haumann, Michael
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine-valine ether cross-link formation in the R2-like ligand-binding oxidase2019In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 24, no 2, p. 211-221Article in journal (Refereed)
    Abstract [en]

    R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O-2) activation, catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O-2, but prefers manganese in the presence of O-2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O-2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O-2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.

  • 21.
    Griese, Julia J.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kositzki, Ramona
    Schrapers, Peer
    Branca, Rui M. M.
    Nordström, Anders
    Lehtiö, Janne
    Haumann, Michael
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 42, p. 25254-25272Article in journal (Refereed)
    Abstract [en]

    Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn(II) and Fe(II) in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R. M., Lehtiö, J., Gräslund, A., Lubitz, W., Siegbahn, P. E., and Högbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 17189-17194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability.

  • 22.
    Griese, Julia J.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Roos, Katarina
    Stockholm University, Faculty of Science, Department of Physics.
    Cox, Nicholas
    Shafaat, Hannah S.
    Branca, Rui M. M.
    Lehtio, Janne
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lubitz, Wolfgang
    Siegbahn, Per E. M.
    Stockholm University, Faculty of Science, Department of Physics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 43, p. 17189-17194Article in journal (Refereed)
    Abstract [en]

    Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn-II < Fe-II < Ni-II < Co-II < Cu-II > Zn-II). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from MnII and FeII in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds FeII over MnII as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.

  • 23.
    Griese, Julia J.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Srinivas, Vivek
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Assembly of nonheme Mn/Fe active sites in heterodinuclear metalloproteins2014In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, no 6, p. 759-774Article, review/survey (Refereed)
    Abstract [en]

    The ferritin superfamily contains several protein groups that share a common fold and metal coordinating ligands. The different groups utilize different dinuclear cofactors to perform a diverse set of reactions. Several groups use an oxygen-activating di-iron cluster, while others use di-manganese or heterodinuclear Mn/Fe cofactors. Given the similar primary ligand preferences of Mn and Fe as well as the similarities between the binding sites, the basis for metal specificity in these systems remains enigmatic. Recent data for the heterodinuclear cluster show that the protein scaffold per se is capable of discriminating between Mn and Fe and can assemble the Mn/Fe center in the absence of any potential assembly machineries or metal chaperones. Here we review the current understanding of the assembly of the heterodinuclear cofactor in the two different protein groups in which it has been identified, ribonucleotide reductase R2c proteins and R2-like ligand-binding oxidases. Interestingly, although the two groups form the same metal cluster they appear to employ partly different mechanisms to assemble it. In addition, it seems that both the thermodynamics of metal binding and the kinetics of oxygen activation play a role in achieving metal specificity.

  • 24.
    Gräve, Kristine
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bennett, Matthew D.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase reveals mechanism of substrate binding and metal catalysis2019In: Communications biology, ISSN 2399-3642, Vol. 2, article id 175Article in journal (Refereed)
    Abstract [en]

    Tuberculosis causes over one million yearly deaths, and drug resistance is rapidly developing. Mycobacterium tuberculosis phosphatidylinositol phosphate synthase (PgsA1) is an integral membrane enzyme involved in biosynthesis of inositol-derived phospholipids required for formation of the mycobacterial cell wall, and a potential drug target. Here we present three crystal structures of M. tuberculosis PgsA1: in absence of substrates (2.9 angstrom), in complex with Mn2+ and citrate (1.9 angstrom), and with the CDP-DAG substrate (1.8 angstrom). The structures reveal atomic details of substrate binding as well as coordination and dynamics of the catalytic metal site. In addition, molecular docking supported by mutagenesis indicate a binding mode for the second substrate, D-myo-inositol-3-phosphate. Together, the data describe the structural basis for M. tuberculosis phosphatidylinositol phosphate synthesis and suggest a refined general catalytic mechanism-including a substrate-induced carboxylate shift-for Class I CDP-alcohol phosphotransferases, enzymes essential for phospholipid biosynthesis in all domains of life.

  • 25.
    Hammerstad, Marta
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Oslo, Norway.
    Rohr, Asmund K.
    Andersen, Niels H.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersson, K. Kristoffer
    The class Ib ribonucleotide reductase from Mycobacterium tuberculosis has two active R2F subunits2014In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, no 6, p. 893-902Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class Ib RNRs require either a diiron-tyrosyl radical (Y center dot) or a dimanganese-Y center dot cofactor in their R2F subunit to initiate ribonucleotide reduction in the R1 subunit. Mycobacterium tuberculosis, the causative agent of tuberculosis, contains two genes, nrdF1 and nrdF2, encoding the small subunits R2F-1 and R2F-2, respectively, where the latter has been thought to serve as the only active small subunit in the M. tuberculosis class Ib RNR. Here, we present evidence for the presence of an active Fe (2) (III) -Y center dot cofactor in the M. tuberculosis RNR R2F-1 small subunit, supported and characterized by UV-vis, X-band electron paramagnetic resonance, and resonance Raman spectroscopy, showing features similar to those for the M. tuberculosis R2F-2-Fe (2) (III) -Y center dot cofactor. We also report enzymatic activity of Fe (2) (III) -R2F-1 when assayed with R1, and suggest that the active M. tuberculosis class Ib RNR can use two different small subunits, R2F-1 and R2F-2, with similar activity.

  • 26.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear-an intricate bioinorganic workaround to use different metals for the same reaction2011In: METALLOMICS, ISSN 1756-5901, Vol. 3, no 2, p. 110-120Article, review/survey (Refereed)
    Abstract [en]

    The ferritin-like superfamily comprises of several protein groups that utilize dinuclear metal sites for various functions, from iron storage to challenging oxidations of substrates. Ribonucleotide reductase R2 proteins use the metal site for the generation of a free radical required for the reduction of ribonucleotides to deoxyriboinucleotides, the building blocks of DNA. This ubiquitous and essential reaction has been studied for over four decades and the R2 proteins were, until recently, generally believed to employ the same cofactor and mechanism for radical generation. In this reaction, a stable tyrosyl radical is produced following activation and cleavage of molecular oxygen at a dinuclear iron site in the protein. Discoveries in the last few years have now firmly established that the radical generating reaction is not conserved among the R2 proteins but that different subgroups, that are structurally very similar, instead employ di-manganese or heterodinuclear Mn-Fe cofactors as radical generators. This is remarkable considering that the protein must exercise a strict control over oxygen activation, reactive metal-oxygen intermediate species and the resulting redox potential of the produced radical equivalent. Given the differences in redox properties between Mn and Fe, use of a different metal for this reaction requires associated adaptations of the R2 protein scaffold and the activation mechanism. Further analysis of the differences in protein sequence between R2 subgroups have also led to the discovery of new groups of R2-like proteins with completely different functions, expanding the chemical repertoire of the ferritin-like superfamily. This review describes the discoveries leading up to the identification of the different Mn-containing R2 protein groups and our current understanding of them. Hypotheses regarding the biochemical rationale to develop these chemically complex alternative solutions are also discussed.

  • 27.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nitrogenase Mechanism. A dynamic tool for nitrogen reduction2014In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 345, no 6204, p. 1568-1568Article in journal (Other academic)
    Abstract [en]

    Even though nitrogen makes up almost 80% of the atmosphere, it is a limiting nutrient for biomass production. The low reactivity of nitrogen gas (N2) is a result of its very strong, unpolarized triple bond. Nitrogenase is the only enzyme known that can break this bond to produce compounds such as ammonia (NH3) for use in biosynthetic pathways. The atomic structure of this amazing system has been known for more than two decades (1, 2), but the chemical mechanism of this central reaction remains unknown. In a biochemical and structural tour de force, on page 1620 of this issue, Spatzal et al. (3) report the crystal structure of carbon monoxide (CO) bound to the catalytic metal cluster of the enzyme. This work revealed an unexpected structural rearrangement of the cofactor.

  • 28.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Radical generation and stabilisation in ribonucleotide reductase R22003Doctoral thesis, monograph (Other academic)
    Abstract [en]

    Diiron carboxylate proteins contain a cofactor that consists of two iron atoms coordinated by carboxylate and histidine ligands. These proteins perform a multitude of chemical reactions in the cell that generally involve activation of molecular oxygen at the diiron site.

    Ribonucleotide reductase is the only enzyme that performs de novo synthesis of all four deoxyribonucleotides, the building blocks of DNA. The R2 protein of Class-I ribonucleotide reductase, which is a diiron carboxylate protein, utilises the high-valent iron-oxygen species generated at the diiron site to produce a stable tyrosyl radical required for enzymatic activity.

    In this work, X-ray crystallography and EPR are used to study the R2 protein from Escherichia coli with the goal of understanding its mechanism of oxygen activation, radical generation and radical stabilisation. Based on these studies a detailed structural mechanism is proposed, which might be common to several oxygen activating diiron carboxylate proteins. The orientation of the active radical species in R2 has also been determined. In addition, these results provide a rationale for the unusual stability of the radical.

    Crystal structures of R2 proteins from two other species, Corynebacterium ammoniagenes and Chlamydia trachomatis, have also been solved.

    The C. ammoniagenes protein has been reported to be manganese-dependent. The results presented here, however, support dependence of iron and not manganese.

    Sequence alignments indicate that the chlamydial R2s lack the, otherwise conserved, radical harbouring tyrosine. This is confirmed by the structure, which also reveals other unique features in the diiron site. Hypotheses regarding the function of the protein and the reason for the differences are presented.

  • 29.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The manganese/iron-carboxylate proteins: what is what, where are they, and what can the sequences tell us?2010In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 15, no 3, p. 339-349Article in journal (Refereed)
    Abstract [en]

    The manganese/iron-carboxylate proteins make up a recently discovered group of proteins that contain a heterodinuclear Mn/Fe redox cofactor. The chemical potential of the heterodinuclear metal site is just starting to be characterized, but available data suggest that it may have capabilities for similarly versatile chemistry as the extensively studied diiron-carboxylate cofactor. The presently identified members of the manganese/iron-carboxylate proteins are all sequence homologues of the radical-generating R2 subunit of class I ribonucleotide reductase, canonically a diiron protein. They are also commonly misannotated as such in databases. In spite of the sequence similarity, the manganese/iron-carboxylate proteins form at least two functionally distinct groups, radical-generating ribonucleotide reductase subunits and ligand-binding Mn/Fe proteins. Here, the presently available sequences for the manganese/iron-carboxylate proteins are gathered, grouped, and analyzed. The analysis provides sequence determinants that allow group identification of new sequences on the single-protein level. Key differences between the groups are mapped on the known representative structures, providing clues to the structural prerequisites for metal specificity, cofactor formation, and difference in function. The organisms that encode manganese/iron-carboxylate proteins are briefly discussed; their environmental preference suggests that the Mn/Fe heterodinuclear cofactor is preferred by extremophiles and pathogens with a particularly high relative presence in Archaea.

  • 30.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersson, M E
    Nordlund, P
    Crystal structures of oxidized dinuclear manganese centres in Mn-substituted class I ribonucleotide reductase from Escherichia coli: carboxylate shifts with implications for O2 activation and radical generation.2001In: J Biol Inorg Chem, ISSN 0949-8257, Vol. 6, no 3, p. 315-23Article in journal (Refereed)
  • 31.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Collins, Ruairi
    van den Berg, Susanne
    Jenvert, Rose-Marie
    Karlberg, Tobias
    Kotenyova, Tetyana
    Flores, Alex
    Karlsson Hedestam, Gunilla B
    Schiavone, Lovisa Holmberg
    Crystal structure of conserved domains 1 and 2 of the human DEAD-box helicase DDX3X in complex with the mononucleotide AMP.2007In: J Mol Biol, ISSN 0022-2836, Vol. 372, no 1, p. 150-9Article in journal (Refereed)
  • 32.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eklund, Malin
    Nygren, Per-Ake
    Nordlund, Pär
    Structural basis for recognition by an in vitro evolved affibody.2003In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 100, no 6, p. 3191-6Article in journal (Refereed)
  • 33.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ericsson, Ulrika B
    Lam, Robert
    Bakali H, M Amin
    Kuznetsova, Ekaterina
    Nordlund, Pär
    Zamble, Deborah B
    A high throughput method for the detection of metalloproteins on a microgram scale.2005In: Mol Cell Proteomics, ISSN 1535-9476, Vol. 4, no 6, p. 827-34Article in journal (Refereed)
  • 34.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Galander, Marcus
    Andersson, Martin
    Kolberg, Matthias
    Hofbauer, Wulf
    Lassmann, Günter
    Nordlund, Pär
    Lendzian, Friedhelm
    Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-A x-ray data.2003In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 100, no 6, p. 3209-14Article in journal (Refereed)
  • 35.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Huque, Yasmin
    Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Nordlund, Pär
    Department of Molecular Biology and Functional Genomics.
    Crystal structure of the di-iron/radical protein of ribonucleotide reductase from Corynebacterium ammoniagenes.2002In: Biochemistry, ISSN 0006-2960, Vol. 41, no 4, p. 1381-9Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) is the enzyme performing de novo production of the four deoxyribonucleotides needed for DNA synthesis. All mammals as well as some prokaryotes express the class I enzyme which is an alpha(2)beta(2) protein. The smaller of the homodimers, denoted R2, contains a di-iron carboxylate site which, upon reaction with molecular oxygen, generates a stable tyrosyl radical needed for catalysis. The three-dimensional structure of the oxidized class Ib RNR R2 from Corynebacterium ammoniagenes has been determined at 1.85 A resolution and refined to an R-value of 15.8% (R(free) = 21.3%). In addition, structures of both the reduced iron-containing, and manganese-substituted protein have been solved. The C. ammoniagenes R2 has been proposed to be manganese-dependent. The present structure provides evidence that manganese is not oxidized by the protein, in agreement with recent biochemical data, and that no obvious structural abnormalities are seen in the oxidized and reduced iron-containing forms, giving further support that the protein is indeed an iron-dependent RNR R2. The di-manganese structure also provides an explanation for the magnetic properties of this site. The structure of the oxidized C. ammoniagenes R2 also reveals an additional water molecule bridging the radical and the iron site, which has not previously been seen in any other R2 structure and which might have important mechanistic implications.

  • 36.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ihalin, Riikka
    Functional and structural characteristics of bacterial proteins that bind host cytokines2017In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 8, no 8, p. 1592-1601Article, review/survey (Refereed)
    Abstract [en]

    Several human pathogens bind and respond to host cytokines, which can be considered a virulence mechanism that communicates defensive actions of the host to the pathogen. This review summarizes the current knowledge of bacterial cytokine-binding proteins, with a particular focus on their functional and structural characteristics. Many bacterial cytokine-binding proteins function in the development of infection and inflammation and mediate adhesion to host cells, suggesting multiple roles in pathogen-host interactions. The regions of the bacterial proteins that interact with host cytokines can display structural similarities to other proteins involved in cytokine signaling. However, there appears to be no central shared structural themes for bacterial cytokine-binding proteins, and they appear to possess structures that are different from the cytokine receptors of the host. Atomic-level information regarding receptor-cytokine interactions is needed to be able to disrupt these interactions and to elucidate the specific consequences of cytokine binding in a pathogen and host.

  • 37.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jäger, Katrin
    Robel, Ivonne
    Unge, Torsten
    Rohayem, Jacques
    The active form of the norovirus RNA-dependent RNA polymerase is a homodimer with cooperative activity2009In: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 90, no Pt 2, p. 281-91Article in journal (Refereed)
    Abstract [en]

    Norovirus (NV) is a leading cause of gastroenteritis worldwide and a major public health concern. So far, the replication strategy of NV remains poorly understood, mainly because of the lack of a cell system to cultivate the virus. In this study, the function and the structure of a key viral enzyme of replication, the RNA-dependent RNA polymerase (RdRp, NS7), was examined. The overall structure of the NV NS7 RdRp was determined by X-ray crystallography to a 2.3 A (0.23 nm) resolution (PDB ID 2B43), displaying a right-hand fold typical of the template-dependent polynucleotide polymerases. Biochemical analysis evidenced that NV NS7 RdRp is active as a homodimer, with an apparent K(d) of 0.649 microM and a positive cooperativity (Hill coefficient n(H)=1.86). Crystals of the NV NS7 homodimer displayed lattices containing dimeric arrangements with high shape complementarity statistics. This experimental data on the structure and function of the NV RdRp may set the cornerstone for the development of polymerase inhibitors to control the infection with NV, a medically relevant pathogen.

  • 38.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Pär
    A protein carboxylate coordinated oxo-centered tri-nuclear iron complex with possible implications for ferritin mineralization.2004In: FEBS Lett, ISSN 0014-5793, Vol. 567, no 2-3, p. 179-82Article in journal (Refereed)
  • 39.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Voevodskaya, Nina
    McClarty, Grant
    Gräslund, Astrid
    Nordlund, Pär
    The radical site in chlamydial ribonucleotide reductase defines a new R2 subclass.2004In: Science, ISSN 1095-9203, Vol. 305, no 5681, p. 245-8Article in journal (Refereed)
  • 40. Ingvarsson, Henrik
    et al.
    Maté, María J
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Portnoï, Denis
    Benaroudj, Nadia
    Alzari, Pedro M
    Ortiz-Lombardía, Miguel
    Unge, Torsten
    Insights into the inter-ring plasticity of caseinolytic proteases from the X-ray structure of Mycobacterium tuberculosis ClpP1.2007In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 63, no Pt 2, p. 249-59Article in journal (Refereed)
  • 41.
    Johansson, Ann-Louise
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carlsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hosler, Jonathan P
    Gennis, Robert B
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proton Uptake and pK(a) Changes in the Uncoupled Asn139Cys Variant of Cytochrome c Oxidase2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 5, p. 827-836Article in journal (Other academic)
    Abstract [en]

    Cytochrome c oxidase (CytcO) is a membrane-bound enzyme that links electron transfer from cytochrome c to O-2 to proton pumping across the membrane. Protons are transferred through specific pathways that connect the protein surface with the catalytic site as well as the proton input with the proton output sides. Results from earlier studies have shown that one site within the so-called D proton pathway, Asn139, located similar to 10 angstrom from the protein surface, is particularly sensitive to mutations that uncouple the O-2 reduction reaction from the proton pumping activity. For example, none of the Asn139Asp (charged) or Asn139Thr (neutral) mutant CytcOs pump protons, although the proton-uptake rates are unaffected. Here, we have investigated the Asn139Cys and Asn139Cys/Asp132Asn mutant CytcOs. In contrast to other structural variants investigated to date, the Cys side chain may be either neutral or negatively charged in the experimentally accessible pH range. The data show that the Asn139Cys and Asn139Asp mutations result in the same changes of the kinetic and thermodynamic parameters associated with the proton transfer. The similarity is not due to introduction of charge at position 139, but rather introduction of a protonatable group that modulates the proton connectivity around this position. These results illuminate the mechanism by which CytcO couples electron transfer to proton pumping.

  • 42.
    Johansson, Ann-Louise
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chakrabarty, Suman
    Berthold, Catrine L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Warshel, Arieh
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects2011In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1807, no 9, p. 1083-1094Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa(3) CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O(2) reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a pump site and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the HID kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from 02 reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F -> O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P -> F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F -> O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.

  • 43.
    Johansson, Ann-Louise
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Collins, Ruairi
    Arner, Elias S. J.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biochemical Discrimination between Selenium and Sulfur 2: Mechanistic Investigation of the Selenium Specificity of Human Selenocysteine Lyase2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 1, p. e30528-Article in journal (Refereed)
    Abstract [en]

    Selenium is an essential trace element incorporated into selenoproteins as selenocysteine. Selenocysteine (Sec) lyases (SCLs) and cysteine (Cys) desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys, respectively, and generally accept both substrates. Intriguingly, human SCL (hSCL) is specific for Sec even though the only difference between Sec and Cys is a single chalcogen atom. The crystal structure of hSCL was recently determined and gain-of-function protein variants that also could accept Cys as substrate were identified. To obtain mechanistic insight into the chemical basis for its substrate discrimination, we here report time-resolved spectroscopic studies comparing the reactions of the Sec-specific wild-type hSCL and the gain-of-function D146K/H389T variant, when given Cys as a substrate. The data are interpreted in light of other studies of SCL/CD enzymes and offer mechanistic insight into the function of the wild-type enzyme. Based on these results and previously available data we propose a reaction mechanism whereby the Sec over Cys specificity is achieved using a combination of chemical and physico-mechanical control mechanisms.

  • 44.
    Johansson, Ann-Louise
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carlsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 22, p. 8912-8917Article in journal (Refereed)
    Abstract [en]

    Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e. g., Asn, slows proton uptake by a factor of similar to 5,000. Here, we show that proton uptake at full speed (similar to 10(4) s(-1)) can be restored in the Asp-132-Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139-Thr) without compensating for the loss of the negative charge. This proton-uptake rate was insensitive to Zn2+ addition, which in the wildtype cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn2+ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139-Thr/Asp-132-Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.

  • 45.
    Johansson, Ann-Louise
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The role of acidic residues at the orifice of a proton pathway in cytochrome c oxidaseManuscript (preprint) (Other academic)
  • 46. Karlberg, Tobias
    et al.
    Collins, Ruairi
    van den Berg, Susanne
    Flores, Alex
    Hammarström, Martin
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holmberg Schiavone, Lovisa
    Uppenberg, Jonas
    Structure of human argininosuccinate synthetase.2008In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 64, no Pt 3, p. 279-86Article in journal (Refereed)
    Abstract [en]

    Argininosuccinate synthetase catalyzes the transformation of citrulline and aspartate into argininosuccinate and pyrophosphate using the hydrolysis of ATP to AMP and pyrophosphate. This enzymatic process constitutes the rate-limiting step in both the urea and arginine-citrulline cycles. Previous studies have investigated the crystal structures of argininosuccinate synthetase from bacterial species. In this work, the first crystal structure of human argininosuccinate synthetase in complex with the substrates citrulline and aspartate is presented. The human enzyme is compared with structures of argininosuccinate synthetase from bacteria. In addition, the structure also provides new insights into the function of the numerous clinical mutations identified in patients with type I citrullinaemia (also known as classic citrullinaemia).

  • 47. Kositzki, Ramona
    et al.
    Mebs, Stefan
    Marx, Jennifer
    Griese, Julia J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schuth, Nils
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stanford University, United States.
    Schünemann, Volker
    Haumann, Michael
    y Protonation State of MnFe and FeFe Cofactors in a Ligand-Binding Oxidase Revealed by X-ray Absorption, Emission, and Vibrational Spectroscopy and QM/MM Calculations2016In: Inorganic Chemistry, ISSN 0020-1669, E-ISSN 1520-510X, Vol. 55, no 19, p. 9869-9885Article in journal (Refereed)
    Abstract [en]

    Enzymes with a dimetalcarboxylate cofactor catalyze reactions among the top challenges in chemistry such as methane and dioxygen (O-2) activation. Recently described proteins bind a manganeseiron cofactor (MnFe) instead of the classical diiron cofactor (FeFe). Determination of atomic-level differences of homo- versus hetero-bimetallic cofactors is crucial to understand their diverse redox reactions. We studied a ligand-binding oxidase from the bacterium Geobacillus kaustophilus (R2lox) loaded with a FeFe or MnFe cofactor, which catalyzes O-2 reduction and an unusual tyrosinevaline ether cross-link formation, as revealed by X-ray crystallography. Advanced X-ray absorption, emission, and vibrational spectroscopy methods and quantum chemical and molecular mechanics calculations provided relative Mn/Fe contents, X-ray photoreduction kinetics, metalligand bond lengths, metalmetal distances, metal oxidation states, spin configurations, valence-level degeneracy, molecular orbital composition, nuclear quadrupole splitting energies, and vibrational normal modes for both cofactors. A protonation state with an axial water (H2O) ligand at Mn or Fe in binding site 1 and a metal-bridging hydroxo group (OH) in a hydrogen-bonded network is assigned. Our comprehensive picture of the molecular, electronic, and dynamic properties of the cofactors highlights reorientation of the unique axis along the MnOH2 bond for the Mn1(III) JahnTeller ion but along the Fe-mu OH bond for the octahedral Fe1(III). This likely corresponds to a more positive redox potential of the Mn(III)Fe(III) cofactor and higher proton affinity of its mu OH group. Refined model structures for the Mn(III)Fe(III) and Fe(III)Fe(III) cofactors are presented. Implications of our findings for the site-specific metalation of R2lox and performance of the O-2 reduction and cross-link formation reactions are discussed.

  • 48. Kutin, Yuri
    et al.
    Srinivas, Vivek
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fritz, Matthieu
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kositzki, Ramona
    Shafaat, Hannah S.
    Birrell, James
    Bill, Eckhard
    Haumann, Michael
    Lubitz, Wolfgang
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stanford University, United States.
    Griese, Julia J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cox, Nicholas
    Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins2016In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 162, p. 164-177Article in journal (Refereed)
    Abstract [en]

    A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of dass Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is undear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of Mn-II and Fe-II. Using EPR and Mfissbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess Mn-II promotes heterobimetallic cofactor assembly. In the absence of Fe-II, R2c cooperatively binds Mn-II at both metal sites, whereas R2lox does not readily bind Mn-II at either site. Heterometallic cofactor assembly is favored at substoichiometric Feu concentrations in R2lox. Fe-II and Mn-II likely bind to the protein in a stepwise fashion, with Feu binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of Mn-II by Fe-II at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular Mn-II/Fe-II concentrations in the host organisms from which they were isolated.

  • 49. Kwak, Young-Keun
    et al.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Colque-Navarro, Patricia
    Möllby, Roland
    Vécsey-Semjén, Beatrix
    Biological relevance of natural alpha-toxin fragments from Staphylococcus aureus2010In: Journal of Membrane Biology, ISSN 0022-2631, E-ISSN 1432-1424, Vol. 233, no 1-3, p. 93-103Article in journal (Refereed)
    Abstract [en]

    Serine proteases represent an essential part of cellular homeostasis by generating biologically active peptides. In bacteria, proteolysis serves two different roles: a major housekeeping function and the destruction of foreign or target cell proteins, thereby promoting bacterial invasion. In the process, other virulence factors such as exotoxins become affected. In Staphylococcus aureus culture supernatant, the pore-forming alpha-toxin is cleaved by the coexpressed V8 protease and aureolysin. The oligomerizing and pore-forming abilities of five such spontaneously occurring N- and C-terminal alpha-toxin fragments were studied. (3)H-marked alpha-toxin fragments bound to rabbit erythrocyte membranes but only fragments with intact C termini, missing 8, 12 and 71 amino acids from their N-terminal, formed stable oligomers. All isolated fragments induced intoxication of mouse adrenocortical Y1 cells in vitro, though the nature of membrane damage for a fragment, degraded at its C terminus, remained obscure. Only one fragment, missing the first eight N-terminal amino acids, induced irreversible intoxication of Y1 cells in the same manner as the intact toxin. Four of the isolated fragments caused swelling, indicating altered channel formation. Fragments missing 12 and 71 amino acids from the N terminus occupied the same binding sites on Y1 cell membranes, though they inhibited membrane damage caused by intact toxin. In conclusion, N-terminal deletions up to 71 amino acids are tolerated, though the kinetics of channel formation and the channel's properties are altered. In contrast, digestion at the C terminus results in nonfunctional species.

  • 50. Lendzian, F.
    et al.
    Voevodskaya, Nina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Galander, M.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The high-valent Fe(III)Fe(IV) center in class Ic ribonucleotide reductase of chlamydia trachomatis: EPR and ENDOR studies2007Conference paper (Other (popular science, discussion, etc.))
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