title: Semiquinone-induced maturation of Bacillus anthracis ribonucleotide reductase by a superoxide intermediate.
abstract: Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides, and represent the only de novo pathway to provide DNA building blocks. Three different classes of RNR are known, denoted I-III. Class I RNRs are heteromeric proteins built up by α and β subunits and are further divided into different subclasses, partly based on the metal content of the β-subunit. In subclass Ib RNR the β-subunit is denoted NrdF, and harbors a manganese-tyrosyl radical cofactor. The generation of this cofactor is dependent on a flavodoxin-like maturase denoted NrdI, responsible for the formation of an active oxygen species suggested to be either a superoxide or a hydroperoxide. Herein we report on the magnetic properties of the manganese-tyrosyl radical cofactor of Bacillus anthracis NrdF and the redox properties of B. anthracis NrdI. The tyrosyl radical in NrdF is stabilized through its interaction with a ferromagnetically coupled manganese dimer. Moreover, we show through a combination of redox titration and protein electrochemistry that in contrast to hitherto characterized NrdIs, the B. anthracis NrdI is stable in its semiquinone form (NrdIsq) with a difference in electrochemical potential of ∼110 mV between the hydroquinone and semiquinone state. The under anaerobic conditions stable NrdIsq is fully capable of generating the oxidized, tyrosyl radical-containing form of Mn-NrdF when exposed to oxygen. This latter observation strongly supports that a superoxide radical is involved in the maturation mechanism, and contradicts the participation of a peroxide species. Additionally, EPR spectra on whole cells revealed that a significant fraction of NrdI resides in its semiquinone form in vivo, underscoring that NrdIsq is catalytically relevant.
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University SE-10691 Stockholm, Sweden and.
authors.forename: Gustav
authors.initials: G
authors.lastname: Berggren
authors.affiliation: Laboratoire de Chimie et Biologie des Métaux (UMR 5249), CEA-Grenoble, 17, rue des Martyrs, F-38057 Grenoble, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Duraffourg
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University SE-10691 Stockholm, Sweden and.
authors.forename: Margareta
authors.initials: M
authors.lastname: Sahlin
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University SE-10691 Stockholm, Sweden and. Electronic address: britt-marie.sjoberg@dbb.su.se.
authors.forename: Britt-Marie
authors.initials: BM
authors.lastname: Sjöberg
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry; D001408:Bacillus anthracis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D004566:Electrodes; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D008280:Magnetics; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011809:Quinones / Q000737:chemistry*; D012262:Ribonucleoside Diphosphate Reductase / Q000737:chemistry* / Q000235:genetics*; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013481:Superoxides / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005609:Free Radicals; D008670:Metals; D011809:Quinones; D013481:Superoxides; D008345:Manganese; C114872:NrdF protein, bacteria; D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase; D010100:Oxygen
keywords: Bacillus; Cyclic Voltammetry; Free Radicals; Manganese; Maturase; NrdF; NrdI; Ribonucleotide Reductase; Semiquinone; Superoxide Ion