title: Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase.
abstract: Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mössbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.
authors.affiliation: Section of Hematology Research, Department of Biochemistry and Molecular Biology, and Biomedical Mass Spectrometry and Functional Proteomics Facility, Mayo Clinic, Rochester, Minnesota 55905, USA.
authors.forename: T
authors.initials: T
authors.lastname: Jovanović
authors.forename: C
authors.initials: C
authors.lastname: Ascenso
authors.forename: K R
authors.initials: KR
authors.lastname: Hazlett
authors.forename: R
authors.initials: R
authors.lastname: Sikkink
authors.forename: C
authors.initials: C
authors.lastname: Krebs
authors.forename: R
authors.initials: R
authors.lastname: Litwiller
authors.forename: L M
authors.initials: LM
authors.lastname: Benson
authors.forename: I
authors.initials: I
authors.lastname: Moura
authors.forename: J J
authors.initials: JJ
authors.lastname: Moura
authors.forename: J D
authors.initials: JD
authors.lastname: Radolf
authors.forename: B H
authors.initials: BH
authors.lastname: Huynh
authors.forename: S
authors.initials: S
authors.lastname: Naylor
authors.forename: F
authors.initials: F
authors.lastname: Rusnak
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D033862:Iron-Binding Proteins; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013482:Superoxide Dismutase / Q000378:metabolism; D033863:Transferrin-Binding Proteins; D014210:Treponema pallidum / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D011994:Recombinant Proteins; D033863:Transferrin-Binding Proteins; D010088:Oxidoreductases; C121849:superoxide reductase; D013482:Superoxide Dismutase; C414179:nlr protein, Desulfovibrio gigas