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.