Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase

Treponema pallidum, the causative agent of venereal syphilis, is a microaer ophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate subst antial 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 sup eroxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-redu cing anaerobes shown to possess superoxide reductase activity. To analyze t he potential role of neelaredoxin in treponemal oxidative defense, we exami ned the biochemical, spectroscopic, and antioxidant properties of recombina nt T, pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. p allidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containi ng, on average, 0.7 iron atoms/subunit. Mossbauer and EPR analysis of the p urified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully o xidized form, obtained by the addition of K-3(Fe(CN)(6)), exhibits an optic al spectrum with absorbances at 280, 320, and 656 nm; the last feature is r esponsible for the protein's blue color, which disappears upon ascorbate re duction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzy matic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consis tent with it being a superoxide reductase. This finding, the first descript ion of a T. pallidum iron-binding protein, indicates that the syphilis spir ochete copes with oxidative stress via a primitive mechanism, which, thus f ar, has not been described in pathogenic bacteria.