Superoxide reductases are a class of non-
haem iron enzymes which catalyse the monovalent reduction of the
superoxide anion O2- into
hydrogen peroxide and water. Treponema pallidum (Tp), the
syphilis spirochete, expresses the gene for a
superoxide reductase called
neelaredoxin, having the
iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp
rubredoxin. Spectroscopic characterization of this 6 kDa
protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric
iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp
rubredoxin, determined using a glassy
carbon electrode, was -76+/-5 mV. Reduced
rubredoxin can be efficiently reoxidized upon addition of Na(2)IrCl(6)-oxidized
neelaredoxin, in agreement with a direct electron transfer between the two
proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized
rubredoxin per one molecule of
neelaredoxin. In addition, in presence of a steady-state concentration of
superoxide anion, the physiological substrate of
neelaredoxin, reoxidation of
rubredoxin was also observed in presence of catalytic amounts of
superoxide reductase, and the rate of
rubredoxin reoxidation was shown to be proportional to the concentration of
neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a
rubredoxin from the
sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a
superoxide-mediated electron transfer between
rubredoxin and
neelaredoxin and confirmed the physiological character of this electron transfer reaction.