Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion \( {\text{O}}^{{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle-}$}}{ \cdot } }}_{2} \) 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, ε=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 Na₂IrCl₆-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.