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

Abstract

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O : 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, e=6.9±0.4 mM À1 cm À1 . Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was À76±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 À1 . 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 À1 . 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.

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

“…[32]. Dv SOR was overexpressed and purified to homogeneity following the previously published procedures [2].…”

“…[32]. After overexpression of the gene in E. coli cells, a typical purification process involved a combination of affinity (Ni nitrilotriacetic acid resin, BioRad) and anion-exchange (MonoQ-HR5/ 5 resin, Pharmacia) chromatographies.…”

“…[32]. Sodium dithionite stock solution of 100 mM was first prepared in 1 M tris(hydroxymethyl)aminomethane (Tris)-HCl (pH 9.0) under argon, and then dilute solutions were made and used within a few hours.…”

“…Therefore, the presence of an electron donor is necessary to regenerate the ferrous active form and complete the catalytic cycle of the enzyme [10,19,[28][29][30][31]. Recently, several groups, including ours, have clearly established rubredoxin as the proximal electron donor to SORs in the case of Tp, Dv, P. furiosus, and A. fulgidus [4,[32][33][34][35][36].…”

“…Last year, we showed, using a different assay, kinetics evidence for a superoxide-mediated electron transfer from Tp (and Dv) rubredoxins to Tp SOR, and calculated the kinetics parameters of the electron transfer reaction [32]. Now, we have extended this study and present kinetics data of the reactions between members of the three different classes of SORs and rubredoxinlike proteins from the same organisms: Dv SOR was chosen as the prototype of class I, Dg SOR as that of [14,61].…”