Fig 1.
(A) The current Sox pathway model. The pathway cycle starts with an unmodified SoxYZ Cys side chain (top of the panel). Released electrons are transferred to cytochrome c. SoxYZ conjugates with longer chain sulfur species are generated by competition between SoxAX and SoxCD for SoxY(S)Z (bottom of the panel). SoxYZ species that have been identified in extracts of thiosulfate-grown P. pantotrophus [5] are marked with an asterisk (*). (B) A revised model for the Sox pathway. The canonical Sox pathway model shown in (A) is modified to accommodate the results of the current work. The reactions in gray allow the activation of unconjugated SoxYZ.
Fig 2.
Generation of SoxYZ-conjugated candidate Sox pathway intermediates.
SoxYZ was treated by the indicated protocols. The products were then analysed by ion exchange chromatography using a 1 mL MonoQ HR column (GE Healthcare) equilibrated in 30 mM Tris-HCl pH 8.0 (top of each panel). The fractions from the IEC separations that are indicated with arrows (A,B) or numbers (C,D) were analysed by ESI-MS and (for D only) by native PAGE (bottom of each panel). ESI-MS samples in (D) were treated with 20 mM iodoacetamide for one hour prior to analysis. The minor ESI-MS peaks marked with an asterisk (*) represent +15 Da adducts of the main peaks. Tetra-preIEC, Tetra-2, and Tetra-5 samples in (D) correspond to, respectively, the tetrathionate-treated SoxYZ before IEC and to the indicated fractions from the IEC separation.
Fig 3.
Thiosulfate oxidation activity of the Sox pathway reconstituted with different SoxYZ-conjugates.
The SoxYZ conjugates were added to reactions mixtures containing SoxAX, SoxB, SoxCD, thiosulfate and cytochrome c. (A) Progression of Sox pathway activity for reconstitution reactions containing 0.1 μM of the indicated SoxYZ conjugates. Defined fractions are those characterized in panels (B) and (C). The reduction of cytochrome c was monitored at 550nm. IEC fractions of the (B) tetrathionate-treated and (C) tetrathionate then sulfide-treated SoxYZ preparations from Fig 2C and 2D, respectively, were assayed for activity. Fraction numbering is the same as in Fig 2C and 2D.
Fig 4.
Thiosulfate-independent cytochrome c reduction.
Reactions contained 0.1μM SoxB, SoxCD and SoxAX, unless indicated, and 35 μM cytochrome c. Any missing Sox components are indicated by a minus sign (e.g. `-SoxCD’). Reactions were initiated in panels (A) and (C) by the addition of 1 μM tetrathionate-then-sulfide-treated SoxYZ from fraction 3 of the IEX separation shown in Fig 3C (designated `SoxY(Sn)Z’), or in panel (B) by the addition of 10 μM tetrathionate-treated SoxYZ from fraction 3 of the IEX separation shown in Fig 3B (designated `SoxY(SSO3)Z’). Where indicated, Sox components or thiosulfate were added to final concentrations of 0.1 μM or 2 mM, respectively. The progress of the reactions was assessed through monitoring the reduction of cytochrome c at 550nm.
Fig 5.
Thermodynamics of the interaction between T. thermophilus SoxYZ and SoxB measured by isothermal titration calorimetry.
Representative titrations are shown but thermodynamic values were determined by fitting to duplicate titrations. (A) SoxY(SO3)Z was titrated into 50 μM SoxB. (B) SoxYC151EZ was titrated into 50 μM SoxB.
Table 1.
Thermodynamic parameters for the interaction of T. thermophilus SoxB with different T. thermophilus SoxYZ conjugates.