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. Author manuscript; available in PMC: 2018 Oct 16.
Published in final edited form as: Nat Chem. 2018 Apr 16;10(5):568–572. doi: 10.1038/s41557-018-0029-4

Figure 3. Dithionite (DT)-free cluster maturation.

Figure 3

(a) Proteins captured on Ni Sepharose resin after incubation of (1) His-tagged MaNifBapo with 35SO32−, (2) His-tagged MaNifB[Fe4S4] with 35SO32−, and (3) His-tagged MaNifB[Fe4S4] with SAM and 35SO32− (left); clusters extracted from samples a, 2 and a, 3 (middle); and activities of C2H2-reduction by clusters extracted from samples a, 2 and a, 3, after maturation and transfer of clusters to NifDK (right). The 35S label and activity were only detected in SAM/SO32−-treated MaNifB[Fe4S4], suggesting incorporation of S from SO32− into a fully-assembled L-cluster. (b) HPLC elution profiles of (1) SAM, SAH and 5’-dAH standards, (2) SAM incubated with MaNifB[Fe4S4], and (3) SAM incubated with MaNifB[Fe4S4] and SO32−, showing the competence of SAM-treated MaNifB[Fe4S4] in methyltransfer and hydrogen abstraction with or without SO32−-treatment. (c, d) EPR spectra of EuII-EGTA-reduced (c) or IDS-oxidized (d) MaNifB[Fe4S4] incubated without SAM (black), with SAM (red), or with SAM and SO32− (green), suggesting formation of an 8Fe core of the L-cluster in SAM-treated MaNifB[Fe4S4] with or without SO32−. (e) Activities of MaNifB[Fe4S4] treated with (1) no additive, (2) SAM, and (3) SAM and SO32− upon maturation and transfer of clusters to NifDK, showing the sole competence of SAM/SO32−-treated MaNifB[Fe4S4] as an M-cluster donor. Activity data were obtained from three independent experiments (n=6) and presented as mean±s.d. (e).