FIGURE 4.

Purification and biochemical properties of yNifB_Mi. (A) SDS-PAGE and Western blot analysis of yNifB_Mi purification. CFE, 65°C heated SB10Y cell-free extract; FT, affinity chromatography flow through; W1-W4 and E1-E2, affinity chromatography wash and elution fractions containing increasing concentrations of imidazole (see Materials and Methods for details). Grey arrow in the Coomassie stained panel points to the position of yNifB_Mi in the gel. (B) SU9 processing site (black arrow) of yNifB_Mi. Underlined sequence indicates the N-terminal amino acids of yNifB_Mi identified by Edman degradation. (C) UV-visible spectra of as isolated, reconstituted, and dithionite (DTH)-reduced reconstituted yNifB_Mi. (D) Typical color of as isolated and reconstituted yNifB_Mi purified preparations. (E) Titration of FeMo-co synthesis and nitrogenase reconstitution assay with yNifB_Mi. The indicated concentrations of yNifB_Mi monomer were used. NifB activity was determined by acetylene reduction assay of reconstituted NifDK from ΔnifB A. vinelandii UW140 cell-free extracts. Data represent mean ± standard deviation (n = 2) at each yNifB_Mi concentration.