FIGURE 4.

Persulfide formation by purified Nfs1 from already bound cysteine: no significant effect of added Yfh1 or Isu1_Sup. Nfs1 (100 ng) or the Nfs1 (K263A) mutant (100 ng) was incubated with [³⁵S]cysteine (0.1 μm; 5 μCi) and PLP (0.15 mm) in HS buffer containing 0.15 m NaCl at 30 °C for 15 min (first step). Proteins were precipitated with ammonium sulfate and centrifuged to remove excess and unbound [³⁵S]cysteine. The protein pellet containing Nfs1 with bound [³⁵S]cysteine was dissolved in HS buffer plus 0.15 m NaCl and 0.15 mm PLP. As indicated, reaction mixtures were supplemented with increasing amounts of Yfh1 or Isu1_Sup (1× = 50 ng). Following addition of Isd11 (100 ng), samples were incubated at 30 °C for 15 min (second step) and subsequently analyzed by nonreducing SDS-PAGE and autoradiography.