Fig. 5.

Screening of minimal components required for cofactor synthesis and apo-NifDK^RS reconstitution. (A) Complete reactions contained molybdate, SAM, homocitrate, NifH^RS, NifB^RS, and apo-NifDK^RS. NifX^Av, NafY^Av, NifEN^Av or RC-NifU^Av were added when indicated. RC-NifU^Av refers to NifU^Av loaded with [Fe₄S₄] clusters formed in vitro by incubation with Fe, cysteine, DTT, and NifS (SI Appendix, Materials and Methods). Reactions were performed at 48 °C. Negative control assays (- NifB) reached 1.60 ± 0.04 nmol C₂H₄ min⁻¹ mg⁻¹ NifDK^RS. Data are average activities ± SD (n = 2). (B) SDS-PAGE analysis of purified Roseiflexus Nif proteins required for the in vitro synthesis of nitrogenase cofactors. NifB^RS preparations contained two additional bands that were identified by mass spectrometry as GroEL (60 kDa) and C-terminal truncated NifB^RS species (30 kDa).