Figure 13.

Assays for in vitro FeMo-co synthesis and apo-MoFe protein reconstitution. This schematic diagram shows the most relevant in vitro FeMo-co synthesis assays described in the literature and labeled to the right as NifB-dependent, NifB-co-dependent, NifEN-dependent, FeMo-co insertion, and MoFe protein activity, relating to the starting component (protein or cluster precursor) being tested. The assays can be performed by mixing cell extracts containing the indicated components or by using highly purified proteins, precursors and chemicals. The most complex assay would be the NifB-dependent (requires competent NifB containing its [Fe–S] cluster complement), which includes NifB, SAM, ΔnifB apo-NifEN, Na₂MoO₄, homocitrate, Fe protein, DTH, ΔnifB apo-MoFe protein, and ATP mix. NifX and NafY are not essential but stimulate FeMo-co synthesis and insertion. Na₂MoO₄ can be replaced by NifQ.