Identification of iron-sulfur centers in the iron-molybdenum proteins of nitrogenase.

The core extrusion method has been applied to the determination of the type ([2Fe-2S], [4Fe-4S]) and number of iron-sulfur centers in the FeMo proteins of the nitrogenases from Clostridium pasteurianum and Azotobacter vinelandii. The method involves extrusion with o-xylyl-alpha, alpha'-dithiol, ligand exchange of the extrusion products with p-CF3C6H4SH (RFSH), and identification and quantitation of the resultant [FenSn(SRF)4]2- complexes (n = 2,4) by 19F NMR spectroscopy. In hexamethylphosphoramide/water, 4:1 (vol/vol), 49-56% of the Fe content was extruded as [Fe4S4(SRF)4]2-, corresponding to 3,4-4.0 Fe4S4 cores per alpha 2 beta 2 subunit complex. The extruded iron does not arise from the FeMo cofactor, separate examination of which detected no extrusion products, and corresponds to 90-103% of noncofactor iron. No significant quantity of Fe2S2 cores was extruded. These results indicate the presence of four [4Fe-4S] centers per alpha 2 beta 2 subunit complex in preparations undepleted in iron. There are two main structural populations of iron atoms in these proteins, those in the cubane-type Fe4S4 cores and those in the FeMo cofactor.