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

Abstract

The core extrusion method was 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 Fe does not arise from the FeMo cofactor, separate examination of which detected no extrusion products, and corresponds to 90-103% of noncofactor Fe. No significant quantity of Fe2S2 cores was extruded. These results indicate the presence of 4 [4Fe-4S] centers per .alpha.2.beta.2 subunit complex in preparations undepleted in Fe. There are 2 main structural populations of Fe atoms in these proteins, those in the cubane-type Fe4S4 cores and those in the FeMo cofactor.