The nitrogenase proteins of Rhizobium meliloti: purification and properties of the MoFe and Fe components.

The alfalfa-Rhizobium meliloti symbiosis contributes a major portion of biologically fixed nitrogen to temperate zone forage crop production. Highly-purified molybdenum-iron (MoFe) and iron (Fe) nitrogenase components were obtained for the first time from extracts of R. meliloti bacteroids. Intact bacteroid cells were isolated anaerobically from 100 g quantities of alfalfa nodules following storage in liquid nitrogen. Centrifuged bacteroid extracts showed a marked reduction in specific activity when assayed at protein concentrations less than 1 mg/ml. Both nitrogenase proteins were resolved and purified to homogeneity as determined spectroscopically and by SDS-PAGE. The purified MoFe protein differed in several respects from previously characterized nitrogenase proteins. Saturation of the acetylene-reducing and proton-reducing activities of the R. meliloti MoFe protein required higher relative concentrations of Fe protein than nitrogenase proteins purified from free living diazotrophs. Electron allocation to dinitrogen reduction was sustained at component ratios similar to those present in bacteroid extracts, suggesting that while the observed saturation effects were not detrimental to physiological function in the symbiotic system, overall activity could be enhanced by higher levels of iron protein. Analyses of the MoFe protein gave 22 Fe, 22 labile sulfide and 1.7 Mo atoms per molecular unit of 215 kDa. Dithionite-reduced MoFe protein contained a spin 3/2 iron centre but had a lower visible absorbance at 360 nm than the equivalent Azotobacter chroococcum component. Amino-acid composition indicated a notably lesser tryptophan content, and cysteine content greater than that of the equivalent tetrameric protein of free living diazotrophs. Ratios of acidic and basic residues were similar to other MoFe proteins. Calculation of hydrophobicity and discriminant parameters gave values midway between those expected for soluble cytoplasmic proteins and peripheral membrane associated proteins. ADP was tightly bound by the dithionite-free MoFe protein containing reduced iron-molybdenum cofactor. The R. meliloti iron protein was found to be a 64 kDa homodimer containing a single 4Fe-4S metal centre.