Purification and properties of the constituents of the nitrogenase complex from Clostridium pasteurianum

A new procedure for a rapid and extensive purification of the FeMo protein and the Fe protein of the nitrogenase complex from Clostridium pasteurianum is described. Specific activities of 345 and 460 nmoles of N-2 reduced per mg of protein per min for the FeMo protein and for the Fe protein, respectively, have been obtained. Preparations of the FeMo protein contained 0.96 atom of molybdenum and 15 atoms of iron per molecule, whereas those of the Fe protein contained 2.86 atoms of iron per molecule. Experiments suggest that a definite association of two Fe proteins and one FeMo protein is functional in the active enzyme complex. No individual role could be ascribed to either of the two proteins, but the fact that hydrogenase inhibits N-2 fixation but not the reductant-dependent adenosine triphosphate hydrolysis supports the idea that there are two distinct sites on nitrogenase, one concerned with N-2 activation and the other with activated electron transport.