LARGE-SCALE ISOLATION AND CHARACTERIZATION OF THE MOLYBDENUM-IRON CLUSTER FROM NITROGENASE

Here we report the large scale isolation and characterization of a spe cies, designated MoFe cluster, that exhibits an S = 3/2 EPR signal, an d the comparison of this entity to isolated FeMo cofactor in N-methylf ormamide and to the active site of the enzyme nitrogenase. MoFe cluste r is isolated from purified nitrogenase by extraction into acidic meth yl ethyl ketone and it is stable in that solvent in the absence of thi ols. As initially isolated, MoFe cluster solutions exhibit an S = 1/2 EPR signal that arises from an oxidized species that can be reduced by dithionite or thiols to an EPR silent state and then to a state that exhibits an S = 3/2 EPR signal. The S = 3/2 signal is as sharp as the signal exhibited by the protein and much sharper than the signal exhib ited by isolated FeMo cofactor. Circular dichroism experiments indicat e that unlike the last two species, MoFe cluster does not contain the endogenous ligand R-homocitrate and thus, the sharpness of the S = 3/2 signal is an intrinsic property of the metal center and does not depe nd upon specific interactions with this organic ligand or with the pro tein. Metal analyses indicate that the metal core responsible for the S = 3/2 signal contains 6 Fe atoms per molybdenum. X-ray absorption sp ectroscopy experiments show that although the molybdenum atom in MoFe cluster retains its pseudo-octahedral geometry, its first coordination shell has one less iron atom than that of FeMo cofactor and there has been a significant change in the long range order of the cluster.