Large scale isolation and characterization of the molybdenum-iron cluster from nitrogenase.
Here we report the large scale isolation and characterization of a species, designated MoFe cluster, that exhibits an S = 3/2 EPR signal, and the comparison of this entity to isolated FeMo cofactor in N-methylformamide and to the active site of the enzyme nitrogenase. MoFe cluster is isolated from purified nitrogenase by extraction into acidic methyl ethyl ketone and it is stable in that solvent in the absence of thiols. 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 exhibited by isolated FeMo cofactor. Circular dichroism experiments indicate 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 depend upon specific interactions with this organic ligand or with the protein. Metal analyses indicate that the metal core responsible for the S = 3/2 signal contains 6 Fe atoms per molybdenum. X-ray absorption spectroscopy 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.
Ma
L
L
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Gavini
N
N
Liu
H I
HI
Hedman
B
B
Hodgson
K O
KO
Burgess
B K
BK
D001665:Binding Sites; D002074:Butanones / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D008983:Molybdoferredoxin / Q000302:isolation & purification / Q000378:metabolism*; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D014233:Tricarboxylic Acids / Q000378:metabolism
D002074:Butanones; D008983:Molybdoferredoxin; D014233:Tricarboxylic Acids; C028143:homocitric acid; C005222:methylethyl ketone; D009591:Nitrogenase