A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 2. Solubilization of FeMoco in a wide range of organic solvents.
While the iron-molybdenum cofactor (FeMoco) of nitrogenase, a constituent of the active site for nitrogen reduction, can be extracted into N-methylformamide (NMF) and pyrrollidinone, the inability to solubilize it in any other organic solvents has hampered further understanding of its structure and chemical properties. A method to solubilize FeMoco, prepared in N,N-dimethylformamide (DMF) with Bu4N+ as counterion [McLean, P. A., Wink, D. A., Chapman, S. K., Hickman, A. B., McKillop, D. M., & Orme-Johnson, W. H. (1989) Biochemistry (preceding paper in this issue)], in acetonitrile, acetone, methylene chloride, tetrahydrofuran, and benzene is reported. FeMoco evaporated to dryness in vacuo dissolves readily in good yield (55-100%) and with no significant loss in specific activity. In addition, FeMoco can be extracted directly into these solvents from MoFe protein bound to a DEAE-Sepharose column if the protein is pretreated with DMF. Methods have also been developed to extract fully active FeMoco into acetone and acetonitrile in the absence of any amide solvents (NMF or DMF). Extraction of FeMoco into acetone (30% yield) involves only pretreatment of column-bound protein with methanol, while extraction into acetonitrile (22% yield) requires pretreatment with methanol followed by THF. We conclude that the presence of a suitable soluble cation confers solubility to the cofactor in many common organic solvents and that the solubility of FeMoco in a given solvent may be independent of the ability of that solvent to extract the cofactor from column-bound protein.
Wink
D A
DA
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
McLean
P A
PA
Hickman
A B
AB
Orme-Johnson
W H
WH
D002848:Chromatography, DEAE-Cellulose; D005288:Ferredoxins / Q000302:isolation & purification*; D008983:Molybdoferredoxin / Q000302:isolation & purification* / Q000378:metabolism; D009591:Nitrogenase / Q000032:analysis* / Q000378:metabolism; D012995:Solubility; D012997:Solvents
D005288:Ferredoxins; D008983:Molybdoferredoxin; D012997:Solvents; D009591:Nitrogenase