PURIFICATION, COMPOSITION, CHARGE, AND MOLECULAR-WEIGHT OF THE FEMO COFACTOR FROM AZOTOBACTER-VINELANDII NITROGENASE

A procedure has been developed for purifying NMF and NMF/DMF solutions of the FeMo cofactor (FeMoco) derived from the molybdenum iron protein of nitrogenase. This procedure consists of anaerobic chromatography of FeMoco solutions on two consecutive anaerobic molecular sizing columns followed by electrophoretic migration through a third sizing column. FeMoco prepared by this procedure is homogeneous as evidenced by chromatographic, electrophoretic, and compositional criteria. The minimal elemental composition was found to be MoFe6S6 using chemical colorimetric, inductively coupled plasma (ICP), and proton induced x-ray emission (PIXE) analytical procedures. Molecular weight measurements of NMF and DMF solutions of FeMoco using calibrated columns containing various molecular sizing matrices gave values of 1395 +/- 130 daltons for the molecular weight of FeMoco. The measured MW of FeMoco is about twice the value expected from the minimal stoichiometry, suggesting that FeMoco may exist as Mo2Fe12S12 in NMF and DMF solutions. The charge of FeMoco in its EPR silent state was determined to be 2 - per Mo by passing NMF solutions of FeMoco containing excess salts of Na+, K+, Rb+, and Mg2+ through long columns equilibrated with pure NMF and then measuring the M/Mo ratio of the emerging FeMoco. Decomposition of purified FeMoco by acid or O2-exposure followed by exhaustive methylation or silanation of the resulting mixture failed to yield any methylated or silanated homocitric acid as measured by tandem gas chromatography-mass spectrometry (GC-MS) analysis. The GC-MS procedure applied to standard homocitric acid samples and various controls readily detects methylated homocitric acid at the sub-nanomole level. We conclude that the minimum molecular formula for active oxidized (EPR silent) FeMoco in NMF and in NMF-DMF mixtures is [Mo2Fe12S12]4-, but that other small organic anions such as NMF- may be present.