Purification and spectroscopic characteristics in N-methylformamide of the Azotobacter vinelandii Fe-Mo cofactor

The iron-molybdenum cofactor from Azotobacter vinelandii can be removed from significant amounts of extraneous iron and other contaminants using anaerobic gel filtration. Electronic absorption spectra of the so-purified FeMoco along with analysis of the so-called 'easily complexed' iron are suggestive that FeMoco occupies at least two different states in N-methylformamide solution. Batch-related differences in spectral characteristics of independently isolated FeMoco samples are demonstrated. Non-cofactor iron, found in unpurified FeMoco, may affect the interpretation of ligand binding and other experiments probing FeMoco structure and reactivity. Oxidized FeMoco is shown to be clearly discernable from the semi-reduced species by means of electronic spectroscopy, and this method now forms a convenient analytical tool for study of the chemistry and electronic structure of FeMoco.