Characterization of molybdenum cofactor from Escherichia coli

Abstract

Mo cofactor activity was found in the soluble fraction of cell-free extracts of E. coli grown aerobically in media supplemented with molybdate. Cofactor was detected by its ability to complement the nitrate reductase-deficient mutant of Neurospora crassa, nit-1, resulting in the in vitro formation of nitrate reductase activity. Acid treatment of E. coli extracts was not required for release of cofactor activity. Cofactor was able to diffuse through a membrane of nominal 2000 MW cutoff and was insensitive to trypsin. The cofactor was associated with a carrier molecule (approximately 40,000 daltons) during gel filtration and sucrose gradient centrifugation, but was easily removed from the carrier by dialysis. The carrier molecule protected the cofactor from inactivation by heat or O2. E. coli grown in Mo-free media, without and with tungsten, synthesized a metal-free empty cofactor and its tungsten analog, respectively, both of which were subsequently activated by the addition of molybdate. Empty and tungsten-containing cofactor complemented the nitrate reductase subunits in the nit-1 extract, forming inactive but intact 7.9S nitrate reductase. Addition of molybdate to the enzyme complemented in this manner restored nitrate reductase activity.