STRUCTURAL AND IMMUNOLOGICAL STUDIES ON THE SOLUBLE FORMATE DEHYDROGENASE FROM ALCALIGENES-EUTROPHUS

During growth with formate as the sole energy source the autotrophic b acterium Alcaligenes eutrophus synthesizes a cytoplasmic formate dehyd rogenase. The enzyme is a molybdo-iron-sulfur-flavo protein and the ma jor NADH-producing system under these growth conditions, although it w as estimated to constitute only 0.65% of the soluble cell protein. An electron microscopic analysis of the purified enzyme revealed that the particle is made up of four nonidentical sub-masses, corroborating pr evious structural data. The NH2-terminal amino acid sequences of the e nzyme subunits exhibited significant similarities to those of only one other heteromeric formate dehydrogenase, the enzyme from the methane- utilizing bacterium Methylosinus trichosporium. Metal analyses yielded 21.5 g-atom iron, 2.18 g-atom nickel, 0.76 g-atom molybdenum, and 0.5 9 g-atom zinc per mol of enzyme. Initial electron paramagnetic resonan ce spectroscopic studies showed at least three distinct signals which appeared upon reduction of the enzyme with NADH or formate. The corres ponding spin systems could be attributed to iron-sulfur centers of the enzyme. Comparative immunostaining and activity-staining experiments using cell extracts from various bacteria established immunological si milarities between the soluble formate dehydrogenase of A. eutrophus a nd the soluble enzymes from all tested facultative autotrophs as well as from M. trichosporium.