PHYSIOLOGICAL AND BIOCHEMICAL-CHARACTERIZATION OF THE SOLUBLE FORMATEDEHYDROGENASE, A MOLYBDOENZYME FROM ALCALIGENES-EUTROPHUS

Organoautotrophic growth of Alcaligenes eutrophus on formate was depen dent on the presence of molybdate in the medium. Supplementation of th e medium with tungstate lead to growth cessation. Corresponding effect s of these anions were observed for the activity of the soluble, NAD+- linked formate dehydrogenase (S-FDH; EC 1.2.1.2) of the organism. Lack of molybdate or presence of tungstate resulted in an almost complete loss of S-FDH activity. S-FDH was purified to near homogeneity in the presence of nitrate as a stabilizing agent. The native enzyme exhibite d an M(r) of 197,000 and a heterotetrameric quaternary structure with nonidentical subunits of M(r) 110,000 (alpha), 57,000 (beta), 19,400 ( gamma), and 11,600 (delta). It contained 0.64 g-atom of molybdenum, 25 g-atom of nonheme iron, 20 g-atom of acid-labile sulfur, and 0.9 mol of flavin mononucleotide per mol. The fluorescence spectrum of iodine- oxidized S-FDH was nearly identical to the form A spectrum of milk xan thine oxidase, proving the presence of a pterin cofactor. The molybden um-complexing cofactor was identified as molybdopterin guanine dinucle otide in an amount of 0.71 mol/mol of S-FDH. Apparent K(m) values of 3 .3 mM for formate and 0.09 mM for NAD+ were determined. The enzyme cou pled the oxidation of formate to a number of artificial electron accep tors and was strongly inactivated by formate in the absence of NAD+. I t was inhibited by cyanide, azide, nitrate, and Hg2+ ions. Thus, the e nzyme belongs to a new group of complex molybdo-flavo Fe-S FDH that so far has been detected in only one other aerobic bacterium.