MOLYBDENUM-INDEPENDENT NITROGENASES OF AZOTOBACTER-VINELANDII - A FUNCTIONAL SPECIES OF ALTERNATIVE NITROGENASE-3 ISOLATED FROM A MOLYBDENUM-TOLERANT STRAIN CONTAINS AN IRON MOLYBDENUM COFACTOR

Nitrogenase-3 of Azotobacter vinelandii is synthesized under condition s of molybdenum and vanadium deficiency. The minimal metal requirement for its synthesis, and its metal content, indicated that the only tra nsition metal in nitrogenase-3 was iron [Chisnell, Premakumar and Bish op (1988) J. Bacteriol. 170, 27-33; Pau, Mitchenall and Robson (1989) J. Bacteriol. 171, 124-129]. A new species of nitrogenase-3 has been p urified from a strain of A. vinelandii (RP306) lacking structural gene s for the Mo- and V-nitrogenases and containing a mutation which enabl es nitrogenase-3 to be synthesized in the presence of molybdenum. SDS/ PAGE showed that component 1 contained a 15 kDa polypeptide which N-te rminal amino acid sequence determination showed to be encoded by anfG. This confirms that nitrogenase-3, like V-nitrogenase, comprises three subunits. Preparations of the nitrogenase-3 from strain RP306 contain ed 24 Fe atoms and 1 Mo atom per molecule. Characterization of the cof actor centre of the enzyme by e.p.r. spectroscopy and an enzymic cofac tor assay, together with stimulation of the growth of strain RP306 by Mo, showed that nitrogenase-3 can incorporate the Mo-nitrogenase cofac tor (FeMoco) to form a functional enzyme. The specific activities (nmo l of product produced/min per mg of protein) determined from activity titration curves were: under N2, NH3 formation 110, with concomitant H -2 evolution of 220; under argon, H-2 evolution 350; under 10% acetyle ne (C2H2) in argon, ethylene (C2H4) 58, ethane (C2H6) 26, and concomit ant H-2 evolution 226. The rate of formation of C2H6 was non-linear, a nd the C2H6/C2H4 ratio strongly dependent on the ratio of nitrogenase components.