Characteristics of NIFNE in Azotobacter vinelandii Strains: IMPLICATIONS FOR THE SYNTHESIS OF THE IRON-MOLYBDENUM COFACTOR OF DINITROGENASE

Abstract

The products of the nifN and nifE genes of Azotobacter vinelandii function as a 200-kDa alpha 2 beta 2 tetramer (NIFNE) in the synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase, the enzyme system required for biological nitrogen fixation. NIFNE was purified using a modification of the published protocol. Immunoblot analysis of anoxic native gels indicated that distinct forms of NIFNE accumulate in strains deficient in either NIFB (delta nifB::kan delta nifDK) or NIFH (delta nifHDK). During the purification of NIFNE from the delta nifHDK mutant, its mobility in these gels changed, becoming similar to that of NIFNE from the delta nifB::kan delta nifDK mutant. While NIFB activity initially co-purified with the NIFNE activity from the delta nifHDK mutant, further purification of NIFNE activity resulted in the loss of the co-purifying NIFB activity; this loss correlated with the change in NIFNE mobility on native gels. These results suggest that the form of NIFNE accumulated in the delta nifHDK mutant is associated with NIFB activity in crude extract but loses this association during NIFNE purification. Addition of the purified metabolic product of NIFB, termed NifB-co, to either NIFNE purified from the delta nifHDK strain or to the NIFNE in crude extract of the delta nifB::kan delta nifDK strain caused a change in the mobility of NIFNE on anoxic native gels to that of the form accumulated in a delta nifHDK mutant. These results support a model where both NifB-co and dinitrogenase reductase participate in FeMo-co synthesis through NIFNE, which serves as a scaffold for this process.