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.
Highlights
Biological nitrogen fixation is the process by which atmospheric nitrogen is reduced to ammonium
As determined by antibody capture quantitation, the amount of protein accumulated in wild-type (DJ) is approximately 5-fold less than the amount accumulated in CA12 (I!.nifHDK) and approximately 15-fold less than the amount accumulated in DJ677 (I!.nifB::kan I!.nifDK). These results suggest that mutants defective in FeMo-co synthesis either synthesize more NIFNE or that NIFNE accumulated in those mutants is relatively more stable than NIFNE accumulated in wild type
It is based on the assumption that when FeMo-co synthesis is blocked by a mutation affecting a critical step in the process, the accumulated NIFNE is expected to harbor the form of immature FeMo-co that is the substrate of the mutationally absent gene product
Summary
Biological nitrogen fixation is the process by which atmospheric nitrogen is reduced to ammonium This process is carried out by the nitrogenase enzyme system, which is composed of two separate protein complexes [1, 2]. Dinitrogenase reductase (component II, iron protein, NIFH), a 65-kDa az dimer of the nifH gene product, serves as a specific electron donor to the other protein complex, dinitrogenase. Dinitrogenase (component I, molybdenum-iron protein, NIFKD) is a 240-kDa a2f32 tetramer of the nifK and nifD gene products. Dinitrogenase requires a unique cofactor, FeMo-co,l which contains iron, molybdenum, acid-labile sulfur, and homocitrate. The role of NIFB in FeMo-co synthesis is undefined, but recently a small molecule containing iron and sulfur, thought to be a FeMo-co precursor produced by the activity of NIFB, termed NifB-co, has been isolated [10]
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