Incorporation of Molybdenum into the Iron-Molybdenum Cofactor of Nitrogenase

Abstract

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase was investigated using 99 Mo to follow the incorporation of Mo into precursors. 99 Mo label accumulates on dinitrogenase only when all known components of the FeMo-co synthesis system, NifH, NifNE, NifB-cofactor, homocitrate, MgATP, and reductant, are present. Furthermore, 99 Mo label accumulates only on the gamma protein, which has been shown to serve as a chaperone/insertase for the maturation of apodinitrogenase when all known components are present. It appears that only completed FeMo-co can accumulate on the gamma protein. Very little FeMo-co synthesis was observed when all known components are used in purified forms, indicating that additional factors are required for optimal FeMo-co synthesis.99 Mo did not accumulate on NifNE under any conditions tested, suggesting that Mo enters the pathway at some other step, although it remains possible that a Mo-containing precursor of FeMo-co that is not sufficiently stable to persist during gel electrophoresis occurs but is not observed. 99 Mo accumulates on several unidentified species, which may be the additional components required for FeMo-co synthesis. The molybdenum storage protein was observed and the accumulation of 99 Mo on this protein required nucleotide.The iron-molybdenum cofactor (FeMo-co) 1 of dinitrogenase ( Fig. 1) constitutes the active site of the nif-encoded, molybdenum-containing dinitrogenase protein in Azotobacter vinelandii and other nitrogen-fixing organisms (1-3). FeMo-co can be isolated by extraction from the purified dinitrogenase protein (2), and the isolated cofactor can be used to activate FeMo-codeficient forms of dinitrogenase (referred to hereafter as "apodinitrogenase") that accumulate in strains unable to synthesize the cofactor (2, 4, 5). FeMo-co consists of Mo, Fe, and S atoms in a 1:7:9 ratio; in addition, the organic acid homocitrate is an integral component of the compound (6), serving as a nonprotein ligand to the molybdenum atom. The structure of FeMo-co in the protein was determined by Kim and Rees (7) and Chan et al. (8).Genetic studies have revealed that functional copies of the nifB, nifN, nifE, nifH, and nifV genes are required for synthesis of FeMo-co in vivo (9 -11); the nifQ gene is also required under conditions of molybdenum limitation (12). The nifKD genes, which encode the subunits of dinitrogenase (NifKD), are not required for FeMo-co synthesis, and thus FeMo-co is not synthesized "in place" but rather is preformed and then transferred to its site in dinitrogenase (13). In the absence of NifKD, completed FeMo-co accumulates on a protein called gamma, which serves as a chaperone/insertase for the maturation of NifKD and for insertion of .An in vitro FeMo-co synthesis system was devised to address the biochemical roles of these genes and to identify other factors required for FeMo-co synthesis (15). In this system, at least homocitrate, molybdenum (supplied as molybdate), MgATP, NifB-co, NifH (dinitrogenase reductase), reductant, ...

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

“…The hypothesis of a FeMo-co biosynthetic factory has been formulated before (67) to explain the observed distribution of radioactively labeled FeMo-co precursors between the NifH, NifEN, NifX, and NafY proteins during the biosynthesis of FeMo-co in vitro (2, 3,57,58). This hypothesis is also consistent with the recently reported distribution of FeMo-co precursors between NifEN and NifH as analyzed by EXAFS (36,38).…”

Purification and Characterization of NafY (Apodinitrogenase γ Subunit) from Azotobacter vinelandii

“…This interaction has not been observed in previous anoxic native gel electrophoresis (33,34) nor in our native gel electrophoresis experiments (data not shown). It is possible that the conditions used to assay the interaction of NifB-co and NafY by fluorescence are milder to the complex than those of native gel electrophoresis.…”

“…50-l samples were subsequently electrophoresed for 20 h in an anoxic native gel that was finally stained with Coomassie Brilliant Blue. NifB-co or labeled molybdenum from a FeMo-co precursor have been found associated to NafY only if all the components necessary for FeMo-co biosynthesis are present in the assay mixture (33,34). These results have been taken as evidence of FeMo-co being synthesized and accumulated on NafY.…”

Accumulation of 99Mo-containing Iron-Molybdenum Cofactor Precursors of Nitrogenase on NifNE, NifH, and NifX ofAzotobacter vinelandii

“…Prior to this study, 99 Mo-labeling of NifNE has not been observed. Previous studies (17,18) have involved the use of crude cell extracts, and two possible reasons for not observing the incorporation of 99 Mo into NifNE could be the interference from other proteins present in the cell extracts, or that the synthesis of FeMo-co is allowed to proceed beyond NifNE. The purified in vitro FeMo-co synthesis reaction is a well-defined system and, being devoid of other extraneous components present in crude enzyme preparations, has facilitated the identification of hitherto undetected incorporation of a FeMo-co precursor into NifNE.…”