Homocitrate is a component of the iron-molybdenum cofactor of nitrogenase

Hoover, Timothy R.; Imperial, Juan; Ludden, Paul W.; Shah, Vinod K.

doi:10.1021/bi00433a004

Cited by 136 publications

(92 citation statements)

References 27 publications

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“…The gene product of nifV is homocitrate synthase (13). Homocitrate is a structural component of FeMo-co (14) and most probably FeV-co, because nifV is also required for full functionality of the vnfdinitrogenase (15). The product of nifH, dinitrogenase reductase, plays multiple roles in the nitrogenase system.…”

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A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Rüttimann-Johnson

Staples

Rangaraj

et al. 1999

Journal of Biological Chemistry

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The vnf-encoded nitrogenase from Azotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the ironmolybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in the vnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe 7-8 S x precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe 3 S 4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from a nifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from the nifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.

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A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Rüttimann-Johnson

Staples

Rangaraj

et al. 1999

Journal of Biological Chemistry

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“…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).…”

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Incorporation of Molybdenum into the Iron-Molybdenum Cofactor of Nitrogenase

Allen¹,

Roll²,

Rangaraj³

et al. 1999

Journal of Biological Chemistry

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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, ...

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“…In the absence of homocitrate or one of its analogs, no FeMo-co is formed in vitro as determined by acetylene and proton reduction assays and by lack of 99Mo incorporation into apodinitrogenase (11). Homocitrate has been shown to be an integral part of the FeMo-co of dinitrogenase, with one mole of homocitrate found for each mole of Mo (12).…”

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“…The crystals of threo diastereomer (120 mg) were dissolved in 0.5 ml of H2O, and the solution was brought to pH 10 with 12 M NaOH, diluted with 4.5 ml of water, and stored at -20°C. The (9,12), along with 0.2 ml of an ATP-regenerating mixture which contained 5 mM sodium dithionite and 0.05 mM sodium molybdate (7) and the indicated concentrations of organic acids. The reaction mixtures were incubated at 30°C for 35 min, after which 0.8 ml of additional ATP-regenerating mixture containing 5 mM sodium dithionite was added, together with purified dinitrogenase reductase (7) and assayed.…”

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Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor.

Madden

Kindon²,

Ludden³

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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In vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase using homocitrate and its analog allows the formation of modified forms of FeMo-co that show altered substrate specificities (N2, acetylene, cyanide, or proton reduction) ofnitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. The (LR,2S)-threo-and (1S,2S)-erythro-fluorinated diastereomers of homocitrate have been incorporated in vitro into dinitrogenase in place of homocitrate. Dinitrogenase activated with FeMo-co synthesized using threo-fluorohomocitrate reduces protons, cyanide, and acetylene but cannot reduce N2. In addition, proton reduction is inhibited by carbon monoxide (CO), a characteristic of dinitrogenase from NifV-mutants. Dinitrogenase activated with FeMo-co synthesized using erythro-fluorohomocitrate reduces protons, cyanide, acetylene, and N2. In this case proton reduction is not inhibited by CO, a characteristic of the wild-type enzyme. Cyanide reduction properties ofdinitrogenase activated with FeMo-co containing either fluorohomocitrate diastereomer are similar, and CO strongly inhibits cyanide reduction. Dinitrogenases activated with FeMo-co containing homocitrate analogs with a hydroxyl group on the C-1 position are much less susceptible to CO inhibition of cyanide reduction. However, proton and cyanide reduction by dinitrogenase containing FeMo-co activated with (1R,2S) threo-isocitrate is only one-third that of dinitrogenase activated with the racemic mixture of isocitrate and shows strong CO inhibition ofsubstrate reduction. These results suggest that CO inhibition of proton and cyanide reduction occurs when the hydroxyl group on the C-1 position of analogs is "trans" to the C-2 carboxyl group (i.e., in the threo conformation). When racemic mixtures ofthese analogs are used in the system, it seems that the erythro form is preferentially incorporated into dinitrogenase. Finally, carbonyl sulfide inhibition of substrate reduction by dinitrogenase is dependent on the homocitrate analog incorporated into FeMo-co.

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Homocitrate is a component of the iron-molybdenum cofactor of nitrogenase

Cited by 136 publications

References 27 publications

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Incorporation of Molybdenum into the Iron-Molybdenum Cofactor of Nitrogenase

Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor.

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