Apodinitrogenase: purification, association with a 20-kilodalton protein, and activation by the iron-molybdenum cofactor in the absence of dinitrogenase reductase

The direction of electron flow through nitrogenase is generally believed to be from the Fe protein to the Pclusters to the FeMo cofactor and then to substrate. In order to examine oxidation states of the P-clusters that might be involved in this pathway, we have constructed a form of the MoFe protein that contains a species called the MoFe cluster Molybdenum nitrogenase is composed of two separate proteins whose complete structures have recently been determined by x-ray crystallography (1-7). The smaller of the two, designated the iron protein (Fe protein), is a dimer of two identical subunits encoded by the nifH gene (1, 8). It contains two binding sites for MgATP and has a single [4Fe-4S] 2ϩ/ϩ cluster. The larger of the two component proteins is designated the molybdenum-iron protein (MoFe protein) and is an a 2 ␤ 2 tetramer with the ␣ and ␤ subunits encoded by the nifD and nifK genes, respectively. It has two types of metal centers, the P-clusters (two per tetramer) that each contain 8Fe and 8S 2Ϫ atoms in the form of bridged [4Fe-4S] clusters and, the iron molybdenum cofactors (FeMo cofactor) (two per tetramer) that have the composition Mo:Fe 7

Section: Resultsmentioning

confidence: 89%

Construction of a Form of the MoFe Protein of Nitrogenase That Accepts Electrons from the Fe Protein but Does Not Reduce Substrate

Ma¹,

Brosius²,

Burgess³

1996

“…It was first observed as a protein that co-purified with apodinitrogenase from nifB mutants (5). Subsequent work has shown that gamma protein is associated with the nifKD gene products in extracts of nifB mutants.…”

Section: Fig 3 Accumulation Ofmentioning

confidence: 99%

“…1) constitutes the active site of the nif-encoded, molybdenum-containing dinitrogenase protein in Azotobacter vinelandii and other nitrogen-fixing organisms (1)(2)(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.…”

mentioning

confidence: 99%

Incorporation of Molybdenum into the Iron-Molybdenum Cofactor of Nitrogenase

Allen¹,

Roll²,

Rangaraj³

et al. 1999

Self Cite

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, ...

“…FeMoco-deficient, but P-cluster containing MoFe proteins have proved to be useful for the study of two major aspects of the nitrogenase research, the maturation of MoFe protein (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and the features of the P-cluster (10, 19 -21). Two types of 100% FeMoco-deficient MoFe proteins, presumably different catalytically and structurally, have been isolated and characterized.…”

mentioning

confidence: 99%

The FeMoco-deficient MoFe Protein Produced by a nifHDeletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

Ribbe¹,

Hu²,

Guo³

et al. 2002

146

The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (⌬nifH MoFe protein) was purified in large quantity. The which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.The metalloenzyme nitrogenase complex catalyzes the biological reduction of dinitrogen to ammonia (for recent reviews, see Refs. 1-6). The enzyme is composed of two separately purifiable proteins, the iron (Fe) protein and the molybdenumiron (MoFe) 1 protein. The Fe Protein is a 60-kDa dimer of two identical subunits encoded by the nifH gene. The two subunits are bridged by a [4Fe-4S] cluster, and each subunit has a binding site for MgATP. The more complicated MoFe protein is a 230-kDa ␣ 2 ␤ 2 tetramer with the ␣ and ␤ subunits encoded by the nifD and nifK genes, respectively. The MoFe protein contains two different types of metal clusters, the [8Fe-7S] cluster (P-cluster) bridged between each ␣␤ subunit pair and the [Mo7Fe-9S-homocitrate] cluster (FeMoco) located within each ␣ subunits. Substrate reduction by the enzyme requires both component proteins, with the Fe protein serving as a specific reductant of the MoFe protein, which in turn provides the site of substrate reduction. To carry out the catalytic function of nitrogenase, the reduced Fe protein first binds two molecules of MgATP and undergoes a conformational change before forming a complex with the MoFe protein. Then, coupled with MgATP hydrolysis, electrons are transferred from the Fe protein to the P-clusters of the MoFe protein within the complex. This process is followed by the dissociation and re-reduction of the oxidized Fe protein and the dissociation of MgADP from the MoFe protein. Finally, the electrons are believed to be transferred from the P-cluster to the FeMoco, where substrate reduction occurs.FeMoco-deficient, but P-cluster containing MoFe proteins have proved to be useful for the study of two major aspects of the nitrogenase research, the maturation of MoFe protein (7-18) and the features of the P-cluster (