Materials and methodsMoFe protein from aerobically grown cells of Azotobacter vinelandii was purified as described previously (1). Crystals were obtained by equilibrating a reservoir solution containing 13% polyethylene glycol 8000, 1 M sodium chloride and 0.1 M Tris-hydroxyethylaminomethane/HCl buffer at pH 8.0 against 8 µl of a 1:1 mixture of 30 mg/ml of MoFe protein and the reservoir solution under strictly anaerobic conditions. For flash-cooling, the crystals were

One of the most complex biosynthetic processes in metallobiochemistry is the assembly of nitrogenase, the key enzyme in biological nitrogen fixation. We describe here the crystal structure of an iron-molybdenum cofactor-deficient form of the nitrogenase MoFe protein, into which the cofactor is inserted in the final step of MoFe protein assembly. The MoFe protein folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits. In this structure, one of the three alpha subunit domains exhibits a substantially changed conformation, whereas the rest of the protein remains essentially unchanged. A predominantly positively charged funnel is revealed; this funnel is of sufficient size to accommodate insertion of the negatively charged cofactor.

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 (

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The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP-AlF(4)(-) structure.