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

Structural data show that the light atom at the center of the nitrogenase active site cofactor is a carbon.

Reduction of N by nitrogenases occurs at an organometallic iron cofactor that commonly also contains either molybdenum or vanadium. The well-characterized resting state of the cofactor does not bind substrate, so its mode of action remains enigmatic. Carbon monoxide was recently found to replace a bridging sulfide, but the mechanistic relevance was unclear. Here we report the structural analysis of vanadium nitrogenase with a bound intermediate, interpreted as a μ-bridging, protonated nitrogen that implies the site and mode of substrate binding to the cofactor. Binding results in a flip of amino acid glutamine 176, which hydrogen-bonds the ligand and creates a holding position for the displaced sulfide. The intermediate likely represents state E or E of the Thorneley-Lowe model and provides clues to the remainder of the catalytic cycle.

Ammonium transporters (Amts) are integral membrane proteins found in all kingdoms of life that fulfill an essential function in the uptake of reduced nitrogen for biosynthetic purposes. Amt-1 is one of three Amts encoded in the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus. The crystal structure of Amt-1 shows a compact trimer with 11 transmembrane helices per monomer and a central channel for substrate conduction in each monomer, similar to the known crystal structure of AmtB from Escherichia coli. Xenon derivatization has been used to identify apolar regions of Amt-1, emphasizing not only the hydrophobicity of the substrate channel but also the unexpected presence of extensive internal cavities that should be detrimental for protein stability. The substrates ammonium and methylammonium have been used for cocrystallization experiments with Amt-1, but the identification of binding sites that are distinct from water positions is not unambiguous. The well ordered cytoplasmic C terminus of the protein in the Amt-1 structure has allowed for the construction of a docking model between Amt-1 and a homology model for its physiological interaction partner, the P II protein GlnB-1. In this model, GlnB-1 binds tightly to the cytoplasmic face of the transporter, effectively blocking conduction through the three individual substrate channels.archaebacteria ͉ membrane proteins ͉ nitrogen metabolism