Crystal Structure of Azotobachter vinelandii Nitrogenase Iron Protein at 2.2 Å Resolution

Biological nitrogen fixation by the two-component metalloenzyme nitrogenase provides an elegant solution to the problem of reducing abundant, but relatively inert, dinitrogen to the biologically usable ammonia needed by all organisms. This oxygen sensitive enzyme, consisting of the separately purifiable nitrogenase iron protein and molybdenum iron protein, couples nucleotide hydrolysis to electron transfer to catalyze the ATP-dependent reaction. Iron protein acts as the sole known biological reductant to molybdenum iron protein, which contains the actual site of substrate reduction. MgATP binding to iron protein induces dramatic conformational changes in the protein's structure required for docking with molybdenum iron protein. Complex formation and dissociation are essential for nucleotide hydrolysis, electron transfer, and substrate reduction. We have determined the crystal structure of Azotobacter vinelandii nitrogenase iron protein at 2.2 Å resolution in the absence of nucleotide. Crystals grew in space group P212121, and represent a new crystal form compared with that of the structure previously determined at 2.9 Å resolution. X-ray diffraction data were collected from a single crystal using cryocrystallographic techniques. The structure was solved by molecular replacement, followed by solvent flattening and noncrystallographic averaging. The current model contains 575 of 578 possible amino acid residues and 372 solvent molecules, and has been refined to R-value of 22.3 % (R-free = 29.0 %) for all data to 2.2 Å, with good stereochemistry. The overall topology of nitrogenase iron protein consists of an Fe4S4 cluster symmetrically ligated by two identical subunits of doubly wound α/β structure similar to those of other nucleotide binding proteins. A detailed description is provided of those structural features important for iron protein function, including nucleotide binding regions, the Fe4S4 cluster environment, intersubunit interactions, and the molybdenum iron protein binding surface. Comparisons are made between the current model and that of C. pasteurianum iron protein, as well as those of two A. vinelandii nitrogenase complexes. Analysis of the various iron protein structures provides a framework for considering the wealth of relevant nitrogenase spectroscopic, biochemical, and genetic information.</p

Structure of ADP·AIF_4 -stabilized nitrogenase complex and its implications for signal transduction

The coupling of ATP hydrolysis to electron transfer by the enzyme nitrogenase during biological nitrogen fixation is an important example of a nucleotide-dependent transduction mechanism. The crystal structure has been determined for the complex between the Fe-protein and MoFe-protein components of nitrogenase stabilized by ADP·AIF_4 –, previously used as a nucleoside triphosphate analogue in nucleotide-switch proteins. The structure reveals that the dimeric Fe-protein has undergone substantial conformational changes. The β-phosphate and AIF_4 – groups are stabilized through intersubunit contacts that are critical for catalysis and the redox centre is repositioned to facilitate electron transfer. Interactions in the nitrogenase complex have broad implications for signal and energy transduction mechanisms in multiprotein complexes

Charges in Hydrophobic Environments: A Strategy for Identifying Alternative States in Proteins

In the V23E variant of staphylococcal nuclease, Glu-23 has a p<i>K</i>_a of 7.5. At low pH, Glu-23 is neutral and buried in the hydrophobic interior of the protein. Crystal structures and NMR spectroscopy experiments show that when Glu-23 becomes charged, the protein switches into an open state in which strands β1 and β2 separate from the β-barrel; the remaining structure is unaffected. In the open state the hydrophobic interior of the protein is exposed to bulk water, allowing Glu-23 to become hydrated. This illustrates several key aspects of protein electrostatics: (1) The apparent p<i>K</i>_a of an internal ionizable group can reflect the average of the very different p<i>K</i>_a values (open ≈4.5, closed ≫7.5) sampled in the different conformational states. (2) The high apparent dielectric constant reported by the p<i>K</i>_a value of internal ionizable group reflects conformational reorganization. (3) The apparent p<i>K</i>_a of internal groups can be governed by large conformational changes. (4) A single charge buried in the hydrophobic interior of a protein is sufficient to convert what might have been a transient, partially unfolded state into the dominant state in solution. This suggests a general strategy for examining inaccessible regions of the folding landscape and for engineering conformational switches driven by small changes in pH. These data also constitute a benchmark for stringent testing of the ability of computational algorithms to predict p<i>K</i>_a values of internal residues and to reproduce pH-driven conformational transitions of proteins