The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

The three-dimensional structure of the hexameric (αβ)6 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex iron-sulfur flavoprotein. In the hexameric species, interprotomeric α-α and α-β contacts are mediated by the C-terminal domain of the α subunit, which is based on a β helical fold so far unique to glutamate synthases. The αβ protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the β subunit, to the FMN on the α subunit, through the low potential [4Fe-4S]1+/2+ centers on the β subunit and the [3Fe-4S]0/1+ cluster on the α subunit. The (αβ)6 hexamer exhibits a concentration-dependent equilibrium with αβ monomers and (αβ)2 dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP+. Hexamerization seems to decrease the catalytic efficiency of the αβ protomer only 3-fold by increasing the Km values measured for l-Gln and 2-OG. However, it cannot be ruled out that the (αβ)6 hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.