EPR investigation of the Mn(II) binding sites in glutamine synthetase (Escherichia coli W). I. High-affinity binding sites.:I. High-Affinity Binding Sites

The high-affinity (n₁, ) Mn (II) binding sites in glutamine synthetase [EC 6.3.1.2] have been examined as a function of Mn (II) concentration and the state of adenylylation by electron paramagnetic resonance (EPR) spectroscopy. Mn (II) EPR spectra are characteristic of paramagnetically dilute samples, and studies of the Mn(II) concentration dependence has indicated that these cations remain magnetically isolated (interaction distance_??_15-17Å) both at different states of adenylylation and in the presence of the inhibitor L-methionine (SR)-sulfoximine (MSOX). An investigation of metal-enzyme complexes at various states of adenylylation has revealed that the n₁, Mn (II) sites are not perturbed by the addition of 5'-adenylyl groups, suggesting that the n₁, Mn (H) sites are well removed from these groups. The small axial distortion (_??_15G) in these complexes is indicative of a nearly isotropic environment for Mn (II), and the invariance of linewidth with adenylylation and incorporation of L-glutamate suggests that the dominant relaxation mechanism for Mn (II) may not involve collisions with solvent molecules. In the presence of MSOX, the linewidth narrows drastically and forbidden transitions are observed. The n₁, Mn (II) site experiences an axial distortion which decreases linearly with increasing adenylylation. In addition, the Mn (II) hyperfine coupling constant, and hence bond ionicity, increases linearly with adenylylation. These results are interpreted in terms of possible longitudinal and transverse distortion mechanisms and their implications for catalysis are discussed. The measured conformational and bonding changes at the n₁, Mn (II) sites may reflect corresponding changes near the γ-carboxyl group of L-glutamate, whose activation is believed to be required for the biosynthetic reaction.