Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf-type (Nii1 and Nii3), and root-type (Nii2 and Nii4). The high-resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions, respectively, revealed that both proteins had very similar structures. The Nii3 structure provided detailed geometries for the [4Fe–4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3-M175G, Nii3-M175E, and Nii3-M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3-Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild-type (Nii3-WT) and the Met175 variants showed that the hydrophobic side-chain of Met175 facilitated enzyme efficiency (kcat/Km). The Nii4-WT has Lys449 at the equivalent position of Gln448 in Nii3-WT. The enzyme activity assay revealed that the turnover number (kcat) and Michaelis constant (Km) of Nii4-WT were lower than those of Nii3-WT. However, thekcat/Km of Nii4-WT was about 1.4 times higher than that of Nii3-WT. A comparison of the kinetics of the Nii3-Q448K and Nii4-K449Q variants revealed that the change inkcat/Km was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low-affinity and Nii4 is the high-affinity aNiR.