Structure–function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high‐resolution structures

Abstract

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 (k cat /K m ). The Nii4-WT has Lys449 at the equivalent position of Gln448 in Nii3-WT. The enzyme activity assay revealed that the turnover number (k cat ) and Michaelis constant (K m ) of Nii4-WT were lower than those of Nii3-WT. However, the k cat /K m 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 in k cat /K m 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.

An alternative plant-like cyanobacterial ferredoxin with unprecedented structural and functional properties

“…NiR reduces nitrite to ammonium in a six-electron reaction, where electrons are provided by Fd red one by one [86][87][88]. The NiR catalytic center is a siroheme, which becomes reduced after single reduction by Fd red .…”

Heme in the marine environment: from cells to the iron cycle

“…41 Ferredoxin nitrite reductases utilize a siroheme, a heme analog, and a [4Fe-4S] cluster as catalytic cofactors. 42 A recent study has convincingly argued that when rapidly relieved of iron stress diatoms appear to partition iron to nitrate and nitrite reductases and other nitrate assimilation proteins while continuing to utilize iron-free photosynthetic proteins. 43 In the same study, iron addition also induced a significant upregulation of the porphyrin biosynthesis pathway, 43 further suggesting that diatoms rapidly partition iron into hemoproteins when relieved of iron stress.…”

X‐Ray Crystal Structure of a Mutant Assimilatory Nitrite Reductase That Shows Sulfite Reductase‐Like Activity

“…The Cl À ion was present because it was present in the reservoir solution used to crystallize Nii3-N226K (LF). This differs from the structure of Nii3-WT, in which a H 2 O molecule binds to siroheme [15]. The binding of the Cl À ion to Nii3-N226K (LF) is supported by the finding that a Cl À ion binds to siroheme in the structure of aSiR from Mycobacterium tuberculosis [9].…”

“…To measure the NO À 2 -reduction rates of Nii3-WT and Nii3-N226K, a method used in our previous study was applied [15]. However, the NO À 2 -reduction rate of Nii3-N226K could not be measured, because the rate slowed gradually over time and finally stopped at the midpoint of the reaction.…”